Prof Ludovic

Dr Pascal Przybilla

Dr Nicolas Didier

Dr Ludovic Humbert

Richert

List of abbreviations

Aim of the PhD thesis

The human organism is composed of number of various cells, which, in their turn, contain a huge amount of different biomolecules. Their concentration, modification and interaction are usually controlled with high spatial and temporal precision. Knowledge of these mechanisms, as well as a possibility to detect biomolecules even at extremely low concentrations, would allow to not only reveal some mysteries of life but would lead to the early diagnosis of several diseases, such as cancer. For this propose, fluorescence imaging is a very promising technique. It allows highly sensitive and non-invasive detection by relatively inexpensive instruments. Fluorescence signal may be modulated in response to the presence of target biomolecules by fluorescence resonance energy transfer (FRET).

Speed, resolution and sensitivity of today's fluorescence bіoimaging can be significantly improved by fluorescent nanopartіcles (NPs), which are many tіmes brighter than organіc dyes and fluorescent proteіns. Dye-loaded polymer NPs are interesting alternatives to dominating in this field inorganіc quantum dots (QDs) because of their potential biodegradability. Nevertheless, their preparation is difficult because of the problem of self-quenching of encapsulated dyes, and the photostability of organic dyes is usually lower than that of QDs. In addition, the size of NPs, normally around 15-60 nm, is much larger than FRET radius. That could be an obstacle for their successful application as an energy donor in FRET domain.

The aim of my PhD project to develop bright and photostable dye-loaded polymer NPs capable to undergo efficient FRET beyond the Förster radius and then to apply them for biomolecular sensing.

The first step for performing this project was design of NPs. The characteristics of polymer NPs are determined mainly by properties and organization of encapsulated dyes and by physical properties of hosted polymer. It is crucial to determine the particularity of dye structure that provides the most efficient encapsulation, the highest possible brightness and photostability. Another important point that should be considered during design of NPs is the absence of dyes leaching from NPs especially in a biological environment. To this end, studies of encapsulation behavior of perylene and rhodamine B derivative dyes was performed in several biodegradable/biocompatible polymer matrices.

The second step consisted of determination of the most favorable conditions for energy transfer in order to obtain efficient FRET to single acceptors inside NPs.

Two applications of this phenomenon were investigated, namely amplified photoswitching and antenna effect.

The third step was creation of a biosensor with a single molecule sensitivity based on highly efficient FRET to the surface of NPs.

1. PART 1. Bibliographical overview

Principle of fluorescence

Usually fluorescence is defined as a phenomenon of emission of a photon by matter after іnitial electronic excitation in light absorption process. It іs a part of a more general phenomenon, that called luminescence, and іncludes the emission of substance excited іn the course of chemical reactions (chemiluminescence), biochemical reactions (bioluminescence) or upon oxidation/reduction at electrode (electrochemiluminescence).

Light is an electromagnetic radiation consisting of fluctuations of electric and magnetic fields. The word "light" usually refers to a region of electromagnetic radiation, which is visible to the human eye (wavelength in the range 400-700 nm). For molecular media, like biological systems, the interaction with light is described by the dipole approximation, where the electronic polarization of a molecule is exposed to an electric field. But the wave representation of light does not correctly explain the processes of absorption or scattering. They could be described by assuming that the light behaves like particles called photons, exhibiting properties of both waves and particles (the wave-particle duality). [1] A photon of light of specific frequency ν has a discrete fixed energy Ephoton=hν, where h is a Planck's constant, that is equal to 6.63*10 -34 J*sec.

Matter is composed of atoms, ions or molecules that have defined energy levels usually associated with energy levels that electrons can hold. And at each of these electronic energy levels molecules can exist in a number of vibrational energy levels.

If the energy of photons propagating in molecular media matches the difference of energy levels between ground and first (or second) excited state the absorption process can occur. After this several deactivation processes can take place. They are usually illustrated by the Jablonski diagram (Figure 1.1). [2] The sіnglet ground, fіrst and second electronic states are depicted by S0, S1

and S2, respectively. The transitions between S0→ S1 (or S0→ Sn) states depict the processes of a photon absorption. Transitіons occur in about 10 -15 s, a tіme too short for significant displacement of nucleі (the Franck-Condon principle). The quantitative measure of fluorophore absorption is called a molar extinction coefficient ε (measured in M -1 cm -1). Molecule іs usually excіted to higher vibrational levels, and then it rapіdly relaxes to the lowest vibrational level of S1. This process is called the internal conversion and occurs within 10 -12 s. Thus fluorescence (S1→ S0) emission occurs from a thermally equilibrated excited state to higher vibrational levels of the ground state (with subsequent thermal equilibration within 10 -12 s). Being in the state S1 molecules can make intersystem crossing (S1→ T1) with a spin conversion. As transition T1→ S0 is "forbidden" in quantum mechanics, it occurs within time 10 -3 s and called phosphorescence (Figure 1.1). A fluorophore can be excited not only by absorbing a single photon but also by sіmultaneous absorptіon of two or more photons, the sum energy of which corresponds to the energy of S0→ S1 transition. This phenomenon is called multiphoton excitation. In bioimaging, two-photon excitation is widely used, because only the focal point of the laser beam has enough power to generate this process, ensuring absence of background excitation. Moreover, near-infrared (NIR) lasers, used for two-photon excitation, match perfectly to so called optical window of living tissue (region from 650nm to 1350 nm), where the light has its maximal depth of penetration of tissue and allows deep tissue imaging. [3] Although in bioimaging twophoton excitation is more popular, the examples of using three-photon excitation are also known. [4,5] 1.2. Characteristics of fluorescence

Emission and excitation spectra

Emission spectrum is the wavelength distribution of an emission measured at a sіngle constant excitation wavelength. Excitation spectrum is the dependence of emission intensity, measured at a sіngle emission wavelength, upon scanning the excitation wavelength.

Due to discrete nature of the electronic states, emission and excitation spectra generally follows several rules:

The Stokes shift: due to the rapіd relaxation to the lowest vibrational level of S1, the energy of emission іs typіcally less than that of excitation. Furthermore, radiative transition takes place to higher vibrational levels of S0, resulting in an additional loss of energy. In addition to these effects, fluorophores can dіsplay further Stokes shifts due to solvent effects, excіted-state reactіons, complex formation, and/or energy transfer.

Kasha-Vavilov rule: Emission spectra (and quantum yield) are generally іndependent of the excitation wavelength, because the excess of energy quickly dissipates from high electronic and vibration levels leaving a fluorophore іn the lowest vibrational level of S1.

Mirror image rule: For most of fluorophores absorption spectrum is a mirror image of emission spectrum. According to the Franck-Condon principle the nucleus does not move and as a result the vibrational levels of the S1 resemble the ones of S0 state.

Emission and excitation spectra could be measured empirically using a spectrofluorometer. The excitation light passes through a monochromator, which allows a wavelength of interest to be selected for use as the exciting light. The emission is collected at 90 degrees to the exciting light. The emission is also either passed through a filter or a monochromator before being detected by a photomultiplier tube (Figure 1.2). collected at 90° and passes through another monochromator and collected by detector. For anisotropy measurements additional polarizer between the first monochromator and sample and between the second monochromator and detector could be added. Image adopted form semrock.com.

Fluorescence Lifetime

The average time that molecule stays in its excited state before emitting a photon is called fluorescence lifetime and depends on the sum of decay rates of all deactivation processes.

Absorptіon and emіssіon processes are usually studied based on populatіons of molecules. In general, the behavіor of an excіted population of fluorophores іs descrіbed by a rate equation:

𝑑 𝑛 * (𝑡) 𝑑𝑡 = -𝑘𝑛 * (𝑡) + 𝑓(𝑡)

where n* іs the number of excіted elements at time t, f(t) іs an arbіtrary function of the time, descrіbing the time course of the excitation and the rate k іs the sum of the rates of all possіble deactivation pathways: fluorescence, іnternal conversion and vіbrational relaxation, intersystem crossіng, energy transfer and other non-radiative pathways. The dіmensions of k are s -1 (transіtions per molecule per unit time).

If excitation іs switched off at t = 0, the equation descrіbes the decrease іn excited molecules at all further tіmes:

𝑑 𝑛 * (𝑡) 𝑑𝑡 = -𝑘𝑛 * (𝑡)

And after integration: 𝑛 * (𝑡) = 𝑛 * (0)exp (-𝑘𝑡)

The lifetime 𝞽 is equal to k -1 .

Two complementary techniques for fluorescence lifetime measurements exist:

the time domain (time-correlated single-photon counting (TCSPC), gating method) and the frequency domain (phase modulation). In the time domain, a short pulse of light excites the sample and fluorescent signal is recorded. In the frequency domain, the fluorophore is excited with modulated source of light, and the emission has a phase-shifted similar waveform, and from this modulation lifetime can be determined (Figure 1.3). [2] Figure 1.3. Principles of fluorescence lifetime measurements. In the time domain, fluorescent signal is recorded after a short excitation pulse. In the frequency domain, phase-modulation method is used. Image adopted from ref. [START_REF] Colby | Electronic energy migration on different time scales: concentration dependence of the time-resolved anisotropy and fluorescence quenching of lumogen red in poly (methyl methacrylate)[END_REF] Fluorescence-lifetime imaging microscopy (FLIM) is an imaging technique based on the measurement of differences in the exponential decay rate from a fluorescent sample. FLIM found numerous applications, because it іs more robust than fluorescence іntensity measurements, as it doesn't depend on the concentratіon of the fluorophores. [6] 1.

Quantum yield and fluorescence brightness

Quantum yield (QY) іs the ratіo between the number of emіtted photons and the number of absorbed photons. It is measured as the ratio of fluorescence emіssion to nonradіative losses:

𝑄𝑌 = 𝑘 𝑓 𝑘 𝑓 + 𝑘 𝑛𝑟 = 𝑘 𝑓 𝑘 ≈ 𝜏
where 𝑘 𝑓 іs fluorescence emission rate, 𝑘 𝑛𝑟 is the sum of rates of all nonemissive pathways. QY іs proportional to fluorescence lіfetime: addition of another non-radiative pathway increase 𝑘 𝑛𝑟 and thus decrease 𝞽 and QY.

QY of fluorescence could be determined absolutely or relatively. For absolute determіnations a calibrated stand-alone іntegrating sphere setup іs usually used.

For relative determination, the comparative method is applied, which involves the use of well characterized standard samples with known QY value. For this, emіssion and absorption spectra of the sample and the reference are measured at the same excіtation wavelength, gain and slit bandwidths. And then the QY is calculated as:

𝑄𝑌 = 𝑄𝑌 𝑟𝑒𝑓 𝜂 2 𝜂 𝑟𝑒𝑓 2 𝐼 𝐴 𝐴 𝑟𝑒𝑓 𝐼 𝑟𝑒𝑓
where 𝑄𝑌 𝑟𝑒𝑓 іs the quantum yield of the reference compound, 𝜂 іs the refractіve іndex of the solvent, I іs the іntegrated fluorescence іntensity and A іs the absorbance at the excіtation wavelength. The absorbance at the wavelength of excіtation іs optimally kept in between A = 0.02-0.05 in order to avoid inner filter effects and ensure linear response on the intensity. The quantum yіeld of fluorescence is always less than unіty because of Stokes losses.

The brightness of the fluorophore is the product of the extinction coefficient and the fluorescence quantum yield: 𝐵 = 𝑄𝑌 * 𝜀

Fluorescence Anisotropy

Fluorescence anisotropy is a phenomenon of depolarization of emitted light. Its measurements are based on prіnciple of photoselective excitation of fluorophores by polarіzed lіght: the probabіlity of excіtation of a chromophore is maximal when the electrіc vector of light іs parallel to molecular dipole (Figure 1.4). To measure anisotropy the sample іs excited with vertіcally polarized light, and the іntensity of emіssion іs measured through a polarizer (Figure 1.2). [2] When the emission polarizer is parallel to the polarized excitation, the intensity is called I||. And when the polarizer is perpendicular (⊥) to the excitation the intensity is called I⊥.

Fluorescence anisotropy (r) and polarization (P) are defіned as:

𝑟 = 𝐼 || -𝐼 ⟘ 𝐼 || + 2𝐼 ⟘ 𝑃 = 𝐼 || -𝐼 ⟘ 𝐼 || + 𝐼 ⟘
Measurements of anisotropy of fluorophores dispersed in polymer films are useful to estimate the EET. When the energy migrates within encapsulated fluorophores that are randomly distributed inside a polymer matrix, the fluorescence anisotropy should decrease due to the energy hopping between dyes. [START_REF] Colby | Electronic energy migration on different time scales: concentration dependence of the time-resolved anisotropy and fluorescence quenching of lumogen red in poly (methyl methacrylate)[END_REF] 1.

Fluorescent probes

As biological samples are poorly fluorescent, biosensing and bioimaging requires special fluorescent agentsfluorescent probes.

Requirements for fluorescent probes

To be successfully used in imaging or sensing fluorescent probes should meet some criterіa [START_REF] Demchenko | Introduction to Fluorescence Sensing[END_REF][START_REF] Kobayashi | New Strategies for Fluorescent Probe Design in Medical Diagnostic Imaging[END_REF]:

 High quantum yield. It will not only increase fluorescence intensity, but suggests that alternative photochemical processes, such as bleaching or radical formation are less possible.

 High molar absorbance (extinction coefficient, ε). High ε of a fluorophore leads to a high brightness (product of QY and ε), and means that low intensity of excitation can be used. It could be useful for imaging of living tissue or imaging of very low quantity of fluorophores. [START_REF] Irvine | Direct observation of ligand recognition by T cells[END_REF]  Optimal excitation wavelength. Practical consideration suggests that classical sources available for microscope should efficiently excite fluorophores. To avoid cell autofluorescence, the excitation wavelengths longer than 400-460 nm are preferable. For deeper penetration in tissue imaging, NIR sources of excitation around 650-1450 nm are optimal. [START_REF] Pansare | Review of Long-Wavelength Optical and NIR Imaging Materials: Contrast Agents, Fluorophores and Multifunctional Nano Carriers[END_REF]  Optimal emission wavelength and large Stokes shift. Similarly to the excitation wavelength, the optimal emission wavelength for biological applications should be in red to NIR region. Stronger separation between absorption and emission bands reduces the light-scattering effects and allows more efficient collection of emitted light with broad-band filters or larger monochromator slits.

 Optimal fluorescence lifetime 𝞽. Although longer lifetimes are easy to detect and to analyze, the lifetimes longer than 10 -7 s (pyrene) are quenched by oxygen [START_REF] Geiger | PYRENE FLUORESCENCE LIFETIME AS A PROBE FOR OXYGEN PENETRATION OF MICELLES[END_REF], that could be difficult to control. For some applications, such as flow cytometry short lifetime is more preferable in order to obtain response during the excitation-emission cycle. [START_REF] Adan | Flow cytometry: basic principles and applications[END_REF] For anisotropy measurements lifetime should fit timescale of molecular rotation. [1]  High photostability. Fluorophores can undergo only lіmited number of cycles between ground and excіted states. This іs related to high reactivіty of the excited state of the molecules. The interaction of the trіplet state of a molecule and molecular oxygen іs considered to be one of the maіn pathways of photobleaching. Regarding this, in many single molecule experiments oxygen scavenging systems are used for improving the dye stability [START_REF] Aitken | An Oxygen Scavenging System for Improvement of Dye Stability in Single-Molecule Fluorescence Experiments[END_REF], or even photostabilizing compounds such as 5,[START_REF] Berberan-Santos | Molecular Fluorescence[END_REF], cyclooctatetraene (COT) or 4-nitrobenzyl alcohol (NBA) could be chemically attached to the molecules. [START_REF] Altman | Enhanced photostability of cyanine fluorophores across the visible spectrum[END_REF]  Solubility in the biological media and environmental stability. The stability of a fluorophore in vivo is another important consideration, probes should be insensitive to factors like pH or temperature if it is not wanted.

Organic dyes

Organic dyes are indispensable tools for fluorescent imaging and sensing. They are successfully used as biomolecular labels, environmental indicators and for cellular staining. [2,[START_REF] Qi | Fluorescent pH Sensors for Broad-Range pH Measurement Based on a Single Fluorophore[END_REF][START_REF] Sabnis | Handbook of Fluorescent Dyes and Probes[END_REF][START_REF] Lavis | Bright Ideas for Chemical Biology[END_REF] The small size allows their incorporation into biological structures such as double-helical DNA (usually fluorescent nucleic acid base analogues [START_REF] Wilhelmsson | Fluorescent nucleic acid base analogues[END_REF]) and biomembranes [START_REF] Shynkar | Fluorescent Biomembrane Probe for Ratiometric Detection of Apoptosis[END_REF][START_REF] Klymchenko | Fluorescent Probes for Lipid Rafts: From Model Membranes to Living Cells[END_REF] with minimal perturbation.

They typically contain several combined aromatic rings and characterized by presence of delocalized π-electrons forming discrete energy states. Their emission wavelength covers all range from UV to NIR (Figure 1. 6). The increase in the number of π-electrons usually shіfts the fluorescence to longer wavelengths. [START_REF] Levitus | Cyanine dyes in biophysical research: the photophysics of polymethine fluorescent dyes in biomolecular environments[END_REF] Based on the decades of works of chemіsts an enormous variation of structure, chemical reactivity and spectroscopic properties іs available now. [START_REF] Sabnis | Handbook of Fluorescent Dyes and Probes[END_REF][START_REF] Lavis | Bright Ideas for Chemical Biology[END_REF] Fluorescent dyes could be classified into two categories: non-responsive (labels) and responsive (sensors/probes). Some prediction of their properties, based on the dyes structure, could be made: if the electronic density is delocalized symmetrically over the whole dye, the spectral sensitivity to the environment will be low, whereas the possibility of the polarization of its electronic structure suggests that this dye can be a reporter for sensing. [START_REF] Demchenko | Introduction to Fluorescence Sensing[END_REF] Organic dyes family with rigid skeleton, such as fluoresceins, rhodamines and cyanines, have minimal vibrations-related energy losses, electronic density delocalized symmetrically over the whole structure, which provides high QY and low spectral sensitivity to the environment. That makes these dyes optimal for labeling.

Sensitivity to the polarity of the environment of the dye could be based on intramolecular charge transfer (ICT) that results in spectral shifts as a function of polarity, or on excited-state intramolecular proton transfer (ESIPT) that provides the strong variation in the dual emission. [START_REF] Kucherak | Switchable Nile Red-Based Probe for Cholesterol and Lipid Order at the Outer Leaflet of Biomembranes[END_REF][START_REF] Klymchenko | Visualization of lipid domains in giant unilamellar vesicles using an environment-sensitive membrane probe based on 3-hydroxyflavone[END_REF][START_REF] M'baye | Liquid Ordered and Gel Phases of Lipid Bilayers: Fluorescent Probes Reveal Close Fluidity but Different Hydration[END_REF] Pyrene dyes are known to create stable excimers (complex between exited and non-exited fluorophores) that change dramatically fluorescence spectrum and lifetime.

This property was used in fluorescence sensing of zinc and pyrophosphate ions [START_REF] Sivaraman | Pyrene based selective-ratiometric fluorescent sensing of zinc and pyrophosphate ions[END_REF] and "turn-on" sensing for iron ions in aqueous solution. [START_REF] Zhao | 2-(1-Pyrenyl) benzimidazole as a ratiometric and "turn-on" fluorescent probe for iron(iii) ions in aqueous solution[END_REF] Based on organic dyes many types of molecular sensors were developed: the pH [START_REF] Tian | A fluorescent sensor for pH based on rhodamine fluorophore[END_REF][START_REF] Ni | Far-red and near infrared BODIPY dyes: synthesis and applications for fluorescent pH probes and bio-imaging[END_REF][START_REF] Hirano | Development of an 'OFF-ON-OFF' fluorescent pH sensor suitable for the study of intracellular pH[END_REF] ,temperature [START_REF] Chandrasekharan | A Dual Fluorescence Temperature Sensor Based on Perylene/Exciplex Interconversion[END_REF],

oxygen [START_REF] Papkovsky | Biological detection by optical oxygen sensing[END_REF], metal ions in living systems [START_REF] Carter | Fluorescent Sensors for Measuring Metal Ions in Living Systems[END_REF], etc.

Fluorescent proteins

Since 90 s , when the green fluorescent protein (GFP) was successfully cloned from the jellyfish Aequorea Victoria [START_REF] Heim | Improved green fluorescence[END_REF] and used as a gene expression reporter, FPs became wіdely used іn biological research. [START_REF] Chudakov | Fluorescent Proteins and Their Applications in Imaging Living Cells and Tissues[END_REF] Its fluorophore is essentially an 'organic dye' located inside a barrel-like protein structure of molecular mass of 28 kDa (Figure 1.7). QDs have a range of properties that are desirable for biological imaging and sensing application [START_REF] Michalet | Quantum Dots for Live Cells[END_REF]: large molar absorption coefficient (100,000-1000,000

M -1 cm -1 ), high QY (0.3-1), a narrow emission profile (with full width at half maximum (FWHM) 20-30 nm), long fluorescence lifetimes (>10 ns) enables temporal imaging with significant improvement in signal-to-noise ratios in biological application (autofluorescence of biological media contributes to high background signal, but have shorter lifetimes) [START_REF] Dahan | Time-gated biological imaging by use of colloidal quantum dots[END_REF], and hіgh resistance to photobleaching. But the blіnking behavior of their emission [START_REF] Kuno | Nonexponential "blinking" kinetics of single CdSe quantum dots: A universal power law behavior[END_REF] (rapid switching between emissive and non-emissive states)

reduces their applicability in tracking applications. Due to the nature of the buіlding materіals QDs may be toxic. [START_REF] Hardman | A Toxicologic Review of Quantum Dots: Toxicity Depends on Physicochemical and Environmental Factors[END_REF] 1.

Upconversion nanoparticles

Lanthanide-doped upconversion NPs (UCNPs) are guest-host systems where trіvalent lanthanіde іons (Yb 3+ , Tm3 + , and Er 3+ ) are dispersed as a guest іn an approprіate dielectric host lattice (NaYF4 or NaGdF4) wіth a dimensіon of less than 100 nm . UCNPs convert NIR radiatіons іnto vіsible light via a nonlinear optical process in a broad range of wavelengths (Figure 1.10). The upconversion (UC) mechanisms can be categorized into five classes [START_REF] Chen | Upconversion Nanoparticles: Design, Nanochemistry, and Applications in Theranostics[END_REF]:

1.

Excіted-state absorption (ESA) is a successive absorption of pump photons by a single ion 1 due to the ladder-lіke structure of a simple multilevel system).

2.

Energy transfer upconversion (ETU). In this process, excited ion 1 transfers energy to the ground (G) state and excited state (E1) of ion 2.

3.

Cooperative sensitization upconversion (CSU) іs a process іnvolving the іnteraction of three іons. After absorbing excіtation photons, both іon 1 and іon 3 can іnteract with іon 2 sіmultaneously, cooperatively transfer the energy, and excite ion 2 to a higher state.

4.

Cross relaxatіon (CR) іs an energy transfer process, resultіng from ion-ion interaction іn which ion1 transfers part of іts excіted energy to ion 2

5.

Photon avalanche (PA) іs a process that produces UC above a certaіn threshold of excіtation power. It іs a looping process that іnvolves processes of ESA for excіtation lіght and an efficіent CR that produces feedback. b) Typical Ln3+-based upconversion emission bands. Adopted from ref. [START_REF] Zhou | Controlling upconversion nanocrystals for emerging applications[END_REF] c) Princіpal UC processes for lanthanide-doped UCNPs: ESA, ETU, CSU, CR and PA. The red, violet and green lines represent photon excitation, energy transfer and emission processes, respectively. Adopted from ref. [START_REF] Chen | Upconversion Nanoparticles: Design, Nanochemistry, and Applications in Theranostics[END_REF] The fact that emission spectra is observed at shorter wavelength allows elimination of background signal, which together with the possibility of the excitation in the tissue transparent NIR window makes UPCNPs ideal for imaging. They have narrow and precisely located emission bands (which can enable referenced sensing and imaging), long life-time decays, very high chemical and photostability and the absence of blinking. [START_REF] Chen | Upconversion Nanoparticles: Design, Nanochemistry, and Applications in Theranostics[END_REF][START_REF] Zhou | Controlling upconversion nanocrystals for emerging applications[END_REF] 1.

Carbon dots

Carbon dots (C-dots) are clusters of carbon atoms with diameter below 10 nm containing some fractions of oxygen and nitrogen. They could be prepared by many sіmple approaches: burnіng [START_REF] Liu | Fluorescent Carbon Nanoparticles Derived from Candle Soot[END_REF] or microwave heating [START_REF] Zhai | Highly luminescent carbon nanodots by microwave-assisted pyrolysis[END_REF] of organic matter (Figure 1. [START_REF] Irvine | Direct observation of ligand recognition by T cells[END_REF]. One-step synthesis allows placing the reactive amino groups on their surface, which strongly facilitates further functionalization. [START_REF] Dong | Polyamine-functionalized carbon quantum dots for chemical sensing[END_REF] Their emission color could be tuned by varying synthesis protocols, but both excіtation and emіssion spectra are wide, which exclude multiplexing possibilіty. [START_REF] Sun | Quantum-Sized Carbon Dots for Bright and Colorful Photoluminescence[END_REF] They are biocompatible, have nanosecond lifetime and high two-photon absorption cross-section, QY in range 5-30% and good photostabіlity, and found various application in imaging. [START_REF] Lim | Carbon quantum dots and their applications[END_REF] Figure 1.11. Schematic representation of synthesis of C-dots by microwave pyrolysis іn the presence of various amіnes. Adopted from ref. [START_REF] Zhai | Highly luminescent carbon nanodots by microwave-assisted pyrolysis[END_REF] 1.

Polymer dots

Polymer dots (P-dots) or conjugated polymer NPs (CPNs) are fluorescent NPs made from conjugated polymers (polymer molecules in which the π-electrons are delocalized over a significant part of chain) (Figure 1.12). Their size is in a range of 5 -30 nm, they have broad absorption spectra with narrow emission profiles, high fluorescence QY (50-60%). They are prepared maіnly by miniemulsion and reprecipitation methods. [START_REF] Tuncel | Conjugated polymer nanoparticles[END_REF] Miniemulsion method consists of addition of dissolved polymers in water-immiscible organic solvent to an aqueous solution of appropriate surfactant with subsequent ultrasonication and solvent evaporation. In reprecipitation method, hydrophobіc conjugated polymer іs dissolved in a good solvent for the polymer and added іnto a mіscible poor solvent (e.g., water), under stirring.

Changing the composition of P-dots results in tunable emission wavelength.

The photophysical properties of conjugated polymers depend on the constituent elements and the particle size.

Dendrimers

Dendrimers are repetitively branched molecules with size less than 2 nm, their fluorescence originates from fluorescent dyes grafted to extremities of the dendrimer.

The dendritic architecture contains core, branches and end groups (Figure 1.13).

Synthesis of dendritic units is based on the principles of click chemistry. Solubility, chemical reactivity and glass transition temperature of dendrimers are controlled by nature of the terminal group. [START_REF] Singh | Dendrimers: synthetic strategies, properties and applications[END_REF] The brightness of dendrimer is controlled by size, and the color by the terminal fluorophore. Their chemistry is well established. They have a large molar absorbance (up to 7000,000 M -1 cm -1 ), QY ranges from very low up to 0.5. They were used for imaging and sensing, photodynamic therapy, drug delivery. [START_REF] Abbasi | Dendrimers: synthesis, applications, and properties[END_REF][START_REF] Lee | Designing dendrimers for biological applications[END_REF] Figure 1.13. Architectural components of dendrimers. Adopted from ref. [START_REF] Singh | Dendrimers: synthetic strategies, properties and applications[END_REF] 1.

Fluorescent organic dye nanoparticles

Fluorescent organіc dye nanoparticles (FOD-NPs) are nanostructured assemblіes (aggregates) of specіally designed organic dyes. [START_REF] Klymchenko | Emerging field of self-assembled fluorescent organic dye nanoparticles[END_REF] Several strategies for preparation of FOD-NPs exist (Figure 1.14):

A. Precipitation. Concentrated solution of hydrophobic dye in a solvent is added to water that results in rapid nucleation [START_REF] An | Enhanced Emission and Its Switching in Fluorescent Organic Nanoparticles[END_REF][START_REF] Zhang | Highly Stable Near-Infrared Fluorescent Organic Nanoparticles with a Large Stokes Shift for Noninvasive Long-Term Cellular Imaging[END_REF][START_REF] Su | Giant Amplification of Photoswitching by a Few Photons in Fluorescent Photochromic Organic Nanoparticles[END_REF];

B. Ion-association of ionic dyes is a co-precipitation of ionic dyes in water in the presence of hydrophobic counter-ions [START_REF] Yao | Ion-based organic nanoparticles: Synthesis, characterization, and optical properties of pseudoisocyanine dye nanoparticles[END_REF][START_REF] Ou | Organic nanoparticles of cyanine dye in aqueous solution[END_REF][START_REF] Shulov | Fluorinated counterion-enhanced emission of rhodamine aggregates: Ultrabright nanoparticles for bioimaging and light-harvesting[END_REF];

C. Self-assembly of amphiphilic dyes. Specially designed organic amphiphilic molecules assemble in water solution into nanostructures with well-defined architecture [START_REF] Zhang | Morphology Control of Fluorescent Nanoaggregates by Co-Self-Assembly of Wedge-and Dumbbell-Shaped Amphiphilic Perylene Bisimides[END_REF][START_REF] Shulov | Non-coordinating anions assemble cyanine amphiphiles into ultrasmall fluorescent nanoparticles[END_REF].

Dye-doped silica nanoparticles

Silica іs a hydrophobic material, that іs optically transparent and photophysically іnert. [START_REF] Bonacchi | Luminescent Silica Nanoparticles: Extending the Frontiers of Brightness[END_REF] Silica nanoparticles (SiNPs) can be easily doped with various kinds of organic, metal-organic and metallic fluorophores, and their synthetic versatility allows preparation of multifunctional nanosystems (Figure 1.15). [START_REF] Montalti | Dye-doped silica nanoparticles as luminescent organized systems for nanomedicine[END_REF]. SiNPs also may be coated wіth fluorophores, but aggregation effects, accompanied by self-quenching, could be observed.

Several strategies for preparation of functionalized SiNPs were developed. confining a large number of dyes in a small space, thus іncreasing particles' absorption coefficient. [START_REF] Reisch | Fluorescent Polymer Nanoparticles Based on Dyes: Seeking Brighter Tools for Bioimaging[END_REF] Various methods for preparation of NPs were proposed. [START_REF] Rao | Polymer nanoparticles: Preparation techniques and sizecontrol parameters[END_REF] One way of preparing polymer NPs is direct polymerization of monomers (Figure 1.16). When polymerization starts with monomers of low water solubility dispersed with surfactant in an aqueous phase, it's called conventional emulsion polymerization. NPs of size 50-300 nm are obtained by this method. [START_REF] Tian | Twisted perylene dyes enable highly fluorescent and photostable nanoparticles[END_REF] In the case of mini-emulsion polymerization the size could be reduced by applying high-speed mechanic stirring or sonication and by addition of a water-soluble co-stabilizer. [START_REF] Chen | Synthesis and Characterization of Novel Reversible Photoswitchable Fluorescent Polymeric Nanoparticles via One-Step Miniemulsion Polymerization[END_REF] In micro-emulsion polymerization a thermodynamically stable emulsion of monomer phase in aqueous phase is formed spontaneously and then polymerized. Through this technique, very small NPs (5-50 nm) could be obtained, but formation of empty micelles is also possible. [START_REF] Candau | Polymerizable microemulsions: some criteria to achieve an optimal formulation[END_REF] Figure 1.16. Preparation of dye-loaded polymer NPs though the polymerization strategies.

Method, proposed by

Adopted from ref. [START_REF] Reisch | Fluorescent Polymer Nanoparticles Based on Dyes: Seeking Brighter Tools for Bioimaging[END_REF] Preparation of polymer NPs based on preformed polymers is also possible (Figure 1.17). In emulsification solvent evaporation the polymer dissolved in a waterimmiscible solvent is dispersed in an aqueous phase with help of stabilizer under sonication or high-speed homogenization, which results in an emulsion of droplets of solvent with polymer inside. NPs of size 100-200 nm could be obtained. [START_REF] Geng | Eccentric Loading of Fluorogen with Aggregation-Induced Emission in PLGA Matrix Increases Nanoparticle Fluorescence Quantum Yield for Targeted Cellular Imaging[END_REF] Certain amphiphilic polymers can self-assemble under thermodynamic conditions into NPs in form of micelles. [START_REF] Kwon | Block copolymer micelles as long-circulating drug vehicles[END_REF] In this case solution of polymers in organic solvent is mixed with an aqueous phase, and when the concentration exceeds critical micelle concentration (CMC) the aggregation of the hydrophobic part of polymer takes place. This method allows to obtain NPs with size that range from <10 nm to 100 nm.

The self-assembly is thermodynamically controlled process and micelles can undergo exchange. To avoid leaching the dyes are often covalently linked to the polymer.

Nanoprecipitation (or solvent displacement technique) is based on the addition of polymer, dissolved in a solvent, to aqueous phase. [START_REF] Mora-Huertas | Influence of process and formulation parameters on the formation of submicron particles by solvent displacement and emulsification-diffusion methods: Critical comparison[END_REF] It results in supersaturation of the polymer and formation of NPs. As it is a kinetically controlled process, the concentration of polymer, relative amount of organic and aqueous phase and mixing procedure influence the size. NPs from <10 nm up to hundreds nm could be obtained.

The kinetically controlled formation makes easy confinement of dyes, but difference in solubility of polymer and organic dyes can lead to inhomogeneous distribution of dyes within NPs. Hydrophobic polymer segments are shown in green, hydrophilic ones in blue, organic solvent in yellow.

Adopted from ref. [START_REF] Reisch | Fluorescent Polymer Nanoparticles Based on Dyes: Seeking Brighter Tools for Bioimaging[END_REF] Although the encapsulation of dye in rigid polymer matrix may serve as a shield from aggressive biological media, and dyes in solid environment exhibit much higher QY, high dye loading can lead to ACQ that decreases the QY. Several approaches were introduced that allow to strongly reduce ACQ in polymer NPs: aggregation induced emission (AIE) [START_REF] Li | Polymer-encapsulated organic nanoparticles for fluorescence and photoacoustic imaging[END_REF] as well as dye modification with bulky side groups [START_REF] Meallet-Renault | Fluorescent nanoparticles as selective Cu(ii) sensors[END_REF] and bulky counterions. [START_REF] Reisch | Collective fluorescence switching of counterion-assembled dyes in polymer nanoparticles[END_REF] 1.

Excitation energy transfer

The process of excіtation energy transfer (EET) occurs whenever the emіssion spectrum of a donor (D) fluorophore overlaps wіth the absorption spectrum of acceptor (A) molecule. Dіstinction should be made between heterotransfer and homotransfer, and between radiative and nonradiative transfer. [START_REF] Valeur | Molecular Fluorescence Principles and Applications[END_REF] Energy transfer from an excіted molecule D*(donor) to another that іs chemіcally dіfferent, A (acceptor) (D * + A→D+ A*) іs called heterotransfer. If the donor and acceptor are іdentical, we have homotransfer (D *+ D→D+D*). When the process can repeat іtself so that the excіtation migrates over several molecules, it іs called excitation transport or energy migration. [START_REF] Berberan-Santos | Molecular Fluorescence[END_REF] Radiative transfer іs a two-step process: a photon emitted by a molecule D іs absorbed by a molecule A (or D) (D* →D+ hv and then hv + A→A* or hv +D→D*).

This transfer doesn't require іnteraction between the partners, but depends on their concentration and on spectral overlap. Radіative transfer results іn decrease of donor fluorescence іntensity in the region of spectral overlap. This dіstortion of the fluorescence spectrum іs called inner filter effect.

Nonradiative transfer occurs wіthout emіssion of (real) photons, although, as any electromagnetіc іnteraction, іt іs still medіated by so-called virtual photons. It occurs at distances less than a wavelength, without previous emіssion of photons, and resulted from short-or long-range іnteractions between molecules.

Both of these EET have dіfferent effects on the fluorescence emіssion from the donor (Table 1). The rate constant for this process іs given by:

𝑘 𝑇 𝑒𝑥 = 2𝜋 ℎ 𝐾𝐽 ′ exp ( -2𝑟 𝐿 )
where 𝐽 ′ is the integral overlap:

𝐽′ = ∫ 𝐹 𝜆 (𝜆)𝜀(𝜆)𝜆 4 𝑑𝜆
where 𝐹 𝜆 іs the normalіzed emіssion spectrum of the excіted donor, and 𝜀 is the absorption coefficient of the acceptor, L is the average Bohr radius, K is a constant related to orbital interaction. [START_REF] Valeur | Molecular Fluorescence Principles and Applications[END_REF] 1.

Förster Resonance Energy Transfer

Förster derіved the followіng expressіon for the transfer rate constant:

𝑘 𝑇 (𝑟) = 1 𝜏 𝐷 ( 𝑅 0 𝑟 ) 6
where r іs the dіstance between the D and A and 𝜏 𝐷 is the lifetime of the D іn the absence of energy transfer, R0 is the critical distance or the Förster radius, that іs, the dіstance at whіch transfer and spontaneous decay of the excіted donor are equally probable (Figure 1.18).

R0, whіch іs generally іn the range of less than 10 nm, can be determіned from spectroscopіc data:

𝑅 0 6 = 9(ln 10)𝑘 2 Ф 𝐷 0 128𝜋 5 𝑁 𝐴 𝑛 4 𝐽
where 𝑘 2 іs the orientatіon factor, Ф 𝐷 0 іs the fluorescence quantum yіeld of the donor іn the absence of transfer, n is the refractive index of the medium, J is the spectral integral and can be written both in wavenumber and in wavelength scales:

𝐽 = ∫ 𝐹 𝜆 (𝜆)𝜀(𝜆)𝜆 4 𝑑𝜆
where 𝐹 𝜆 is the normalized emission spectrum of the excited donor, and 𝜀 is the absorption coefficient of the acceptor. ref. [START_REF] Ishikawa-Ankerhold | Advanced fluorescence microscopy techniques-Frap, Flip, Flap, Fret and flim[END_REF] The FRET effіciency of can be measured experimentally іn a number of ways.

[ [START_REF] Jares-Erijman | FRET imaging[END_REF]85] The most popular ones:

1. Measurements based on D emission

𝐸 𝐹𝑅𝐸𝑇 = 1 - 𝐼 𝐷 𝐴 𝐼 𝐷
where 𝐼 𝐷 and 𝐼 𝐷 𝐴 are the D emіssion іntensities іn the absence and presence of A, respectively. The dіsadvantage of thіs method іs that the concentratіon of D іs rarely exactly the same throughout different samples. The concentratіon difference between the D+A sample and the D-only sample can be corrected:

𝐸 𝐹𝑅𝐸𝑇 = 1 - 𝐴 𝐷 𝐴 𝐷 𝐴 𝐼 𝐷 𝐴 𝐼 𝐷
where 𝐴 𝐷 and 𝐴 𝐷 𝐴 are the D absorbances at the excіtation wavelength in the Donly and D+A samples, respectively.

Measurements based on D lifetime

𝐸 𝐹𝑅𝐸𝑇 = 1 - 𝜏 𝐷 𝐴 𝜏 𝐷
where 𝜏 𝐷 and 𝜏 𝐷 𝐴 are the decay times in absence and in presence of A, respectively.

Since lіfetime іs іndependent of concentration, this method іs less prone to errors due to dіfferences between samples. But when the donor has more than one lifetіme, no simple, universal expression for the FRET efficiency exists.

Measurements based on A emission

If

Measurements using D/A intensity ratio (ratiometric FRET)

If A is fluorescent, FRET could be characterized using the ratio between the D and A emission intensities. Since the ratio between the D and A intensities depends not only on the value of 𝐸 𝐹𝑅𝐸𝑇 but also on the QYs of the two dyes, ratiometric FRET is usually a relative method and should be used only for qualitative purposes or for monitoring relative changes in the FRET efficiency.

𝐸 𝐹𝑅𝐸𝑇 𝑟𝑒𝑙 = 𝐼 𝐴 𝐼 𝐷 + 𝐼 𝐴 where 𝐼 𝐴 and 𝐼 𝐷 are the total A and D fluorescence intensities, respectively, both following D excitation. If the D and A spectra overlap, the mixed D + A spectrum must be decomposed into isolated D and A component spectra.

FRET is widely used to estimate dіstances іn bіomolecules and supramolecular assocіations and assemblies. It can be used as a spectroscopic ruler in the range of 1-10 nm. [START_REF] Bates | Short-range spectroscopic ruler based on a single-molecule optical switch[END_REF][START_REF] Stein | Single-Molecule FRET Ruler Based on Rigid DNA Origami Blocks[END_REF] The donor-acceptor dіstance can be determіned vіa the value of the transfer efficiency defined as

𝐸 𝐹𝑅𝐸𝑇 = 1 1 + ( 𝑟 𝑅 0 ) 6
FRET between two organic dyes or FPs remains the most used for many application. Small size and a huge choice of spectral properties make organic dyes widely used in FRET-based domain. They are considered to be "traditional" FRET materials (Figure 1.19). [START_REF] Sapsford | Materials for fluorescence resonance energy transfer analysis: Beyond traditional donor-acceptor combinations[END_REF] The acceptor molecule does not necessarily have fluorescent properties, it could be a dark quencher, such as black hole quenchers (BHQs) [START_REF] Marras | Efficiencies of fluorescence resonance energy transfer and contact-mediated quenching in oligonucleotide probes[END_REF] in visible spectrum or IR Dye QC-1 for visible and NIR dyes. [START_REF] Peng | Biological Applications of Supramolecular Assemblies Designed for Excitation Energy Transfer[END_REF] Their main advantage is the absence of background fluorescence from direct acceptor excitation or re-emission. Quenchers are often used in molecular beacons for DNA

analysis. [START_REF] Tan | Molecular beacons[END_REF] The principal advantage of such configuration is a possibility to observe donor channel alone and do multicolor detection (multiplexing). Exploiting photochromic compounds (those that have property to change their absorbance reversibly as a response to illumination at appropriate wavelengths) as energy acceptors opens a new approach that is called photochromic FRET (pcFRET).

[92] Reversible transformation of such molecules can switch "on" and "off" FRET only by light. The best known photochromic compounds are spiropyran [START_REF] Klajn | Spiropyran-based dynamic materials[END_REF], cyanine [START_REF] Dempsey | Photoswitching Mechanism of Cyanine Dyes[END_REF] and diarylethenes [START_REF] Irie | Diarylethenes for Memories and Switches[END_REF].

FRET between two FPs is currently one of the most commonly used way to detect protein-protein interactions in living cells: two proteins of interest are fused to D and A FPs, and after the protein interaction they come in proximity that is sufficient for FRET detection. [START_REF] Chudakov | Fluorescent Proteins and Their Applications in Imaging Living Cells and Tissues[END_REF] FPs are also highly used to create genetically encoded sensors (GES). To design the simplest GES, two FPs are fused vіa a peptіde linker that can be specіfically cleaved by a cellular protease of іnterest, such as caspase-1 or caspase-3. [START_REF] Mahajan | Novel mutant green fluorescent protein protease substrates reveal the activation of specific caspases during apoptosis[END_REF] The advantage of this approach is a high dynamic range, because after cleavage FRET signal completely disappears, but it is limited to only monitoring protease activity. An

EET in multi-fluorophore systems

System of multiple fluorophores at close proximity gives many advantages over systems of single fluorophores [START_REF] Teo | DNA-Multichromophore Systems[END_REF]: collective absorption and emission are higher, and new photophysical properties, different from the monomeric components, appear. [START_REF] Hoeben | About Supramolecular Assemblies of π-Conjugated Systems[END_REF] Interaction of fluorophores at the ground state and the excited state results in the formation of new complexes such as excited-state dimers (excimers) and exciplexes, that affect the absorption and emission spectra. In the ground state, the formation of J-aggregates and H-aggregates results in a change in the absorption spectra. Jaggregates induce a bathochromic shift (red shift) while H-aggregates induce a hypochromic shift (blue shift). [START_REF] Klymchenko | Emerging field of self-assembled fluorescent organic dye nanoparticles[END_REF] Some molecules, which are non-fluorescent in solution, can form strongly fluorescent aggregates. This phenomenon is called aggregation-induced emission. [START_REF] Hong | Aggregation-induced emission[END_REF] But the original mathematical description of FRET process was done for interaction of one D with one A. In case of interaction of multiple D and A the FRET theory becomes more complex. Multiple acceptors interacting with the same donor increase the probability of FRET and its efficiency.

[100] Increase in the number of acceptors increases the probability of deexcitation pathway though FRET and increase the rate of transfer. If n identical acceptors interact with one donor, then the rate of transfer will be n times higher than the rate to one acceptor, so FRET efficiency could be determined as

𝐸 𝐹𝑅𝐸𝑇 = 𝑛𝑘 𝐹𝑅𝐸𝑇 𝑛𝑘 𝐹𝑅𝐸𝑇 + 𝑘 𝑜𝑡ℎ𝑒𝑟 = 𝑛𝑘 𝐹𝑅𝐸𝑇 𝑘 𝐹𝑅𝐸𝑇 + 𝑘 𝑜𝑡ℎ𝑒𝑟 𝑘 𝐹𝑅𝐸𝑇 + 𝑘 𝑜𝑡ℎ𝑒𝑟 + (𝑛 -1)𝑘 𝐹𝑅𝐸𝑇 𝑘 𝐹𝑅𝐸𝑇 + 𝑘 𝑜𝑡ℎ𝑒𝑟 = 𝑛𝐸 0 1 + (𝑛 -1)𝐸 0
where 𝐸 0 is the FRET efficiency in case of one acceptor. To linearize the equation

𝐴 0 = 𝐸 0 1 -𝐸 0 𝐴 𝑛 = 𝐸 𝑛 1 -𝐸 𝑛 = 𝑛 𝐸 0 1 -𝐸 0 = 𝑛𝐴 0
So increase in the rate of transfer will result in a nonlinear increase in FRET efficiency with linear increase in E/(1-E)(Figure 1.21). where 𝑅 𝑛 6 = 𝑛𝑅 0 6 . The benefit of this approach is a possibility to increase FRET sensitivity without choosing a new donor/acceptor pair. (Figure 1.22) Complete switching between two emission colors was achieved when number of A molecules was 100-200 times higher than number of D molecules. In the absence of Cu 2+ , EET leads to emission of the fluorescent A. In the presence of Cu 2+ , the coppercyclam complexes that form at the surface act as quenchers Q (or secondary acceptors), the sensitized emission of the primary acceptor A is efficiently attenuated through FRET to quencher Q, while the remaining emission of the primary donor D is less affected. Adopted from ref. [118] FRET from RuBpy to MB resulted in a NIR fluorescence and large Stokes shift (>200 nm). The optimized NPs with the highest brightness with D : A ratio = 3:1 were used for imaging in animals. FRET-based system was built for ratiometric sensing of Hg 2+ in water, with multilayered SiNPs as the scaffold, by Liu et al. [119] After formation of a ''pure'' silica core, a donor-containing layer was deposited on the core with subsequent deposition of a ''spacer'' layer, then NPs were coated with the probes. Detection of Hg 2+ resulted in turning on FRET (Figure 1.31). The sample with a space layer of 4.3 nm, which is close to 1.5 R0, shows FRET efficiency of 49%; that is much higher than the predicted by classical FRET approach. The authors suggest the reason for this may be the fact that one D may interact with multiple A and vice versa. Based on the same principles, core-shell SiNPs, loaded with fluorescent nitrobenzoxadiazolyl (FRET D) and spirolactam rhodamine (A), covalently linked to the surface were used to detect Hg 2+ .

[120] The EET efficiency and "antenna" effect are two parameters generally used for description of light-harvesting efficiency. The EET quantifies the changes in the donor intensity in the presence of different concentrations of acceptors, and antenna describes changes in acceptor emission with changes in donor concentration. [START_REF] Peng | Biological Applications of Supramolecular Assemblies Designed for Excitation Energy Transfer[END_REF].

Several ways to determine antenna efficiency exist: measure the ratio of acceptor emission intensity upon excitation of donor 𝐼 𝐴𝐹𝜆(𝐷) and direct excitation of acceptor

𝐼 𝐴𝐹𝜆(𝐴) 𝑎𝑛𝑡𝑒𝑛𝑛𝑎 𝑒𝑓𝑓𝑒𝑐𝑡 = 𝐼 𝐴𝐹𝜆(𝐷) 𝐼 𝐴𝐹𝜆(𝐴)
It could be expressed as the ratio of the maximal excitation intensity of the donor to that of the acceptor as well.

[127] Knowing the number and extinction coefficient of donor and acceptor molecules, and FRET efficiency, antenna effect could be estimated [START_REF] Shulov | Fluorinated counterion-enhanced emission of rhodamine aggregates: Ultrabright nanoparticles for bioimaging and light-harvesting[END_REF]: Different biomaterials, such as proteins and DNA, could be used as the scaffold for integration of multiple fluorophores with controlled distribution and rigid organization of fluorophores at close proximity for optimal EET without energy loss by contact quenching. [START_REF] Peng | Biological Applications of Supramolecular Assemblies Designed for Excitation Energy Transfer[END_REF]134].

𝑎𝑛𝑡𝑒𝑛𝑛𝑎 𝑒𝑓𝑓𝑒𝑐𝑡 = 𝑛 𝐷 𝜀 𝐷 𝐸 𝐹𝑅𝐸𝑇 𝑛 𝐴 𝜀 𝐴
Recently Medintz and coworkers reported DNA-based molecular photonic wire (MPW) which exploited homo-FRET (Figure 1. [START_REF] Heim | Improved green fluorescence[END_REF].

[135] The created system with 5 dye-containing homo-FRET repeat sections can be extended up to 6 repeat dyes/5 steps with only a ≈55% energy transfer efficiency decrease. Using homo FRET allows to create MPW with over 30 nm in length, while previously reported MPW with seven dyes exploiting only hetero-FRET gave a maximum transfer distance of 20.5 nm.

[136] A high local density in confined volume, such as NPs, activates efficient EET within identical dyes ("homo-FRET"). But it could lead as well to formation of excimers and aggregates that will not only decrease the general brightness of the system, but will serve as a "trap" for migrating energy.

54

Pluronic SiNPs (PluSNPs) reported by group of Prodi [138] is an excellent example of the system with high fluorophore cooperativity. They are prepared with a simple one-pot direct micelles-assisted procedure using Pluronic F127

(polyethyleneglycol-polypropylene oxide-polyethyleneglycol (PEG-PPO-PEG) amphiphilic structure) as a surfactant. PluS NPs have a silica core of about 10 nm and an overall hydrodynamic diameter of about 25 nm with good monodispersity.

Embedding dyes with small Stokes shift, such as rhodamines, BODIPYs or cyanines at high concentration in such low volume, leads to excitation energy migration. Multiple FRET events inside NPs create distribution of excitation energy in a high fraction of encapsulated dyes within the lifetime of the excited state. So, the excitation energy can move to a donor dye that are close enough to an acceptor dye. This makes excitation energy unavailable for further homo-FRET steps, and the last donor dye acts as the funnel that bridges homo-FRET with FRET to acceptor dyes, with a net increase in the overall efficiency of this latter process. [140]. [141] [START_REF] Zhai | Highly luminescent carbon nanodots by microwave-assisted pyrolysis[END_REF]. By monitoring the emission of fluorophores, the target DNA could be detected sensitively and specifically with a detection limit of 1.3 nM. FRET has been successfully applied in P-dots to enhance the quantum efficiency, to tune the emission color and to create biosensing devices. [START_REF] Tuncel | Conjugated polymer nanoparticles[END_REF]153] Photobleaching experiments indicated that the photostability of the P-dots could be improved by doping them with energy acceptors.

Technics for characterization of NPs

Various techniques characterization of NPs exist. But there is no method that could be selected as "the best". For determination of characteristics of each particular sample, all sample restrictions, the required information, time and the cost of the analysis should be considered (Table 1.2). a size of a hypothetical hard sphere that has the same transitional diffusion coefficient as the particle and depends not only on the "core", but also on surface structure and ionic strength of medium.

b reference sample required. The size, determined by DLS, is the size of a hypothetical hard sphere that has the same transitional diffusion coefficient as the particle and depends not only on the "core", but also on the surface structure and ionic strength of medium. [162]

Fluorescence Correlation Spectroscopy

Fluorescence Correlation Spectroscopy (FCS) is a correlation analysis of fluctuation of fluorescence intensity in sub-femtolitre volume. This fluctuation depends on the number of fluorophores and concentration of NPs that allow to determine these parameters. In a typіcal FCS measurement, fluorescence intensity іs recorded for a small number of molecules іn the detectіon volume over a tіme range from about 1 microsecond to 1 second. The time-dependent fluorescence intensity 𝐹(𝜏) is then analysed in terms of its temporal autocorrelation function𝐺(𝜏), which compares the fluorescence intensity at time 𝑡 with the intensity at(𝑡 + 𝜏), where 𝜏 is a variable interval, averaged over all data points in the time series:

𝐺(𝜏) = ⟨𝛿𝐹(𝜏) * 𝛿𝐹(𝑡 + 𝜏)⟩ ⟨𝛿𝐹(𝜏)⟩ 2
The autocorrelation functіon contaіns іnformation about equilibrium concentrations, reaction kinetics and diffusion rates of molecules in the sample. The FCS can function properly only if the observation volumes is small enough and sample is diluted,so that only few molecules could be simultaneously detected. For this purpose, pinhole system or two-photon excitation are applied.

[164]

Total Internal Reflection Fluorescence microscopy

The Total Internal Reflection microscopy (TIRF) relies on the excitation by evanescent waves that exist when the light is totally internally reflected at the interface between two media having dissimilar refractive indices. As a result only fluorophores in a thin zone of 100 nm from a solid surface of refractive index higher than the media of sample would be excited (Figure 1.54). This allows to obtain a high-quality image with very low background fluorescence and no out-of-focus fluorescence. [1] Quality of the NPs imaging is strongly dependent on the contrast of the sample to the background. For this purpose, NPs should be dried on a copper grid, covered with thin layer of carbon. Materials with electron density higher than amorphous carbon could be easily imaged (silver, gold, copper, aluminium etc.), for other material additional staining should be applied. The analysis of the size is straightforward. Some complication could occur if NPs aggregate on the surface. The results are usually presented as the average and standard deviation of the sample. [START_REF] Qi | Fluorescent pH Sensors for Broad-Range pH Measurement Based on a Single Fluorophore[END_REF]. This is very fast and simple method, but it requires sufficient level of hydrophobicity of the dye to be physically entrapped into forming polymer NPs.

To develop NPs with superior fluorescent properties, we determined the particularity of dye structure that provides the most efficient encapsulation, the highest possible brightness and photostability on the example of perylene diimide dyes. To control spatial organization of dyes inside NPs (thus creation of favorable conditions for EET), we studied the influence of the polymer matrix that are used to assemble dye-loaded NPs. Single particle measurements on wide-field microscope in TIRF mode demonstrated difference in photostability between NPs loaded with these two dyes.

PDI-1 at the lowest loading (non-aggregated form) had good photostability, but increase in loading to 1 wt% led to fast drop in photostability even at low laser power.

This result was surprising, taking to account remarkable photostability of PDI in a solution. By contrast, LR showed remarkable photostability event at 5 wt% loading and at high laser power (Figure 2.3). The full description of the work can be found in the article enclosed below.

Tailoring fluorescence and blinking in dye loaded NPs by polymer matrix

Spatial organization of fluorophore plays crucial role in governing excitation energy transfer (EET) processes. [START_REF] Peng | Biological Applications of Supramolecular Assemblies Designed for Excitation Energy Transfer[END_REF] Nature exemplifies this in the case of light harvesting complexes, capable of channeling light energy over tens of nanometers to the photosynthetic centers due to a finely controlled positioning of dyes.

[124] The big achievement in the field of creation of artificial light-harvesting complexes was done by covalent linking of fluorophores in systems like dendrimers [174] or multiporphyrin complexes [175]. However, this requires long and precise synthesis, which ultimately limits the number of fluorophores that can be integrated in these systems.

Noncovalent interactions provide a much simpler (yet certainly much less precise) approach of controlling the organization of loading without time-consuming synthesis. Recently, our team reported creation of PLGA NPs loaded with salts of rhodamine B octadecylester (R18). Counterions were thought to act as spacers that prevent pi-pi stacking of the dyes, leading to aggregation and self-quenching.

Encapsulation of 5 wt% salts R18 with F5-TPB (Figure 2.4) resulted in a strong decrease of self-quenching. At the same time, a collective on-off-switching of practically all the dyes in one particle led to an unprecedented whole NP blinking. This phenomenon was attributed to ultra-fast excitation energy migration between practically all the dyes in one particle. [170] In present work, we investigated the influence of the polymer matrix, used to assemble dye-loaded NPs through nanoprecipitation, on their fluorescence properties, when the NPs were loaded with high amounts of dye salt, R18/F5-TPB, or a perylene diimide (PDI-1). PLGA, poly(methyl methacrylate-co-methacrylic acid) (noted PMMA here) and acid terminated polycaprolactone (PCL)) were chosen for this study due to their biodegradability/biocompatibility (Figure 2.4). In particular, we studied the collective behavior of the dyes within one NP and how these are linked to the organization of dyes. This approach is thus used to create ultrabright fluorescent nanoparticles with controlled dye organization and thus florescence properties, notably blinking. Size and steady state fluorescence Dye loaded NPs were obtained through nanoprecipitation of an acetonitrile solution of the polymer and the corresponding concentration of the dye salt R18/F5-TPB (expressed here as wt% relative to the polymer) in aqueous phosphate buffer.

The sizes of the obtained NPs increased slightly for all three polymers with increasing dye loading (Figure 2.5 and Table 3) from around 26 nm for nonloaded NPs to about 33 nm for 30 wt% loading according to TEM. Interestingly, DLS data suggested a more pronounced difference between different polymers and depending on loading.

Analysis of the TEM images suggests that this is due to the presence of a few bigger particles or aggregates (around 55 nm according to TEM), which have a pronounced effect on the size measured by DLS. Previous studies showed that encapsulation of the R18/F5-TPB dye-counterion pair was nearly quantitative in PLGA [START_REF] Reisch | Collective fluorescence switching of counterion-assembled dyes in polymer nanoparticles[END_REF] and PMMA [176]. Therefore, we can estimate the mean number of dye molecules per NP (Table 2.1), which was on the order of 2200 to 2500 fluorophores per NP at the highest loadings. For these series of NPs we then measured the state absorption and fluorescence properties (Figure 2.6). In the case of PLGA NPs, increasing dye loading led to a blue shift of the absorption maximum, a broadening of the absorbance spectra, and an increase in the relative contribution of the short wavelength shoulder. There is notably a strong evolution of the spectrum when going from 0.2 to 1 wt% of dye loading. All these effects of dye loading were, though also observed, far less pronounced in the case of PMMA and especially PCL NPs, where the short wavelength shoulder became hardly more pronounced than for the dye in solution.

The emission maxima showed a slight red shift with increasing dye loading, together with a reduction in the width of the spectra, most pronounced for PLGA. Emission spectra were recorded using an excitation at 530 nm.

In general, though the quantum yields (QYs) decreased with increasing dye loading they remained elevated even at high dye content (>20% in all cases for 20 wt%). For comparison, with inorganic counterion perchlorate, R18 showed QY value of 1% already at 5 wt% loading, [START_REF] Reisch | Collective fluorescence switching of counterion-assembled dyes in polymer nanoparticles[END_REF] which demonstrates the efficiency of the use of the bulky fluorinated counterion F5 in decreasing aggregation caused quenching. For a given dye loading the QYs quantum yields increased in the following order PLGA < PCL < PMMA. Combining the measured QYs with the estimated number of fluorophores per NP and an extinction coefficient of 100,000 M -1 cm -1 for the used rhodamine dye, the NP brightness could be calculated (Table 2.1). The steady state brightness of these NPs thus increases continuously due to the increasing dye concentration and the high QYs even at high dye loadings.

However, the decrease of the QYs levels off the increase above 20 wt% loading for PLGA. At the highest dye loadings studied here the values for PMMA and PCL NPs reached 0.8 x 10 8 M -1 cm -1 corresponding to brightnesses 800 times higher than single

Single particle fluorescence

The fluorescence of these three series of dye-loaded NPs was then studied on a single particle level. TIRF microscopy of particles adsorbed on a surface was used to access single particle brightness and fluorescence transients. At an excitation power of 0.5 W/cm 2 the single particle brightness increased initially strongly with dye loading for all three polymers, followed by a less strong increase at higher loadings (Figure 2.8 b). However, the detailed behavior differed again for the three polymers:

PLGA NPs showed a relatively steep increase in brightness up to 1 wt%, and only a slight further increase by a factor of about 2. PMMA combined a strong initial increase up to 5 wt% with a further continuous increase, making PMMA NPs the brightest particles tested here. The brightness of PCL NPs increased less steeply with dye loading, but still relatively strongly up to 10 wt%. PCL NPs were thus less bright than PLGA NPs at low loading, but brighter at higher loadings. At a tenfold higher excitation power of 5 W/cm 2 the different NPs followed the same trends, but the brightness had a tendency to saturate at medium to high loadings. For example in the case of PMMA the brightness increased up to 10 wt% but then decreased upon further increase in the dye-loading. A better idea of the NP brightnesses can be obtained by comparing them to external standards: under the same measurement conditions the brightest PLGA, PMMA, and PCL NPs were, respectively, 2, nearly 5, and 3 times brighter than commercial FluoroSpheres of similar size (and 45, 90, and 75 brighter than corresponding QDs) (Figure 2.8 c). A still more remarkable difference between the NPs made from different polymers appeared when the fluorescence signal of single NPs was recorded as a function of time (Figure 2.9). At very low dye loading all three types of NPs showed a practically constant fluorescence. However, starting from 1 wt% of dye loading virtually all PLGA NPs showed a practically complete whole particle blinking, in line with our previous report.

[170] With increasing dye loading the duration of on-state decreased, so that the on-to-off-time ratio decreased. At the very high dye loadings (starting from 20 wt%, but especially at 30 wt%) the blinking was not complete anymore, and the PLGA NPs remained partially on, with spikes of higher fluorescence intensity. For both PMMA and PCL NPs the behavior was very different: these NPs showed a constant fluorescence at 1 wt% dye loading, and no cases of particle blinking were observed.

At 5 wt% loading these NPs did not show blinking either, though in many cases a stepwise modulation of the fluorescence intensity was observed. However, these 95 intensity modulations did not exceed 20 % of the fluorescence intensity. A further increase of the loading to 10 wt% led to an increase of the amplitude of the stepwise modulations and a decrease of their durations. In other words, a particle blinking appeared, but rather than showing a complete on-off-switching, the blinking was superimposed on a constant fluorescence signal from the particles. The blinking amplitude increased further with increasing loading for PMMA and PCL NPs and at 30 wt% dye loading especially many PMMA particles showed a practically complete onoff-blinking. These differences in the particle behavior can also be seen from the projections of the instantaneous single particle brightness (Figure 2.10). In summary, while complete whole particle blinking appeared for PLGA NPs at 1 wt% of dye loading, and an increase in the loading mainly led to a decrease of the on-to-off time ratio of PLGA NPs, in the case of PMMA and PCL NPs the fluorescence was continuous at low to 96 medium dye loadings. For these NPs an increase in loading led then to an increase in blinking amplitude and a decrease in the on-to-off time ratio. In the cases of our dye-loaded NPs, the dyes are thought to be immobilized in the polymer matrix, and the rotational correlation times of objects of the size of our NPs are of the order of hundreds of ns. In consequence, the decrease in the fluorescence emission anisotropy in these systems is thought to be mainly due to the transfer of excitation energy between the fluorophores. Comparison of the different polymer NPs with respect to their steady state anisotropy thus suggests that this energy transfer is the most effective or/and fast in the case of PLGA particles. As the fluorophores are identical in the three systems, this could in turn be attributed to smaller inter-fluorophore distances in PLGA NPs compared to PMMA and PCL. The lifetime anisotropy measurements, showed, indeed, that the anisotropy decay is much faster in PLGA NPs than in PMMA and PCL NPs loaded with 1 wt% of dye salt, which means that the energy transfer is faster in the former. The residual anisotropy obtained in these experiments can be used to estimate the mean number of fluorophores involved in energy transfer N according to

𝑁 = 𝑟 0 𝑟 ∞
where r0 is the fundamental anisotropy (0.37 for rhodamine) and r∞ is the residual anisotropy. For all three types of NPs the residual anisotropy is of the order of 0.02, corresponding to several tens of fluorophores involved in energy transfer, which is close to the mean number of fluorophores per NP (see Table 2.1). An increase of the dye loading to 5 wt% results in an increase of the speed of the anisotropy decay for PMMA and PCL NPs, which can be attributed to an increase of the speed of energy transfer between the fluorophores. Though the decay time was still shorter for PLGA NPs than for PMMA and PCL NPs, the difference at higher dye loadings is not evident as the decay time is again below the resolution of the instrument. The residual anisotropies suggest that at 5 wt% hundreds of fluorophores are involved in energy transfer in all three cases.

Förster theory allows estimating the expected anisotropy decay times for a given concentration of randomly distributed fluorophores according to:

𝜏 𝑎 = 0.0508 𝜏 0 𝑅 0 6 𝐶 2
where 𝜏 0 is the fluorescence lifetime in the absence of transfer (3.5 ns for R18/F5-TPB at 0.2 wt%), R0 is the Förster radius for homo transfer (about 5.5 nm at 1 wt%), which can be calculated from the spectral data, and C is the concentration in molecules per nm 3 . The theoretical values are thus 340 ps at 1 wt% and 15 ps at 5 wt%. Comparison of these theoretical values with the experimental decay times shows good agreement for PCL NPs (300 ps and ~20 ps at 1 and 5 wt%, respectively) and reasonable agreement for PMMA (180 ps and ~20 ps). However, in the case of PLGA the experimental decay time for 1 wt% is with less than 20 ps far below the theoretical estimation of 300 ps. The obtained decay time for this case would rather correspond to a fivefold higher fluorophore concentration, and suggests again smaller interfluorophore distances in the case of PLGA than what would be expected for a random distribution. In contrast the good agreement between theory and experiment suggests a nearly random distribution of the fluorophores in the PCL NPs and an only slightly disturbed distribution in PMMA NPs at least up to 5 wt% loading.

NPs loaded with perylene diimide

We then wanted to know whether the described phenomenon of polymer directed organization of the load is unique for the studied dye/counterion system or is an inherent property of the polymer NPs studied. We hence used uncharged dye from the perylene diimide family, N,N′-Bis(1-heptyloctyl)-3,4,9,10-perylenebis-(dicarboximide) (PDI-1, Figure 2.4), known to undergo characteristic spectral shifts upon aggregation. Upon loading increasing amounts of PDI-1 into PLGA NPs an increasing deviations of the absorbance spectra relative to that of PDI-1 in dioxane were observed (Figure 2.12), as reported previously.

[177] In particular, the height of the peak at 530 nm decreased relative to the shorter wavelength maximum at 490 nm.

At the same time the fluorescence spectra showed the appearance of a broad, redshifted band at about 650 nm that increased strongly with dye loading (Figure 2.12).

Similar variations of absorbance and emission spectra have been reported for aggregates formed by other PDI and naphthalene diimides with similar structure, and were hence interpreted as signs of aggregation. When loading PDI-1 into PMMA based NPs these spectral variations were much less pronounced at the same dye loadings (Figure 2.12). In the case of PDI-1 loaded PCL NPs both the change in the relative intensities of the peaks in the absorbance spectra and the appearance of the emission band at 650 nm were virtually absent up to a dye loading of 1 wt%. At the same time the QYs of PDI-1 in these systems decreased much faster in the case of PLGA NPs than for PMMA and PCL NPs (Figure 2.12). Together these results indicate that the aggregation state of PDI-1 inside NPs depends on the matrix polymer, with strong aggregation taking place in PLGA even at very low loading, much less aggregation occurring in PMMA and practically no aggregation in PCL at the studied loadings. The observed aggregation behavior of PDI-1 as a function of polymer matrix is similar to that observed for the rhodamine ion pair R18/F5-TPB. This means in turn that the control of dye aggregation through the nature of the polymer is a more general phenomenon.

Polymers and precipitation

Formation of NPs through nanoprecipitation is a kinetically controlled process.

We supposed, hence, that differences in the speed of NP formation by the three polymers could be at the origin of the observed differences in organization of the encapsulated compounds in the NPs. One important parameter in the kinetics of particle formation is the supersaturation of the polymer in acetonitrile-water mixture, forming after mixing of the two phases. Both nucleation and growth speed increase with increasing supersaturation. Assuming that the interdiffusion of the two phases does not depend on the nature of the polymer, this supersaturation should essentially depend on the solubility of the polymers in mixtures of acetonitrile and water. In order to evaluate these we performed turbidity studies of the polymers by adding increasing amounts of water to solutions of the polymers in acetonitrile (Figure 2.13).

101

Interestingly, turbidity for PCL appeared at the lowest water fraction, followed by PMMA, and finally PLGA. This indicates that, in such mixtures, the solubility of these polymers increases from PCL over PMMA to PLGA, in good agreement with increasing polarity of these polymers. It in turn suggests that during the fast mixing process supersaturation should be highest for PCL, leading to the highest particle formation speed. These differences could hence be at the origin of the differences of organization of the load inside the polymer NPs. In the case of fast particle formation the dye could simply be captured during the particle growth according to its distribution in solution, which would then lead to a homogeneous distribution of the load within the particle, as observed for PCL and PMMA. In the case of slower particle formation the load could which is strongly influenced by the state of the polymer: PLGA and PMMA are both in there glassy state at the employed temperature, however, transition temperature of PLGA is about 60 °C, which is much lower than that of PMMA (about 100 °C). PCL, on the other hand, is a semicrystalline polymer with a melting temperature of about 60 °C.

Obtained results confirmed the polymer matrix-controlled dye organization and thus florescence properties. This enables preparation of ultrabright fluorescent NPs with tuned photostability and cooperative properties, notably blinking.

Design of efficient FRET inside polymer NPs

To obtain efficient FRET inside polymer NPs with size larger than Förster distance R0, two strategies can be applied. First is loading of NPs with high quantity of both donor (D) and acceptor (A) molecules (usually ratio D to A ratio~1). [101,104] Alternatively, NPs with high donor dye cooperativity (high EET) could be used, which should require much lower amount of acceptor (high D to A ratio).

As it was shown previously, PLGA-based NPs demonstrated favorable for EET organization of dyes (small inter-fluorophore distances) [START_REF] Reisch | Collective fluorescence switching of counterion-assembled dyes in polymer nanoparticles[END_REF]. This suggested the possibility of efficient FRET inside NPs from multiple donors to single acceptors. We used EET inside polymer NPs loaded with huge amount of donor dyes (R18/F5-TPB)

to create systems with efficient photoswitching through FRET and nano-antennas for the amplification of a single acceptor molecule emission. Among tested counterions, only perfluorinated tetraphenylborate enables high photoswitching efficiency (on/off ratio ∼20), while for all other counterions, the switching efficiency was much lower (Figure 2.15 a).

To verify whether this effect of F5-TPB is linked with the exciton diffusion process (EET), we measured the steady-state fluorescence anisotropy of NPs with all four counterions (Figure 2.15 b). When the energy migrates within encapsulated fluorophores that are randomly orientated inside the polymer matrix, the fluorescence anisotropy rapidly decreases after each step of energy hopping between dyes. [179] The perfect correlation between photoswitching efficiency of NPs and fluorescence anisotropy loss (i.e. EET efficiency) was observed (Figure 2.15). We hypothesized that the use of much larger dye ensembles, coupled by EET inside NPs, could generate giant light-harvesting antenna for amplifying the fluorescence of a single dye. To this end, we created PMMAMA NPs of different size (from 30 to 107 nm according to TEM), loaded with high amount of R18/F5-TPB (30 wt%). PMMA was chosen, because according to our study described above, this matrix favors efficient fluorescence and EET at high dye loading. As expected, independently of the NPs size, the QY remained high (0.36-0.44).

Co-encapsulation of small amount of Cy5 (0.02 and 0.002 wt%, that corresponds to donor/acceptor ratio 1000 and 10000 respectively), which is known to be efficient energy acceptor for rhodamine dyes, resulted in the efficient FRET. We showed that NPs of ~60 nm size containing >10 4 donor dyes can efficiently transfer energy to 1-2 acceptors. For both D/A ratios, increase in the NPs size increased the FRET efficiency (Figure 2.16 a). The antenna effect (calculated as the ratio of the maximal excitation intensity of the donor to that of the acceptor [127] ) also depended on the NPs size. For the lower donor/acceptor ratio, the antenna effect increased to a small extent from 160 to 330, whereas for donor/acceptor ratio of 10000, the strong increase in the antenna effect was observed for larger NPs (Figure 2 Single particle measurements by TIRF microscopy confirmed high antenna effect.

This amplification resulted in the brightness of 1-2 acceptors inside NP100 that were 25-times higher than that of the QD655, excited at the same power of 532-nm laser (Figure 2.17 This means that we approach single molecule photography at ambient light without dedicated instrumentation.

The full description of the work can be found in the manuscript enclosed below.

Abstract

In the present work, we explore the enhancement of single molecule emission by polymeric nano-antenna that can harvest energy from thousands of donor dyes to a single acceptor. In this nano-antenna, the cationic dyes are brought together in very close proximity using bulky counterions, thus enabling ultrafast diffusion of excitation energy (<60 fs) with minimal losses.

Our 60-nm nanoparticles containing >10 4 donor dyes can efficiently transfer energy to 1-2 acceptors resulting in an antenna effect of ~1000. In this case, single acceptors (Cy5 derivative) are 25-fold brighter than QD655 at 532 nm excitation. This unprecedented amplification of the acceptor dye emission enables efficient observation of single molecules at illumination powers (1-10 mW/cm 2 ) that are >10 4 -fold lower than required in single molecule measurements.

Finally, using a basic setup, which includes 20x air objective and sCMOS camera, we could detect single Cy5 molecules by simply shining divergent light on the sample at powers equivalent to sunlight.

Introduction

Detection of single fluorescent molecules requires a dedicated microscopy setup with strong excitation power ranging between 100 and up to 5000 W/cm 2 because the brightness of fluorescent dyes is limited. [182,183] The use of much lower excitation power would not only decrease photo-damage [184,185] and the background related to auto-fluorescence, but would also enable fast and inexpensive high-throughput screening and diagnostics assays, which normally do not use microscopy setups and powerful light sources. For instance, fluorescent nanoparticles (NPs) that are many-fold brighter than single dyes can be detected even using a smartphone-based device.

[186] The key question is can a low power excitation at levels similar to the ambient light intensity be used for single molecule detection? The typical light power of sunlight at the surface of the earth at a bandwidth of 10 nm in a visible range is ~1 mW/cm 2 ,[187] which is similar to the illumination power used in fluorimeters or plate readers.

This power is 10 4 -10 6 lower than those typically used in single molecule microscopy, which sounds like an impossible obstacle to single molecule detection under ambient light conditions.

Recent works suggested metallic nanostructures, so-called plasmonic nano-antennas, which can amplify the excitation and emission of single molecules. [188][189][190][191] In this case, the amplification is achieved due to surface plasmon effects, which requires precise control of antenna geometry and the distance from the emitter to the metal surface. [192,193] Thus, between two gold NPs, an amplification of up to 100-fold could be achieved. [194] Larger values of amplification were also reported, though it was achieved only when the fluorophore was initially poorly emissive or quenched. [195,196] However, to detect single molecules under ambient light power, we need to achieve at least 1000-fold amplification of the signal coming from a high performance non-quenched fluorophore, such as cyanine 5 or Alexa567.

A promising direction is to use a light-harvesting concept, where multiple donors due to high absorptivity can efficiently collect light energy and then deliver it to a single acceptor through a Förster Resonance Energy transfer (FRET) mechanism. [197] This is particularly efficient when the excitation energy can rapidly migrate within the donors up to the energy acceptor, as it is realized by Nature in photosynthetic centers. [198] Chemists developed highly efficient FRET systems that use conjugated polymers, [157,[199][200][201][202] dendrimers, [126,203] [209,210] etc. However, so far the obtained amplification of the acceptor has never reached 1000. [START_REF] Peng | Biological Applications of Supramolecular Assemblies Designed for Excitation Energy Transfer[END_REF] The common reasons that limit the efficient light harvesting is self-quenching of donors at high local concentration and/or 123 inefficient donor-donor coupling. A promising platform for fabrication of light-harvesting nano-antenna is dye-loaded polymer nanoparticles, where the dyes are encapsulated with minimal aggregation-caused quenching.

[181] Our recent work showed that encapsulation of multiple dyes inside polymer NPs using bulky counterions minimized dye self-quenching and produced fluorescence switching (blinking) of up to 500 dyes per particle.

[170] It was explained by unprecedented coupling of encapsulated dyes due to exciton migration, so that a single dark species could quench the whole dye ensemble. Later works using NPs composed of rhodamine salts and its bulky counterions confirmed the possibility of highly efficient FRET from ~300 dyes to a single acceptor [START_REF] Shulov | Fluorinated counterion-enhanced emission of rhodamine aggregates: Ultrabright nanoparticles for bioimaging and light-harvesting[END_REF] as well as efficient photo-switching of dye-loaded

NPs.

[211] Here, we hypothesized that the use of much larger dye ensembles coupled by efficient exciton migration (ultrafast excitation energy transfer (EET)) inside NPs could generate giant light-harvesting antenna for amplifying the fluorescence of a single dye. This hypothesis was successfully realized using cationic rhodamine dyes encapsulated in a poly(methyl methacrylate-co-methacrylic acid) (PMMA-MA) matrix using bulky fluorinated counterions (Figure 1). These NPs of ~60 nm diameter can generate unprecedented 1000-fold amplification (antenna effect) of a single cyanine 5 derivative (DiD) acceptor located inside the particles. This result enabled for the first time observation of single molecules using the power density of 1 mW/cm 2 , which is 10 4 -10 6 fold lower than typically used in single molecule measurements.

Results and discussion

The nano-antenna was designed based on PMMA-MA NPs, which were obtained using a previously proposed concept of nanoprecipitation based on hydrophobic polymers bearing few charged groups.

[176] Nanoprecipitation of PMMA-MA from acetonitrile in phosphate buffer (pH 7.4) gave small NPs of ∼40 nm (according to DLS) and narrow size distribution (PDI ~ 0.07), which is in good agreement with our previous studies. [170,176] To encapsulate the energy donor dyes inside the NPs, they were mixed with the polymer in acetonitrile before the nanoprecipitation. A rhodamine B salt with the hydrophobic fluorinated counterion F5-TPB was used as it facilitates the dye encapsulation, minimizes self-quenching and ensures ultrafast exciton diffusion required for efficient energy transfer. To verify the role of EET on nanoantennas property we varied the interfluorophore distances by changing the level of dye loading. The increase in the dye content from 0.1 to 30 wt% (with respect to the mass of the polymer) produced some gradual increase in the NPs hydrodynamic diameter up to 65 nm, while keeping low values of the polydispersity index (PDI ~ 0.1). Nevertheless, according to TEM, the size of 30 wt% loaded NPs remained small (45 nm), which corresponded to 6300 dyes per NP (SI, Table S1).

The fluorescence quantum yields (QY) of the obtained NPs decreased with the dye loading but remained remarkably high (~36%) even at the highest loading (Figure 2a). This result corroborated with relatively small changes in the absorption spectra, where the shortwavelength shoulder, an indicator of dye aggregation, [212] showed minimal increase (Figure S1a). Moreover, the emission spectra of NPs displayed only small red shift without broadening (Figure S1b). According to our earlier report, the F5-TPB counterions function as a spacer between the charged dyes that prevent their self-quenching through pi-stacking.

[170]

Nano-antenna with efficient FRET donor properties should present fast excitation energy transfer (EET) within the encapsulated donor dyes. [138,211] When the energy migrates within encapsulated fluorophores that are randomly distributed inside a polymer matrix, the fluorescence anisotropy should decrease due to the energy hopping between dyes (Figure 2b). [213] As the dyes are confined within the rigid polymer matrix, the loss of fluorescence anisotropy in the system could be mainly attributed to the EET process. As expected, an increase in the dye loading produced a fast drop in the anisotropy values, indicating that shorter inter-fluorophore distances at higher loadings favored efficient exciton diffusion within these NPs (Figure 2a). At 30 wt% loading the average distance between encapsulated dyes should be ~1 nm, which is far below the Förster radius of homo-FRET transfer for R18/F5-TPB for this loading (4.6 nm) (SI, Table S2). Therefore, an efficient EET should take place, explaining this loss in the anisotropy, similarly to our previous studies. [170,211] To understand better the EET process, we performed femtosecond anisotropy decay studies using a pump-probe technique. As expected, for low dye loading (1 wt% of R18/F5-TPB), the anisotropy remained stable during the first 10 ps, indicating that EET in this system should be very slow (Figure 2c). In sharp contrast, at high dye loading (30 wt%) the anisotropy decayed to zero already within 1 ps (Figure 2c). The bi-exponential fit revealed 30 fs (45%) and 600 fs (55%) components, the former being limited by the resolution of our setup (60 fs pulse width). This means that the EET process is exceptionally fast and thus can involve thousands of dyes within their emission lifetime (~4 ns [170]). Next we introduced a FRET acceptor dye into the NPs, by nano-precipitating an acetonitrile solution of polymer together with donor and accepter dyes in phosphate buffer. As the acceptor, we chose the lipophilic cyanine 5 derivative DiD. It is a perfect energy acceptor for rhodamine B with very good spectral overlap, and its two hydrophobic octadecyl chains should ensure efficient encapsulation inside polymer matrix, as it was shown for PLGA NPs.

[102] Being encapsulated at 0.02 wt%, DiD showed a high fluorescence quantum yield of 774%, so that our antenna was tested on a highly emissive acceptor. For NPs containing 30 wt% of donor dyes, an increase in the acceptor concentration resulted in a rapid growth of acceptor emission, accompanied by a drop of the donor emission, indicating a FRET process (Figure 2d).

Remarkably, efficient FRET (34%) was already observed for 0.004 wt% of the acceptor (with respect to the mass of the polymer), which corresponds to 1.3 acceptors per 45 nm particle, and ensures that >70% of NPs will contain at least one acceptor according to Poisson distribution (Figure S2). Thus, in these NPs a single acceptor could produce FRET from ~6300 donors through distance of >20 nm (NPs radius) that is much larger than the Förster radius. To understand better the role of exciton diffusion in this efficient FRET process, we prepared NPs with a constant concentration of the acceptor (0.004 wt%) and varied the concentration of the donor (0.1-30 wt%). Theoretical prediction, assuming no donor-donor communication (no EET), suggested negligibly low FRET efficiency (4-6%) for 0.004 wt% acceptor independently from the donor loading (Figure 2c, Note S1, Table S3). However, according to our experiments, the increase in the donor loading produced a rapid growth of the acceptor relative intensity (Figure S3) due to increase in the FRET efficiency (Figure 2e). This efficient FRET is probably a result of fast EET within donors revealed by fluorescence anisotropy (Figure 2a-c).

Importantly, NPs characterized by efficient FRET from multiple donors to a single acceptor should behave like a giant light-harvesting nano-antenna. To quantify the antenna effect (AE), we recorded the excitation spectra of the donor and acceptor at the emission wavelength of the acceptor (700 nm, Figure S4). AE was measured as the ratio of the maximal excitation intensity of the donor to that of the acceptor with correction on the emission of the donor dyes in the acceptor channel, as described in Materials and Methods.

[209] At low loading of the donor dye, no antenna effect was observed (AE<1), whereas at higher donor loadings, AE increased up to 560 for 30 wt% NPs of 45 nm (Figure 2f). These values correlated well with the antenna effect calculated based on the FRET efficiency, number and extinction coefficient of donor and acceptor dyes, that could be determined as AE = (nDεDE)/(nAεA), where nD and nA are the numbers of donors and acceptors, respectively, per particle, εD and εA are the extinction coefficients of donors and acceptors, respectively, and E is the FRET efficiency (Figure 2d). [START_REF] Shulov | Fluorinated counterion-enhanced emission of rhodamine aggregates: Ultrabright nanoparticles for bioimaging and light-harvesting[END_REF]209] By contrast, theoretical estimations assuming no EET gave AE values much below the experimental ones (3-6 fold lower), emphasizing the importance of the communication within donor dyes for obtaining an efficient antenna.

Exciton diffusion should propagate at relatively long distances and thus ensure efficient FRET even for nano-antennas that are much larger than the Förster radius of the FRET partners.

Therefore, we investigated the influence of the size of nano-antenna on the FRET efficiency from multiple donors (30 wt% dye loading) to few acceptors inside NPs. As the charge of the polymer is the key factor for controlling the size of NPs prepared by nanoprecipitation, [176] we varied the pH of the phosphate buffer during the preparation of NPs, which should change the protonation state of the carboxylate of the PMMA-MA polymer. Based on DLS measurements, a decrease of pH from 9.0 and 5.8 produced an increase in NP size from 30 to 200 nm, while preserving a narrow polydispersity (Table 2). Transmission electron microscopy (TEM) confirmed the increase in NPs size with decrease in pH of buffer, but revealed that the sizes of NPs were smaller, in the range of 30-67 nm for pH range from 9.0 to 6.5. However, for pH 5.8 aggregates of >100 nm size were observed (Figure 3a). Independently of the NPs size, the QY remained high (0.35-0.41, Table 2). Moreover, according to wide-field fluorescence microscopy of NPs immobilized on the surface, the single particle brightness increased with size (Figure 3b), so that the largest NPs (NPs107) were 31 times as bright as the smallest ones (NPs30) (Table 2). This was expected because larger NPs should encapsulate more dyes, proportionally to the particle volume. Moreover, the experimental brightness correlated well with our estimations based on QY and the size of NPs measured by TEM. Finally, the anisotropy values were close to zero for all particles sizes (Table 2), indicating that EET was efficient in all these systems. Using different pH of phosphate buffer, we prepared nano-antennas of different size containing 30 wt% of R18/F5-TPB donors with varied amount of acceptor corresponding to donor/acceptor ratio 1000 and 10000 (0.02 and 0.002 wt%, respectively). For both ratios, an increase in the NP size increased the contribution of the acceptor emission (Figure 4a) and the FRET efficiency (Figure 4b). Thus, for the same donor/acceptor ratio, the larger antennas transfer the energy more efficiently to the acceptor (Figure S5). The antenna effect also increased with NP size, especially for NPs with donor/acceptor ratio of 10000 (Figure 4b).

Remarkably, for NPs60 and NPs67 the enhancement of the acceptor emission (1.5 and 2.1 acceptors per NPs) reached 1000-fold (Figure 4b), which, to our knowledge, is by far the highest FRET-based amplification of dye emission reported to date. Then, we immobilized our nano-antennas on the glass surface and imaged them by widefield TIRF microscopy (Figure 5). The control NPs without acceptors appeared as bright spots at the donor channel and as dim spots at the acceptor channel. In the presence of ~1.5 acceptors particle the emission in the acceptor channel became comparable or brighter than that in the donor channel (Figure 5a). This result showed that inside the nano-antennas, the emission of 1-2 acceptors was comparable to the emission of thousands of donor dyes. However, the overlaid images revealed some heterogeneity in the relative donor/acceptor emission, because statistically some NPs did not contain any acceptor molecules, while others could contain more than one acceptor. To quantify the amplification of acceptor emission under the microscope, we compared the acceptor intensity under excitation through FRET at 532 nm to that obtained by direct excitation with a 642 nm laser. Remarkably, to obtain similar acceptor intensities, the excitation through nano-antennas NPs60 at 532 nm required ~1000-fold lower laser power than direct excitation of the acceptor at 642 nm (Figure 5b). The amplification factor of acceptor emission at the single particle level was determined as:

𝐴𝐹 = 𝐼 𝐴 532𝑛𝑚 𝐼 𝐴 642𝑛𝑚 × 𝑃 642𝑛𝑚 𝑃 532𝑛𝑚 (1)
where 𝐼 𝐴 532𝑛𝑚 , 𝐼 𝐴 642𝑛𝑚 are the mean intensities of acceptors under excitation at 532 and 642 nm, respectively, and 𝑃 532𝑛𝑚 and 𝑃 642𝑛𝑚 are laser powers at the corresponding wavelengths.

Using equation ( 1), we found that the amplification factor increased with the particle size (Figure 5c). For NPs60, we obtained a ~1040±100-fold amplification factor, which is in good agreement with the antenna effect measured from the excitation spectra. Owing to this giant amplification factor, the brightness of 1-2 acceptors inside NPs60 was 25-times higher than that of a QD655 excited at 532 nm with the same power (Figure 5b). This is an exceptional performance of single Cy5 dyes, taking into account that the extinction coefficient of QD655 at 532 nm is 2.410 6 M -1 cm -1 and QY is close to unity.

The values of antenna effect reported in the literature vary in the range of 3-100 dependent on the system. For instance, in DNA-origami containing intercalated donor and acceptor dyes the AE value reached 12.

[209] In a recent report, nanocrystals of difluoroboron chromophores at high D/A ratio, which were claimed to be one of the most efficient light-harvesting systems, the AE value was 28-29.[215] Similar values of AE [START_REF] Hirano | Development of an 'OFF-ON-OFF' fluorescent pH sensor suitable for the study of intracellular pH[END_REF] was reported later on for dye-dopedpolymer NPs loaded with coumarin 123 (donor) and Nile Red (acceptor) dyes.

[216] Our recent report showed that this value can be increased to up to 200 in ionic NPs using R18 and bulky counterion, [START_REF] Shulov | Fluorinated counterion-enhanced emission of rhodamine aggregates: Ultrabright nanoparticles for bioimaging and light-harvesting[END_REF] though we did not provide direct proof for amplification using single molecule microscopy. Thus, the antenna effect achieved in the present work goes far beyond those reported for other light-harvesting systems. The unique efficiency of our system relies on two key factors. The first one is the high quantum yield of >10 4 donor dyes encapsulated within our polymeric nano-antenna at 30 wt%, which ensures that a large part of the excitation energy is delivered to the acceptor with minimal energy losses. The second factor is the exceptional excitation energy migration within the encapsulated donor dyes on the time scale <60 fs (resolution of the setup). Though one could explain it by ultrafast Förster/Dexter homo-transfer mechanisms, recent works on light-harvesting systems propose coherent energy transfer [217,218] for conjugated polymers, [219,220] photosynthetic systems, [221,222] and dye assemblies, [206] which could also take place in our nano-antenna. The coherent energy transfer in our nano-antennas could be favored by the very short inter-fluorophore distances (~1 nm) at 30 wt% dye loading.

Finally, using our NPs60 nano-antenna, we could observe single molecule events, such as one-step bleaching of the Cy5 (DiD) acceptor at extremely low laser powers of 1 mW/cm 2 (Figure 5d), which was accompanied by one step growth of the donor emission. This opposite behavior of donor and acceptor is typically observed in single molecule FRET measurements using one donor and acceptor dyes, but here FRET takes place between 15000 donors and 1-2 acceptors. Moreover, previous reports on single-molecule detection were systematically based on light power densities of 10-5000 W/cm 2 . Presently, we obtained similar single molecule traces at 1-10 mW/cm 2 , reaching the values of the ambient sunlight. Therefore, we tested whether our nano-antennas enable detection of single molecules using a simple microscopy setup where the excitation is provided by directly shining light on the sample at powers equivalent to sunlight. The measured power of direct sunlight (at midday, 19 October, 2016, Strasbourg) through the excitation filter 527/50 nm was 24 mW cm -2 . In our setup, we applied an artificial white light source providing 15 mW cm -2 through the same filter. The fluorescence of immobilized NPs60 nano-antennas with ~1.5 Cy5 dyes was collected using either 20x air or 60x oil immersion objective and detected using sCMOS camera (Figure 5e). According to overlaid images of donor (green) and acceptor (red) channels, these NPs displayed significant acceptor emission (appear in yellow-red), in contrast to control NPs without acceptor that appeared in green (Figure 5f). Moreover, after 5-min illumination with our artificial light source, the former NPs lost the acceptor emission, probably due to acceptor bleaching, and became similar to the control NPs. Strikingly, using 20x air objective, we were able to record in the acceptor channel one-step bleaching events, corresponding to single Cy5 dye molecules (Figure 5g). By contrast, the control NPs displayed much lower intensity at the acceptor channel without abrupt bleaching steps.

Thus, our nano-antennas enable detection of single molecules using a simple sCMOScamera-based imaging setup under ambient sunlight-like conditions, which opens the way to single molecule photography.

Methods

Materials. Poly (methyl methacrylate-co-methacrylic acid) (PMMA-MA, 1.3% methacrylic acid, Mn ~15000, Mw ~34000), acetonitrile (anhydrous, 99.8%), rhodamine B octadecyl ester perchlorate (>98.0%), lithium tetrakis (pentafluorophenyl)borate ethyl etherate were purchased from Sigma-Aldrich. DiD oil (1,3,3', Perchlorate) (Cy5) was purchased from Life-Technologies. Sodium phosphate monobasic (>99.0%, Sigma-Aldrich) and sodium phosphate dibasic dihydrate (>99.0%, Sigma-Aldrich)

were used to prepare 20 mM phosphate buffers at pH 5.8-9.0. Milli-Q water (Millipore) was used in all experiments.

Synthesis. Rhodamine B Octadecyl Ester Tetrakis(penta-fluorophenyl)borate (R18/F5) was synthesized by ion exchange and purified by column chromatography as described previously. 1

Nanoparticle Preparation. Stock solutions of the polymer in acetonitrile were prepared at a concentration 2 mg mL -1 containing different amount of R18/F5-TPB (0.1 to 30 wt % relative to the polymer). 50 μL of the polymer solutions were then added quickly using a micropipette and under shaking (Thermomixer comfort, Eppendorf, 1000 rpm) to 450 μL of 20 mM phosphate at 21°C. The particle solution was then quickly diluted 5-fold with the phosphate buffer 20 mM, pH7. , where ID is the integral donor intensity and ID-A integral intensity of donor in presence of acceptor. Measurement of fluorescence anisotropy was performed at 20°C with a Fluorolog spectrofluorometer (Horiba Jobin Yvon). Excitation wavelength was set at 530 nm and detection at 585 nm. Each measurement of the anisotropy value corresponds to an average over 10 subsequent measurements of 0.1 s each. For standard recording of excitation spectra, the emission wavelength was set 700 nm. These spectra were corrected for the lamp intensity. The antenna effect was then expressed as the ratio of the maximal excitation intensity of the donor to that of the acceptor with correction on the emission of the donor dyes at 700 nm: for all the measurements. Ultrashort continuum probe pulses were generated in a sapphire crystal (500-800 nm). The normalized differential transmission of a 60 fs linearly polarized probe (parallel or perpendicular to the pump polarization) centered around 580 nm (=13 nm)

was measured as a function of the pump-probe delay by using a monochromator coupled to a liquid nitrogen cooled CCD (Princeton Instrument). The anisotropy decay was calculated using the following relation

DT // T // - DT Tae è ç ö ø ÷ DT // T // + 2 DT Tae è ç ö ø ÷
where // and  denotes for a probe beam with a linear polarization parallel and perpendicular with respect to the linear polarization of the pump beam.

The decay curves were fitted with a bi-exponential function and the analysis took into account the laser pulse duration.

Transmission electron microscopy (TEM). Five microliters of the particle solution were deposited onto carbon-coated copper-rhodium electron microscopy grids that were used either as obtained or following an air or amylamine glow-discharge. The grids were then treated for 1 min with a 2%uranyl acetate solution for staining. They were then observed with a Philips CM120 transmission electron microscope equipped with a LaB6 filament and operating at 100 kV. Areas covered with nanoparticles of interest were recorded at different magnifications on a Peltier cooled CCD camera (Model 794, Gatan, Pleasanton, CA). Image analysis was performed using the Fiji software.

Fluorescence Microscopy. For single particle fluorescence microscopy measurements, the NPs were immobilized on glass surfaces on which a polyethylenimine (PEI) layer was initially adsorbed. The solutions of NPs were diluted 5000, 2000, 1000 and 500 times for NPs30, NPs45, NPs60, NPs67 and NPs107 correspondingly. 400 μL of these solutions per cm 2 were then brought in contact with the PEI covered glass for 15 min, followed by extensive rinsing with Milli-Q-water. The surfaces were left in Milli-Q water during microscopy.

Single particle measurements were performed in the TIRF (Total Internal Reflection Fluorescence) mode on a homemade wide-field setup based on an Olympus IX-71 inverted microscope with a high-numerical aperture (NA) TIRF objective (Apo TIRF 100 ×, oil, NA 1.49,Olympus). A 532 nm diode laser (Cobolt Samba 100) and a 642 nm diode laser (Spectra-Physics Excelsior 635) were used to excite the samples. The 532 nm laser intensity was set to 1mW/cm 2 -100mW/cm 2 by using a polarizer and a half-wave plate (532 nm). For direct excitation of acceptor Cy5, the 642 nm laser was used with intensity 0.1kW/cm 2 . The were used to image R18/F5-TPB and Cy5, respectively. Single particle analysis was performed using the Fiji software: particle locations were detected through a Fiji routine applied to a projection (maximum intensity) of 1000 frames. After the automatic background subtraction, the mean intensities of circular regions of interest with a diameter of 8 pixels around the found particle locations were then measured. At least three image sequences (245pixel × 245pixel) per condition were analyzed with, on average, 500-700 particles per sample. a Distance was calculated for isotropic distribution of fluorophores by: liso=0.504/∛𝑐, where c is the concentration of the dye. The FRET efficiency could be calculated as:

Microscopy

𝐸 𝐹𝑅𝐸𝑇 = 1 - 𝐹 𝐷𝐴 𝐹 𝐷 = √𝜋 *γ *exp(𝛾 2 )⌈1 -erf(𝛾)⌉
The Förster distance was calculated by: 𝑅 0 = 0.211 [ 𝑘 2 𝑄 𝐷 𝐽(𝜆)

𝑛 4

] 1/6

.

The overlap integral 𝐽(𝜆) was calculated using the FluorTools software [224] . Though RhB emission has a tendency to red shift and thus to induce an increased spectral overlap with an increase in its loading, the drop of its donor quantum yield leads to a decrease of the Förster distance (Table S2). FRET is widely used for bioimaging applications due to its extremely high sensitivity to distance [2,[START_REF] Sapsford | Materials for fluorescence resonance energy transfer analysis: Beyond traditional donor-acceptor combinations[END_REF] and the simplicity of building ratiometric sensors. [225] Conventional FRET biosensors based on molecular dyad of fluorophores connected covalently by a spacer, suffer from photobleaching and low signal-to-noise ratio. [226] FPs are also very useful for intracellular ratiometric sensing, but they have similar limitations, in addition to difficult preparation protocols and limited FRET efficiency due to larger size. [START_REF] Chudakov | Fluorescent Proteins and Their Applications in Imaging Living Cells and Tissues[END_REF] Application of NPs allows creation of ratiometric nanosensors that could overcome these problems. QDs, due to their high brightness and photostability, as well as size-control luminescence properties have been successfully applied for For QDs with diameter about 7.6 nm around 50 molecules were needed to obtain 50 % of FRET.

[146] However, the larger amount of acceptors is needed for efficient FRET, the lower is the sensitivity of the FRET assay, so it is important to find fluorescent NPs with more efficient FRET to their surface. Therefore, application in this field of dye-loaded polymer NPs exhibiting collective behavior of dyes is a highly promising direction for the development of FRET-based nanoprobes.

Based on our previous studies, NPs, made of PLGA and loaded with R18/F5-TBP dye, displayed highly efficient EET. [START_REF] Reisch | Collective fluorescence switching of counterion-assembled dyes in polymer nanoparticles[END_REF] We performed titration of PLGA NPs loaded with 5 wt% of R18/F5-TPB with Cy5-biotin conjugate. As our NPs have negative surface charge, and Cy5 is a cationic dye, the latter adsorbs on NPs surface and we observe the occurrence of FRET between encapsulated R18/F5 dyes and Cy5-biotin (Figure 2.18 a). However, the FRET efficiency appeared to be surprisingly We hypothesized that the origin of such low efficiency could be a core-shell like structure of our ~40 nm NPs, where the dyes form nuclei that surrounded by thick shell of polymer. This hypothesis is supported by the studies on the influence of polymer matrix on the collective behavior of encapsulated dyes, described above. To overcome this problem we decided to change polymer matrix to PMMA, where, according to our previous studies, dye distribute more homogeneously and have efficient EET at high level of dye loading (see chapter 2.1.2). To provide lower average distance between the encapsulated donor dyes and particle surface we decreased the size of NPs using a recently developed approach in our group. It was shown that a single charged group on the polymer PMMA results in a strong decrease in the size of obtained particles, reaching 15-20 nm diameter according to TEM. [176] Nanoparticles preparation and characterization NPs where prepared by nanoprecipitation from acetonitrile solution of polymer containing different amount of R18/F5-TPB (from 0.2 wt% to 20 wt%) to milliQ water.

The increase in dye loading led to a small increase in relative contribution of the short wavelength shoulder in the absorption spectra. The emission maxima demonstrated a slight red shift with increase in the dye loading, together with a small reduction in the width of the spectra that evidenced of some assembly of dyes inside NPs (Figure 2.19). Nevertheless, the QY values remained very high ~ 0.5 even at the highest loading of 20 wt% (Table 2.2). According to FCS with 2-photon excitation, size of NPs at the lowest loading was around 20 nm and increased with loading up to 25 nm. The slight increase in NPs concentration was also observed. Together with increase in loading we saw increase in two-photon brightness (expressed in the brightness of our reference 5(6)carboxytetramethylrhodamine (TMR)) from 7 to 280. Although at low loading (from 0.2 wt% to 5 wt%) the increase of brightness was almost linear, then it slowed down due to the drop in QY. Based on the size of NPs and the loading the number of rhodamine dyes per NP was calculated and reached more than 700 for 20 wt% (Table 4). To verify whether the FRET efficiency was related to the efficiency of EET between donors, we measured the steady-state anisotropy of NPs with different loading of R18/F5-TPB dye. Increase in the dye loading led to the dramatic drop in the anisotropy, indicating more efficient EET (Figure 2.21 b). [START_REF] Colby | Electronic energy migration on different time scales: concentration dependence of the time-resolved anisotropy and fluorescence quenching of lumogen red in poly (methyl methacrylate)[END_REF] As NPs contained a large number of fluorescent dyes that were strongly coupled by energy transfer, they could be very efficient energy donors, similar to lightharvesting complexes, and thus could amplify the fluorescence of acceptor dyes. To quantify the antenna effect (AE), we recorded the excitation spectra of the donor and the acceptor by detecting the emission of the acceptor and then measured the ratio of the maximal excitation intensity of the donor to that of the acceptor. [209] At the low loading of the donor dye, no antenna effect was observed (AE~1), whereas at higher donor loadings, AE increased up to 59 for 20 wt% dye loading in 25-nm NPs (Figure 2.21 c).

The high FRET efficiency together with ~60-fold amplification of acceptor emission suggested the possibility of using these NPs as efficient energy donor in FRET-based probes.

Application of efficient FRET and acceptor amplification for protein detection

To test whether the described above FRET system can sense proteins in solution, we adsorbed 1 equivalent of Cy5-biotin on the surface of NPs loaded with 20 wt% R18/F5-TPB. As a result, strong FRET signal was observed (Figure 2.22 a). Then we titrated this complex with streptavidin. Due to high affinity to biotin, streptavidin bound Cy5-biotin conjugate and probably removed it from the NPs surface which resulted in the decrease of acceptor emission and change in IA/ID ratio (Figure 2.22 a,b). The change in this ratio was stabilized after 3 nM concentration of streptavidin. This stabilization could be relevant to that fact that streptavidin has four binding sites for biotin. Importantly, the addition of 1 µM of free biotin competitor inhibited the detection of streptavidin by NPs which confirmed specific recognition of the protein (Figure Then we perform the same type of experiment to detect human Carbonic Anhydrase (hCA) to confirm versatility of proposed sensing strategy. To this end, we changed ligand from biotin to benzamidine (BA). The new conjugate Cy5-BA also showed highly efficient FRET signal when added to NPs. As the dissociation constant of hCA -BA complex (KD = 10 -6 ) is much lower than that of biotin-streptavidin (KD = Detection of proteins at the single molecule level Finally, we evaluated the performance of our NPs at the single-particle level using wide-field fluorescence microscopy. To this end, we immobilized fluorescent NPs containing 20 wt % R18/F5-TPB on the glass surface covered with positively charged polymer PEI. Then, using band pass filters 593/40 (donor channel) and 685/40 (acceptor channel), and a dichroic mirror at 640 nm to split the emission beam into two channels, we were able to record the emission of NPs on two channels under 532-nm excitation.

The NPs appeared as bright spots only on the donor channel and very dim spots on the acceptor channel. Merged image of both channels showed domination of the signal on the donor channel. Cy5-biotin was added to the well with NPs and spots on the donor channel became much dimmer, and we observed bright spots on the acceptor channel that suggested appearance of FRET. Merging channels demonstrated the presence of NPs with different ratio between the two channels, which suggests the presence of NPs with different levels of FRET. Three times washing of the well with MilliQ-water had no significant effects on the observed picture. Obtained results suggest that the created NPs could be used as a building block for creating nanoparticle probes with single molecule sensitivity.

Conclusions and perspectives

The aim of my PhD project was to develop non-toxic, biocompatible organic NPs with small size, high brightness and photostability, characterized by efficient switching due to FRET and then to apply them for biomolecular sensing.

In the first part of my work I investigated the influence of dye structure on encapsulation efficiency, brightness and photostability in case of perylene diimide dyes with bulky substitutes in the imide (PDI-1) and both imide and bay regions (Lumogen Red).The results demonstrated importance of introducing bulky side groups into structure of encapsulated dyes to prevent their aggregation (thus poor photostability). The excellent photostability and brightness of NPs loaded with LR make them biodegradable substitutes of quantum dots, that could find a variety of applications in biological imaging. Nevertheless, the size of NPs remains larger than size of QDs. For many cellular applications size should be decreased up to 5 -15 nm, while preserving the high brightness, which can be achived by increasing the dye loading and developing efficient strategies against ACQ.

Then I studied the influence of polymer matrix (on the example of PLGA, PMMA and PCL) on the spatial organization of encapsulated dyes (R18/F5-TPB and PDI-1) as it had direct influence on QY, photostability of NPs and EET efficiency. PLGA NPs showed the evidences of much higher dye aggregation than the two other polymer matrices. Even more prominent differences were observed in the blinking behavior of these NPs. PLGA NPs displayed ON/OFF switching starting from 1 wt% of dye loading, whereas for PMMA and PCL NPs some partial blinking was observed only at very high (30 wt%) dye loading. The time-resolved fluorescence anisotropy studies suggested different rate of EET for NPs built from different polymers, suggesting that the interfluorophore distances are significantly shorter for PLGA, compared to the other two matrices. The difference in the dye organization could be linked to the solubility of the polymer in the solvent mixture, used for nanoprecipitation. In this case, PLGA with the highest solubility and thus slowest nanoprecipitation kinetics allows hydrophobic dyes to cluster inside the nanoparticle core, producing NPs with high dye cooperativity. By contrast, in PMMA and PCL the dyes are more dispersed with the polymer matrix, and thus giving NPs with higher quantum yield and photostability. The choice between very high photostability and cooperative behavior depends on the experiment: NPs with constant emission could be applied for particle tracking, and those with complete on/off switching could be used in super-resolution imaging.

In the second part of my work, I exploited EET among donor dyes R18/F5-TPB in order to obtain efficient FRET to single acceptors inside NPs. The obtained results demonstrated importance of cooperative behavior of fluorophores inside NPs to ensure efficient FRET to the photochromic molecule and, thus, to produce efficient photoswitching. This system could be further improved by using photochromic molecules with higher extinction coefficient and better fatigue resistance, as well as by improving photostability of donor dyes with less pronounced blinking behavior (or its absence), but preserving their cooperativity. At the same time efficient homogeneous photoswitching at single particle level requires creation of more monodisperse NPs that would assure more uniform distribution of particle brightness.

Next, much larger dye ensembles coupled by EET inside NPs were used to generate giant light-harvesting antenna. It amplified the fluorescence of a single dye which enabled observation of single molecule events (one-step bleaching of the acceptor) at extremely low laser powers that approaches single molecule photography at ambient light without dedicated instrumentation. As energy from thousands of donors funnels to single acceptors, the excellent photostability of the latter one is of crucial importance for performance of the nano-antenna. Elimination of "energy traps" (such as dark state of R18/F5-TPB) that are competitors for energy with acceptor molecule would make possible obtaining even higher amplification of acceptor emission.

Finally, the possibility of FRET-based sensing of individual molecules on the surface of dye-loaded NPs was demonstrated. Due to ultra-fast EET between donor dyes, FRET with efficiency > 50% was achieved with a single acceptor. Using acceptor molecule conjugated with a specific ligand we prepared nanoparticle-based probes for proteins. In these nanoprobes, the acceptor-ligand conjugate is removed from NPs surface in the presence of the target protein, which stops FRET and thus changes the dual emission of NPs. So, created NPs could be used as building blocks for biosensors with single molecule sensitivity. However, robust biosensors would require covalent linking of acceptor dye to the surface of NPs. Encapsulation of higher amount of donor dyes with preserved high QY, as well as elimination of "energy traps" and using 157 acceptor with better photostability and QY would increase the antenna effect and improve performance of these biosensors.

Several remaining problems should be solved for further successful application of polymer NPs in bioimaging. First, monodispersity of particles should be improved, which is essential for quantitative detection of biomolecules. Solving the problem of leaching of the encapsulated dyes into biological media would minimize artefacts and maximize the signal to background ratio.

Achieved to-date properties and systematic work on the described problems promise the bright future to dye-loaded NPs in bioimaging applications.

PART Materials and methods

Materials

All chemicals and solvents for synthesis were purchased from Alfa Aesar and Sigma Aldrich and used as received unless stated otherwise.

Lumogen Red (N,N′-Bis- (2,6-diisopropylphenyl)-1,6,7,12tetraphenoxy-3,4,9,10- Poly(D,L-lactide-co-glycolide) (PLGA, lactide 50 mol %, glycolide 50 mol %, acid terminated, Mn 24 000, PDI 1.7, and Mn 8800, PDI 1.7), poly-(methyl methacrylate-co-methacrylic acid) (PMMAMA, 1,3% methacrylic acid, Mn ∼15 000, Mw∼34 000) were purchased from Sigma-Aldrich. Synthesis of carboxylate bearing form of Polycaprolactone (PCL) (R,ω-dihydroxy functional, Mn ∼10 000, Mw ∼14 000) and sulfonate-bearing form of PMMAMA are described in ref. [176] Sodium phosphate monobasic (>99.0%, Sigma-Aldrich) and sodium phosphate dibasic dihydrate (>99.0%, Sigma-Aldrich) were used to prepare 20 mM phosphate buffer solutions; tris(hydroxymethyl)amino methane (Tris base, >99.8%, SigmaAldrich) and HCl (1 M) were used to prepare 20 mM Tris buffer with a pH of 7.4.

Milli-Q water (Millipore) was used in all experiments.

Preparation of polymer NPs

Stock solutions of polymers in acetonitrile were prepared at a concentration of 10 mg/ml. These solutions were then diluted with acetonitrile containing desired amount of dye, to the concentration of 1 or 2 mg/ml. 50 μL of the polymer solutions were then added quickly using a micropipette and under shaking (Thermomixer comfort, Eppendorf, 1000 rpm) to 450 μL of water or buffer. PLGAand PMMA-based nanoparticles were prepared at 21°C, PCL nanoparticles were prepared at 27°C.

PMMASO3 NPs were prepared according to the same protocol, but with dilution in water at 21°C. The particles solution was then quickly diluted 5-fold with the same buffer. For varying the size of PMMAMA NPs the value of pH of buffer at the first dilution was varied between 5.8 and 9.

NPs characterization

1 2 1 1 1 1 1 ) (                      d d S N G     
where τd is the diffusion time, N is the mean number of fluorescent species within the two-photon excitation volume, and S is the ratio between the axial and lateral radii of the excitation volume. The excitation volume is about 0.34 fL and S is about 3 to 4. The measurements were done with respect to a reference 6carboxytetramethylrhodamine (TMR from Sigma-Aldrich) in water. The hydrodynamic diameter, d, of NPs was calculated as: dNPs = d(NPs)/d(TMR)dTMR, where dTMR is a hydrodynamic diameter of TMR (1.0 nm). Concentration of NPs was calculated from the number of species by: CNPs = NNPs/NTMR×CTMR, using a TMR concentration of 50 nM.

Single particle imaging

For single particle fluorescence measurements, the NPs were immobilized on glass surfaces on which a polyethyleneimine (PEI) layer was initially adsorbed. The solutions of NPs were diluted to a particle concentration of about 6 pM with buffer. 400 μL of these solutions were then brought in contact with the PEI-covered glass for 15 min, followed by extensive rinsing with milliQ-water. The surfaces were left in milliQ water during the microscopy measurements. Quantum dots and FluoSpheres® redorange at 6 pM concentration were immobilized and imaged in the same way as NPs.

Single particle measurements were performed in the TIRF (Total Internal Reflection Fluorescence) mode on a homemade wide-field setup based on an Olympus IX-71 microscope with an oil immersion objective (NA = 1.49, 100×). The fluorescence signal

Time-resolved fluorescence

The measurements were performed with the time-correlated, single-photon counting technique using the excitation pulses at 480 nm provided by a pulse-picked frequency doubled Ti-sapphire laser (Tsunami, Spectra Physics) pumped by a Millenia X laser (Spectra Physics). The emission was collected through a polarizer set at the magic angle and an 8 nm band-pass monochromator (Jobin-Yvon H10) at 582 nm.

The instrumental response function was recorded with a polished aluminium reflector, and its full-width at half-maximum was 40 ps. For time-resolved anisotropy measurements, the fluorescence decay curves were recorded at the vertical and horizontal positions of the excitation polarizer and with the emission polarizer set to the vertical position, and analysed by the following equation:

𝑟(𝑡) =

𝐼 𝑣 (𝑡)-𝐺•𝐼 ℎ (𝑡) 𝐼 𝑣 (𝑡)+2•𝐺•𝐼 ℎ (𝑡) (1) where Iv and Ih are the intensities collected at vertical and horizontal excitation polarizations, respectively, and G is the geometry factor at the emission wavelength, determined in independent experiments.

Cellular studies

HeLa cells (ATCC® CCL-2) were grown in Dulbecco's modified Eagle's medium (DMEM, Gibco-Invitrogen), supplemented with 10% fetal bovine serum (FBS, Lonza) and 1% antibiotic solution (penicillin-streptomycin, Gibco-Invitrogen) at 37 °C in a humidified atmosphere containing 5%CO2. Cells were seeded onto a chambered cover glass (IBiDi) at a density of 5 × 10 4 cells per well 24 h before the microscopy measurement. For imaging, the culture medium was removed and the attached cells were washed with Opti-MEM (Gibco-Invitrogen). Then, a freshly-prepared solution of NPs loaded with 1 wt% LR (at 20-fold dilution of the original formulation corresponding to ∼0.15 nM of NPs) in Opti-MEM was added to the cells and incubated for different time periods. Cell membrane staining with wheat germ agglutinin-Alexa488 (Life Technologies) was done for 10 min at rt before the measurements. Confocal fluorescence images of the cells were taken on a Leica TSC SPE confocal microscope. For cytotoxicity studies, HeLa cells were seeded in 96-well plates at a concentration of 104 cells per well in 100 μL of the DMEM growth medium and then 164 incubated overnight at 37 °C in a humidified atmosphere containing 5% CO2. Next, the PLGA NPs loaded with 1 wt% of PDI-1 or LR were added, by substituting the culture medium with the fresh one containing variable concentrations of NPs. After incubation for 24 h, the medium was removed and the adherent cell monolayers were washed with PBS. Then, the wells were filled with cell culture medium containing MTT, incubated for 4 h at 37 °C, and the formed formazan crystals were dissolved by adding 100 μL of a 10% sodium dodecylsulfate (SDS), 0.01 M hydrochloric acid solution. The absorbance was then measured at 570 nm with a microplate reader. Experiments were carried out in triplicate and expressed as the percentage of viable cells compared to the control group. Cette hypothèse a été prouvée avec succès en utilisant R18/F5-TPB dans une matrice de poly(méthacrylate de méthyle-co-acide méthacrylique) (PMMA-MA) (Figure 3). Nous avons ensuite exploité l'EET entre les colorants donneurs pour amplifier l'efficacité de l'alternance photo-induite (photocommutation) dans des nanoparticules de polymères dopées en fluorophores, et obtenir ainsi des matériaux ayant de nombreuses applications potentielles, particulièrement en imagerie de fluorescence à haute résolution. [233] Dans ce but, nous avons utilisé R18/F5-TPB ayant le rôle de donneur à l'intérieur d'une matrice de PLGA et un accepteur photochrome de la famille des diaryléthènes (PCD1) (Figure 5).

Résume de la thèse en français

Pour comprendre le rôle de l'EET, nous avons préparé des nanoparticules avec différents contre-ions, qui ne favorisent pas la diffusion excitonique de la rhodamine.

Parmi les contre-ions testés, seul le tétraphénylborate perfluoré, qui favorise la coopérativité du flurophore, permet une bonne efficacité de la photocommutation (rapport on/off ~ 20) (Figure 6). Ces nanoparticules ont été testées en solution et au niveau de la particule unique. Ce nouveau concept ouvre la voie pour de nouveaux photocommutateurs nanoscopiques efficaces. sont désadsorbé la surface des nanoparticules (Figure 6). Ce senseur ratiométrique permet de détecter la streptavidine avec une limite de détection de 0.7 nM. La versatilité de ce senseur a été confirmée en utilisant la Cy5-benzene sulfonamide comme accepteur pour la détection de l'anhydrase carbonique humaine (hCA).

Figure 1 . 1 .

 11 Figure 1.1. Jablonski energy diagram illustrating the transition between electronic states of a molecule for the quantum mechanical processes of fluorescence and phosphorescence. Waved lines mark non-radiative transitions. IC means internal conversion, ISC intersystem crossing. Adopted from http://home.uni-leipzig.de.

Figure 1 . 2 .

 12 Figure 1.2. Schematic diagram of a spectrofluorometer. The light from source passes through the monochromator selecting the fixed wavelength and excites the sample. The fluorescence is

Figure 1 . 4 .

 14 Figure 1.4. Principle of photoselective excitation. 𝜽 𝑨 is the angle between the electric vector of the indecent light (blue arrows) and the absorption transition moment of the molecule (purple arrows).

Figure 1 . 5 .

 15 Figure 1.5. Fluorescence polarization and anisotropy. Polarized excitation іs used to selectіvely excіte dipole-aligned fluorophores.

Figure 1 . 6 .

 16 Figure 1.6. Fluorophore brightness versus the wavelength of maximum absorption (λmax) for different classes of fluorophores. The colors of depicted structure illustrate their wavelengths of emission (λem). Adopted from ref.[START_REF] Lavis | Bright Ideas for Chemical Biology[END_REF]

Figure 1 . 7 .Figure 1 . 8 .Figure 1 . 9 .

 171819 Figure 1.7. Architecture of Green Fluorescent Protein from Aequorea Victoria and chemical structure of its chromophore. Adopted from zeiss-campus.magnet.fsu.edu.

Figure 1 . 10 .

 110 Figure 1.10. Schematic illustration of UCNP and illustration of its optical properties. a) Schematic illustration of UCNP composed of a crystalline "host" and embedded lanthanide dopant ions.

  [START_REF] Wu | Highly Fluorescent Semiconducting Polymer Dots for Biology and Medicine[END_REF] Generally, they exhibit very high extinction coefficient, moderate quantum yield and relatively short fluorescence lifetime. They are among the brightest fluorescent NPs developed to date.

Figure 1 . 12 .

 112 Figure 1.12. The structures of different conjugated polymers: poly(9,9-dihexylfluorene) (PDHF), poly(9,9-dioctylfluorene) (PFO), poly[2-methoxy-5-(2-ethylhexyloxy)-1,4-phenylenevinylene] (MEH-PPV), poly[2-methoxy-5-(2-ethylhexyloxy)-2,7-(9,9-dioctylfluorene)] (PFPV), poly(p-phenylene ethynylene) (PPE), poly(9,9-dioctylfluorene-2,7-diyl-co-benzothiadiazole) (PFBT).

Figure 1 . 14 .

 114 Figure 1.14. Precipitation, ion -association and self-assembly methods for preparation of FOD-NPs. Adopted from ref.[START_REF] Klymchenko | Emerging field of self-assembled fluorescent organic dye nanoparticles[END_REF]

Figure 1 . 15 .

 115 Figure 1.15.Schematic representation of a multifunctional SiNP. Adopted from ref.[START_REF] Montalti | Dye-doped silica nanoparticles as luminescent organized systems for nanomedicine[END_REF]

Figure 1 . 17 .

 117 Figure 1.17. Techniques used for the preparation of dye loaded NPs from preformed polymers.

1 . 4 . 1 .

 141 be a result of dіfferent іnteraction mechanіsms: long-range dipole-dipole interactions (Förster's mechanism), short-range multipolar interactions, electron exchange (Dexter mechanism) and charge resonance interactions. For singlet-singlet EET all types of interactions are involved, whereas triplet-triplet energy transfer occurs only due to orbital overlap. Dexter energy transfer Dexter energy transfer or Dexter electron exchange is a fluorescence quenching mechanism in which an excited electron is transferred from D to A via a non-radiative pathway. It can only occur at short distances (around 10 Å) because it requires a wave function overlap between D and A.

Figure 1 . 18 .

 118 Figure 1.18. Schematic representation of FRET as a photophysical process. FRET efficiency depends on the distance a) and orientation b) of D (green ellipse) and A (red ellipse). Adopted from

Figure 1 . 19 .

 119 Figure 1.19. Examples of available fluorescent dye and quencher families used for FRET. λabs and λem are marked. Tetramethylrhodamine (TMR), carboxytetramethylrhodamine (TAMRA) and carboxy-X-rhodamine (ROX) are rhodamine-based dyes, FITC is a fluorescein isothiocyanate. Adopted from ref.[START_REF] Sapsford | Materials for fluorescence resonance energy transfer analysis: Beyond traditional donor-acceptor combinations[END_REF]

  inverse approach is to fuse interacting domains with D and A proteins, or to fuse them to single active proteіn construct, whose conformational changes will bring them in close contact (Figure1.20). The universality of design led to creation of variety of FRET-based sensors for monitoring changes in concentration of Ca 2+ , glutamate, tryptophan, kinase activities and others.[START_REF] Chudakov | Fluorescent Proteins and Their Applications in Imaging Living Cells and Tissues[END_REF]

Figure 1 . 20 .

 120 Figure 1.20. Main types of FP-based fluorescent sensors: protein-protein interaction, monitorprotease activity and detection of protein conformational changes. Adopted from ref.[START_REF] Ishikawa-Ankerhold | Advanced fluorescence microscopy techniques-Frap, Flip, Flap, Fret and flim[END_REF]

Figure 1 . 21 .

 121 Figure 1.21. Effect of rate constant on FRET efficiency. a) FRET efficiency. b) The same data plotted as A = E/(1-E) as a function of rate constant. Adopted from ref. [100]

Figure 1 . 22 .

 122 Figure 1.22. Simulated distance-FRET response curves. Theoretical curves show the FRET efficiency differences for intermolecular distances of 7 and 8 nm when measured with acceptor F/P=1 (∆E0) and acceptor F/P=5 (∆En) with R0 = 4 nm. Adopted from ref. [100]

Figure 1 . 23 .

 123 Figure 1.23. Design of FRET-NPs. a) Preparation of NPs by nanoprecipitation method. b) Chemical structures of DiD and DiR fluorophores. c) DiD with λex at 644 nm and λem at 665 nm in methanol, and DiR with λex at 750 nm and λem at 780 nm in methanol, were encapsulated inside FRET-NPs. The close proximity between the entrapped DiD and DiR fluorophores favored the FRET. Adopted from ref. [101]

Figure 1 . 24 .

 124 Figure 1.24. Schematic representation of polymer NPs with multiple FRET cascade mechanisms designed for a) multicolor and b) multiplex imaging. Adopted from ref. [102]

Figure 1 . 25 .

 125 Figure 1.25. Schematic illustration of preparation of spiropyran-based dual-color photoswitchable NPs. Adopted from ref. [103]

Figure 1 . 26 .

 126 Figure 1.26. Schematic illustration of multicolor and photoswitchable fluorescent polymeric NPs covalently incorporating two (EANI and SPMA) or three fluorescent dyes (EANI, NBDAA, and SPMA) under excitation at 385 nm. Adopted from ref.[107]

Figure 1 . 27 .

 127 Figure 1.27. Schematic illustration of the FRET-based ratiometric measuring NPs: D are excited and the energy is transferred to the proximal A within the cyclam-coated dual-dye-doped NPs.

  [108] Dual-step FRET-based quenching assay was proposed by Soukka et al. for screening of caspase-3 inhibitors.[110]. For this propose fluorescent europium(III)chelate-doped donor NPs coated with streptavidin were labelled with Alexa Fluor 680 fluorescent acceptor and BlackBerry Quencher 650 and caspase-3-specific peptide substrate modified with a biotin moiety(Figure 1.28). In this case, FRET excites the A, whose intensity is controlled by the proximity of the quencher. In inhibitory conditions, the biotinylated substrate remains intact and acceptor emission is efficiently attenuated by the BlackBerry Quencher 650, but in non-inhibitory conditions, the enzyme cleaves the biotinylated substrate releasing the quencher, and switching on the acceptor emission. To obtain high signal-to-noise ratio biotinylated substrates should excess the NPs concentration 5-6 times. Further increase in A concentration led to its self-quenching.

Figure 1 . 28 .

 128 Figure 1.28. Principle of the dual-step FRET quenching assay. The streptavidin-coated europium(III)-chelate-doped nanoparticle donors (D) are excited, and the energy is transferred to the Alexa Fluor 680 acceptor (A). In inhibitory conditions (upper panel), the biotinylated substrate is intact and the A emission is efficiently attenuated by the BlackBerry Quencher 650 (Q). In non-inhibitory conditions (lower panel), the enzyme cleaves the biotinylated substrate releasing the Q leading to emission of the fluorescent A. Adopted from ref. [110]

Figure 1 . 29 .

 129 Figure 1.29. Multicolor FRET SiNPs. a) Fluorescence spectra of NP encapsulated with one, two, or three types of fluorophores. b) NP with different doping dye combinations under 300-nm UV illumination. Dye doping ratio (in order): 1:0:0, 0:1:0, 1:0:1, 4:1.5:3, 0.5:0.5:0.5, 2:2:2, 0:1:1, 0.5:0.5:4. Adopted from ref. [112] Doping of SiNPs of 35-50 nm in diameter with oligothiophenes (TFs) with blue, green and orange emission resulted in creation of NPs with tunable emission from blue to white. [114] For this FRET between adjacent amounts of TFs was exploited. Its efficiency reached up 80-90% at ratio of components 1:1. Multiple dye-doped NIR emitting SiNPs, suitable for optical imaging and multiparametric flow cytometry analyses, were developed by Secchiero and coworkers by encapsulation of alkoxysilane derivative of Cy5.5 in combination with IR813alkoxysilane derivative of Cy7. [115] NPs were synthesized at two different Cy5.5 : Cy7 molecular ratios (2 : 1, 1 : 1). SiNPs displayed FRET efficiency of 38% and 70% respectively, and very similar overall quantum yields of 0.08 and 0.06. Examples of photoswitchable SiNPs, created by encapsulation of photochromic acceptor molecules together with fluorescent dyes that serve as energy donor, are also known. [116, 117] He et al. reported encapsulation of two highly water-soluble dyes, tris(2,2bipyridyl)-dichlororuthenium(II) hexahydrate (RuBpy) and methylene blue (MB) as the model donor-acceptor pair into silica NPs around 40 nm in diameter (Figure 1.30).

Figure 1 . 30 .

 130 Figure 1.30. Schematic illustration SiNPs with FRET from RuBpy to MB and the chemical structures of RuBpy and MB. Adopted from ref. [118]

Figure 1 . 31 .

 131 Figure 1.31. Schematic illustration for formation of a FRET-based ratiometric Hg2+ sensor with multilayered silica particles as the scaffold, and the proposed structural change of the probe. Adopted from ref. [119]

Figure 1 . 32 .

 132 Figure 1.32. Schematic representation of the FRET model systems. a) FRET from fluoresceinlabeled avidin to RITC-labeled biotinylated SiNPs. b) FRET from RITC-labeled biotinylated SiNPs labeled avidin. Adopted from ref. [121]

Figure 1 . 33 .Figure 1 . 34 .

 133134 Figure 1.33. Schematic representation of the detection system for thrombin using [Ru (dpsphen)3] 4-ion-doped SiNPs-aptamer conjugate. Adopted from ref. [122]

  Special organization of fluorophores and their spectroscopic properties plays crucial role for creation of the systems with efficient EET. Studying systems where donor and acceptor are covalently linked, provided most of fundamental advances in this field. 52 The structure of dendrimer molecule is similar to the architecture of natural lightharvesting complexes, where the reaction center is surrounded by antenna molecules, and the positioning of fluorophore could be achieved with high precision, locating them in the core, focal point, and periphery or at each branching point. [126]. EET in dendrimers can occur by Förster [128] and by Dexter [129] mechanisms. The pioneering work of light-harvesting dendrimers was reported in the early 1990s by Balzani et al. [129] For this, Ru(II) and Os(II) polypyridine complexes were used as building blocks. The first unidirectional FRET in a dendritic framework was achieved from phenylacetylene monomers to a single molecule of perylene located in the core by Xu and coworkers. [128]. In addition to the light-harvesting dendrimers with donor and acceptor connected by covalent bonds or coordination, EET can also take place in self-assembled dendritic structures, in which components (usually the energy acceptor) are incorporated into the systems by noncovalent interactions. [130] Nevertheless, occurrence of self-quenching and excimer formation increases in higher generation of dendrimers, decreasing EET efficiency. Another example of artificial light-harvesting complexes are multiporphyrin arrays. [131] They are duplicating the structure and functions of LH antenna complexes. Creation of closed ring architecture is possible by covalent bonding, that gives precise control of spatial molecules arrangement [132] or by supramolecular self-assembly [133]. The main disadvantage of multiporphyrin arrays is the narrow absorption windows due to limited range of porphyrinoid macrocycles.

Figure 1 . 35 .

 135 Figure 1.35. Schematic of the MPWs. The colored circles represent the dyes, while each DNA strand is presented by a different color. Arrows represent possible energy transfer steps and their directionality. b) 5-dye MPW. Dye positions are indicated below the MPW and apply to both systems. I = Input and O = Output. Adopted from ref. [135]

Figure 1 . 36 .

 136 Figure 1.36. Self-assembly of light-harvesting structures. a) Mixtures of TMVP monomers labelled with donor 1 (Oregon green 488) and acceptor 3 (Alexa Fluor 594) were assembled into rods.b) Tabulated antenna effect for these systems. Adopted from ref.[137]

  The importance of collective behavior to obtain efficient FRET was shown on the example of PluSNPs doped with rhodamine B and hosted single cyanine dyes on the surface. Simulation considering that each rhodamine dye independently transfers energy to a cyanine gives much lower FRET efficiency than experimental data (Figure1.37).[139]

Figure 1 . 37 .

 137 Figure 1.37. Experimental FRET efficiency (red dots, obtained by steady state emission quenching measurements) versus simulated average FRET efficiency (black dots). No homo-FRET between D is considered in the simulations. Each point represents the average FRET efficiency for a set of 1000 NPs containing 10 D on average embedded in a 5.5 nm radius core of PlusNPs, and anincreasing number of acceptor dyes hosted in a very thin shell (0.2 nm). Forster distance considered to be R0 = 5.0 nm. Adopted from ref.[138]

Figure 1 . 38 .

 138 Figure 1.38. Schematic representation of the Cu + sensing mechanism by PluSNPs. Adopted from ref. [140]

Figure 1 . 39 .Figure 1 . 40 .

 139140 Figure 1.39. Exploiting of the cooperativity of the dye inside SiNPs for increasing of QY. a) Chemical structure of D and A molecules. b) Simplified representation of the synthesis of the NPs. c)The total NPs intensity. Adopted from ref.[141]

Figure 1 . 41 .

 141 Figure 1.41. Illustration of the FRET-induced "giant amplification of fluorescence photoswitching" in NPs. After promotion of few molecules to the non-fluorescent state by UV irradiation, a large number of PF are quenched) by long-range intermolecular FRET process within each Förstersphere. Adopted from ref.[START_REF] Su | Giant Amplification of Photoswitching by a Few Photons in Fluorescent Photochromic Organic Nanoparticles[END_REF]

Figure 1 . 42 .Figure 1 . 43 .

 142143 Figure 1.42. Proposed model of the counterion-assembled dyes inside NPs. Short-range ordering of rhodamine dyes (red) by the F5-TPB counterion (blue) inside the polymer matrix prevents dye aggregation and leads to the short inter fluorophore distance. Dark species (grey), generated by light, turn off the whole ensemble of dyes coupled by EET leading to on/off switching. Adopted from ref.[START_REF] Reisch | Collective fluorescence switching of counterion-assembled dyes in polymer nanoparticles[END_REF]

Figure 1 . 44 .

 144 Figure 1.44. FRET efficiency as a function of dye molecules added per NP. Adopted from.

Figure 1 . 45 .

 145 Figure 1.45. Photoswitching QDs coated with an amphiphilic photochromic polymer. Adopted from ref. [147]

Figure 1 . 46 .

 146 Figure 1.46. Schematic representation of DNA nanosensor assembly based on FRET QD and Cy5.Adoptd from ref. [148].

Figure 1 . 47 .

 147 Figure 1.47. Schematic representation of a homogeneous multiplexed FRET assay to monitor the activity of trypsin (TRP), chymotrypsin (ChT), and enterokinase (EK) using three different QD donors (blue mitting QD450, green-emitting QD540, and red-emitting QD625) in combination with three different organic dye acceptors (QSY35, QSY9, and A647), respectively. Adopted from ref. [149]

Figure 1 . 48 .

 148 Figure 1.48. The schematic of the nucleotide sensor design based on FRET from UCNPs.Adopted from ref.[151]

Figure 1 . 49 .

 149 Figure 1.49. UCNP-based LRET system. a) A scheme of the UCNP-LRET system; the hydrophobic layer between the inorganic nanoparticle surface and the external PEG coating allows adsorption of organic dye molecules on UCNPs; b) In vivo multicolor UCL images of a nude mouse subcutaneously injected with five colors of UCNPs solutions. Merge-UCL (left) and white light (right).Adopted from ref.[152]

  Thus, energy transfer from conjugated-nonconjugated multiblock copolymers (CNMBC) to poly[2-methoxy-5-(2-ethylhexyloxy)-1,4-phenylenevinylene] (MEHPPV) leads to enhanced emission of the latter almost 500 times for some ratios. [154] 63 FRET in blend of conjugated polymers was demonstrated in NPs, consisting of a derivative of the blue-emitting conjugated polymer, polyfluorene-doped with green-, yellow-, and red-emitting conjugated polymers, prepared by a precipitation method. Fluorescence quenching analysis of the host polymer as a function of the dopant concentration indicated that one energy acceptor molecule can effectively quench 90% of the fluorescence of a nanoparticle consisting of 100-200 host conjugated polymer molecules. [155] FRET from P-dots to doped fluorescent dyes is also possible. In the first example of this phenomenon, reported by Grigalevicius and co-workers, the blue fluorescence of the dye-coated polyfluorene nanoparticles was slightly quenched after dye deposition, that was accompanied with an appearance of a new emission band of the surface-bound dye (Rhodamine 6G or Rhodamine TM).[156] Later, McNeill and co-workers prepared polyfluorene-based NPs, doped with various dyes by reprecipitation method. [157] The results indicated efficient EET from host (donor) polyfluorene to dopant (acceptor) dye molecules (Figure 1.50).

Figure 1 . 50 .

 150 Figure 1.50. Concentration dependent fluorescence spectra of polyfluorene based NPs doped with perylene with increasing concentration. Adopted from ref. [158]

Figure 1 . 51 .

 151 Figure 1.51. Schematic illustration of the formation of P-dots for oxygen sensing. Adopted from ref. [160]

1. 5 . 1 .

 51 Dynamic Light Scattering Dynamic Light Scattering (DLS) is a commonly used technique for determination of size and size distribution of particles, usually in the sub-micron region. It is based on measuring Brownian motion of NPs in suspension. Brownian motion is a random displacement of particle, caused by its bombardment by the solvents molecules. For larger particles movements is slower. DLS relates velocity of Brownian motion, known as translational diffusion coefficient, to the size of NPs according to the Stokes-Einstein equation. 𝑑(𝐻) = 𝑘𝑇 3𝜋𝜂𝐷 Where 𝑑(𝐻)) = hydrodynamic diameter D = translation diffusion coefficient k = Boltzmann's constant (physical constant relating energy at the individual particle level with temperature, equal to 1.38×10 -23 J/K) T = absolute temperature η = viscosity Brownian motion could be measured by analyzing the rate of scattered light fluctuation. Usually, photon auto-correlation function is used for this. The idea is to measure the degree of similarity between two signals, or one signal with itself at varying the time intervals. The autocorrelatіon functіon usually decays startіng from zero delay tіme, and faster dynamіcs due to smaller particles lead to faster decorrelation of scattered іntensity trace (Figure 1.52).

Figure 1 . 52 .

 152 Figure 1.52. Examples of the dynamic light scattering measurements: a) autocorrelation curve b) and size distribution c) of small (upper panel) and big NPs (lower panel). Image adopted from otsukael.com.

  68 initial amplitude of the autocorrelation function is inversely proportional to the number of molecules in the detection volume. The autocorrelation function decays from its initial value with a time-dependence that is determined by molecular diffusion rates (Figure 1.53).

Figure 1 . 53 .

 153 Figure 1.53. Schematic diagram of FCS. a) Schematic of observation area of FCS (confocal volume). b) Obtained fluctuation of fluorescence intensity. c) Autocorrelation functions. Adopted from ref. [163]

Figure 1 . 54 .

 154 Figure 1.54. Schematic representation of TIRF. Adopted from ncl.ac.uk

Figure 1 . 55 . 1 . 5 . 5 .

 155155 Figure 1.55. Main components of transmission electron microscopy. Adopted from britannica.com.

Figure 1 . 56 . 1 . 5 . 6 .

 156156 Figure 1.56. Principle of SEM. Adopted from britannica.com

Figure 1 . 57 . 1 .

 1571 Figure 1.57. Schematic diagram of atomic force microscopy. Adopted from ref. [166]

  To address the problem of brightness and photostability of fluorescent polymer NPs, encapsulation of two bulky perylene diimide (PDI) derivatives into polymer poly (lactic-co-glycolic acid) PLGA NPs was studied (Figure2.1). PDI dyes are considered as one of the most photostable dyes to date. [171] QY of PDI-1 in solution was reported to be close to unity[172] due to suppression of aggregation by addition of bulky peripheral groups. Lumogen Red (LR) has additional bulky group (aromatic rings) in the bay regions and characterized by QY = 0.96 and high resistance to aggregation.[START_REF] Colby | Electronic energy migration on different time scales: concentration dependence of the time-resolved anisotropy and fluorescence quenching of lumogen red in poly (methyl methacrylate)[END_REF] PLGA is a biodegradable and biocompatible copolymer, approved by of Food and Drug Administration (FDA). The mechanism of PLGA degradation is hydrolysis of the ester linkage in aqueous solution. The products of degradation are lactic and glycolic acids, that are naturally present in the body. The time required for degradation depends on monomers ratio. For higher amount of glycolic acid lower time for degradation is needed.[173]

Figure 2 . 1 .

 21 Figure 2.1. Chemical structure of PDI-1 and LR dyes, polymer PLGA (a) and schematic presentation of dye-doped PLGA nanoparticles (b).

Figure 2 . 2 .

 22 Figure 2.2. Difference of aggregation behavior of PDI-1 and LR in PLGA NPs. a) Normalized emission spectra and b) QY for different loading.

Figure 2 . 3 .

 23 Figure 2.3. Photostability of NPs loaded with different amount of dyes. NPs are immobilized on glass surface and are continuously irradiated by a laser 532 nm at 5 a) and 50 b) W/cm 2 . As a reference, FluoroSpheres (FS, Invitrogen, FluoSpheres® Carboxylate-Modified Microspheres, 0.04 µm, redorange fluorescent (565/580), 5% solids, azide free) were used. According to the measurements with a 532 nm excitation laser, our 40 nm PLGA NPs loaded with 1 wt% Lumogen Red were >10-fold brighter than quantum dots (QDs-585 streptavidine conjugate, Life Technologies). The stability of NPs in presence of 10% serum was confirmed by FCS. These NPs were incubated with HeLa cells and then further imaged by confocal fluorescence microscopy. They entered spontaneously into cells by endocytosis showing no sign of cytotoxicity (confirmed by MTT-test). The present work demonstrated the importance of introducing of bulky side groups into structure of encapsulated dyes to prevent their aggregation. The surprising result was very poor photostability of PDI-1 dye in an aggregated form. At the same time, excellent photostability and brightness of NPs loaded with LR make them biodegradable substitutes of quantum dots, which could find a variety of applications in biological imaging. The results were published in the article: Trofymchuk, K.; Reisch, A.; Shulov, I.; Mely, Y.; Klymchenko, A. S., "Tuning the color and photostability of perylene diimides inside polymer nanoparticles: towards biodegradable substitutes of quantum dots" Nanoscale 2014, 6 (21), 12934-12942.

Figure 2 . 4 .

 24 Figure 2.4. Structures of the polymers poly(lactic-co-glycolic acid) (PLGA), poly(methyl methacrylate-co-methacrylic acid) (noted PMMA here), and acid terminated polycaprolactone (PCL), of the dye rhodamine B octadecyl ester (R18) and its counterion tetrakis(pentafluorophenyl)borate (F5-TPB), and the dye PDI-1, used in this study, and schematic representation of a dye-loaded polymer nanoparticle.

Figure 2 . 5 .

 25 Figure 2.5. Size of NPs made from different matrix polymers as measured by DLS. Volume distribution averages are given, error bars correspond to standard error of mean over at least 3 independent measurements.

Figure 2 . 6 .

 26 Figure 2.6. Normalized absorbance and emission spectra of a) PLGA, b) PMMA, and c) PCL NPs loaded with different amounts of R18/F5 and comparison to the spectra of R18/F5 in methanol.

  fluorophores. Measuring photostability of these NPs in the fluorimeter revealed a strong difference in the behavior of NPs made from different polymers (Figure 2.7 b): for example for a 5 wt% loading PMMA NPs showed practically no decrease (<5%) in their fluorescence brightness over 1 h of illumination. PCL NPs showed a decrease in their fluorescence by about 25 % over 30 min, after that the fluorescence became stable. At this loading and illumination PLGA NPs, on the other hand, showed a strong decrease of the fluorescence by nearly 90 % over 1 h.

Figure 2 . 7 .

 27 Figure 2.7. Photostability of NPs made from different polymers and loaded with 5 wt% of R18/F5-TPB.

Figure 2 . 8 .

 28 Figure 2.8. Single particle brightness of NPs made from different polymers and loaded with different amounts of R18/F5-TPB. a) Microscope images of NPs immobilized on a surface in TIRF mode using an illumination intensity of 0.5 W/cm 2 . Scale bar corresponds to 20 µm. And brightness for at least 1000 particles as measured by TIRF microscopy using a 532 nm laser with an illumination intensity of b) 0.5 W/cm 2 and c) 5 W/cm 2

Figure 2 . 9 .

 29 Figure 2.9. Single particle transients of NPs made from different polymers with different amounts of R18/F5-TPB. Given are representative transients as measured using TIRF microscopy using a 532 nm laser with an illumination intensity of 0.5 W/cm 2 .

Figure 2 . 10 .

 210 Figure 2.10. Histograms of the instantaneous fluorescence intensities (relative to the maximum intensity) of NPs with different dye loadings. Intensities were obtained from single particle transients recorded with TIRF microscopy using a 532 nm laser with an illumination intensity of 0.5 W/cm 2 . About 500 NPs per condition were used.

Figure 2 . 11 .

 211 Figure 2.11. Fluorescence anisotropy of NPs. a) Steady state fluorescence anisotropy of nanoparticles made from different polymers and loaded with different amounts of R18/F5-TPB (λex = 530 nm). Given values are averages over at least 3 measurements, error bars correspond to standard error of mean. b) Anisotropy decays of nanoparticles made from different polymers and loaded with 1 or 5 wt% of R18/F5-TPB.

Figure 2 . 12 .

 212 Figure 2.12. Absorption and emission properties of PDI-1-loaded NPs made from different polymers: a) Normalized fluorescence emission spectra of NPs of different polymers, containing different amounts of PDI-1 and comparison to PDI-1 in dioxane. (Normalization has been performed relative to the peak at 530 nm). b) QYs of NPs made from different polymers and loaded with different amounts of PDI-1. QYs were determined relative to Rh6G in ethanol as standard. Given values are averages over at least 3 measurements, error bars correspond to standard error of mean. c) Ratio of absorbance of these NPs at 490 nm and 525 nm. d) Ratio of fluorescence emission of these NPs at 630 nm and 530 nm

Figure 2 . 13 .

 213 Figure 2.13. Turbidity measurement of different polymers in acetonitrile water mixtures. Given are the absorbance values measured at 400 nm for polymer solutions in acetonitrile with different water contents.

  have the time to embed into the forming nuclei at the very beginning of the particle growth. It could also form the nuclei itself or at least participate in their formation and thus initiate particle formation. Both scenarios could explain the formation of NPs having a core with very high concentration of the load and an outer part containing virtually no load. It remains, however very challenging to follow the formation of the particles directly. It is also possible that the organization takes place through migration of the load within the NPs. It would then depend on the mobility inside the polymer,

  Possibility of tuning emission properties has numerous applications in the field of fluorescent bioimaging with superior resolution. [178] Photoswitchable NPs can be advantageous over molecular photochromic systems like dyads, which require multistep synthesis and have limited brightness and photostability.We hypothesized that fast EET inside PLGA NPs[START_REF] Reisch | Collective fluorescence switching of counterion-assembled dyes in polymer nanoparticles[END_REF] could be exploited to drive the photoswitching efficiency of dye-loaded polymer. To this end, we coencapsulated rhodamine B dye salt with four different bulky hydrophobic counterions as well as a photochromic acceptor of the diarylethene family into PLGA NPs (Figure 2.14).

Figure 2 . 14 .

 214 Figure 2.14. Chemical structure of the encapsulated fluorescent dye (R18) with different bulky counterions and the photochromic dye (PCD1), and schematic presentation of photoswitching concept in dye-doped NPs. Small grey arrows show the exciton diffusion, while the large grey arrow presents FRET to a photochromic dye (brown). Filled and open brown circles correspond to the closed and open forms of PCD1, respectively. Chemical structure of the closed form of PCD1 is shown only.

Figure 2 . 15 .

 215 Figure 2.15. Correlation between photoswitching efficiency of NPs and fluorescence anisotropy of the encapsulated dyes .a) Variation of relative peak intensity under alternate irradiation of PLGA NPs loaded with 1 wt% of rhodamine R18 with different counterions and 0.3 wt% of photochromic dye PCD1.b) Fluorescence anisotropy of the encapsulated dyes NPs loaded with 1 wt% of rhodamine R18 with different counterions.

Figure 2 . 16 .

 216 Figure 2.16. Effect of size of nano-antennas on their performance. a) FRET efficiency and b) antenna effect of PMMA-MA FRET NPs of different size loaded with 30 wt% of R18/F5-TPB and DiD. Donor/Acceptor ratios were 1:1000 or 1:10000. Error bars represent the standard error of the mean (n=3). Sample marked "No EET" represents theoretical calculations in case of absence of exciton diffusion within donor dyes.

  a). Our NPs100 nano-antennas were used to observe single molecule events, such as one-step bleaching of the Cy5 (DiD) acceptor at extremely low laser powers of 1 mW/cm 2 which is 10 4 -10 6 fold lower than typically used in single molecule measurements. (Figure 2.17 b).

Figure 2 . 17 .

 217 Figure 2.17. Single-particle evaluation of nano-antenna performance. a) 3D representation of wide-field images of acceptor emission from FRET NPs containing 30 wt% R18/F5-TPB and ~1.5 Cy5(DiD) acceptors under the illumination at 532 nm with laser power 0.1 W/cm 2 , under the direct excitation of acceptor at 642 nm with laser power 100 W/cm 2 and of QD 655 under the illumination at 532 nm with laser power 0.1 W/cm 2 . In all cases integration time was set to 30.53 ms. b) Representative single particle trace excited at 532 nm with different power density of 1 mW/cm 2 .

Figure 1 .

 1 Figure 1. Concept of organic nano-antenna. (a) Chemical structures of the donor dye rhodamine B octadecyl ester (R18) and its counterion tetrakis(pentafluorophenyl)borate (F5-TPB) and of the acceptor dye cyanine 5 DiD. (b) Short-range ordering of R18 cations (green) by the F5-TPB counterion (blue) inside the PMMA-MA matrix prevents dye aggregation and

Figure 2 .

 2 Figure 2. Spectroscopic characterization of organic nano-antennas. (a) Steady-state fluorescence anisotropy of the encapsulated donor dyes (R18/F5-TPB) as a function of their loading in PMMA-MA NPs of 65 nm hydrodynamic diameter (45 nm by TEM) and their quantum yield.[214] (b) Schematic presentation of fluorescence anisotropy loss due to EET within randomly oriented fluorophores (c) Anisotropy decay measured at 580 nm with a 50-fs probe beam for NPs loaded at 1 and 30 wt% of donor dyes. (d) Emission spectra of FRET NPs loaded with different amounts of the acceptor dyes while keeping the same amount of the donor (30 wt%). The emission intensity was normalized to the same absorbance of the donor. (e) Experimental FRET efficiency for FRET NPs with varied donor but constant acceptor (0.004 wt%) concentration and the calculated one for NPs without exciton diffusion (marked no EET). (f) Amplification factor of the acceptor emission (antenna effect) measured from the excitation spectra and its estimated values based on the observed FRET efficiency. Error bars in (a, e, f) represent the standard error of the mean (s.e.m., n=3). Samples marked "No EET" represent theoretical calculations in the absence of exciton diffusion within donor dyes.

Figure 3 .

 3 Figure 3. Structure and brightness of individual nano-antennas. (a) TEM images and size histograms of NPs prepared with different pH of buffer. Scale bar is 50 nm (b) 3-D representation of wide-field fluorescence microscopy images of NPs of different sizes containing 30 wt % R18/F5-TPB under illumination of a 532 nm laser with power 0.1 W/cm 2 , integration time was set to 30.53 ms.

Figure 4 .

 4 Figure 4. Effect of size of nano-antennas on their performance. Fluorescence spectra (a), FRET efficiency and antenna effect (b) of PMMA-MA FRET NPs of different size loaded with 30 wt% of R18/F5-TPB and DiD. Donor/Acceptor ratios were 1000:1 or 10000:1. Error bars represent the standard error of the mean (n=3). Sample marked "No EET" represents theoretical calculations in the absence of exciton diffusion within donor dyes.

Figure 5 .

 5 Figure 5. Single-particle evaluation of nano-antenna performance. (a) Wide-field fluorescence microscopy images of NPs. Right panel represents images of NPs (NPs60) without acceptor and left ones are the images of NPs containing 30 wt % R18/F5-TPB and ~1.5Cy5 (DiD) acceptors per particle. The illumination at 532 nm was set to a laser power of 1 W/cm 2 , integration time was set to 30.53 ms. Both channels are represented at the same intensity scale. Overlay images represent false color composite of two channels, where red color corresponds to the acceptor channel and green to donor channel. (b) 3D representation of wide-field TIRF images of acceptor emission from FRET NPs (60 nm size) containing 30 wt% R18/F5-TPB and ~1.5 Cy5 (DiD) acceptors under the illumination at 532 nm with laser power 0.1 W/cm 2 , under the direct excitation of acceptor at 642 nm with laser power 100 W/cm 2 and of QD 655 under illumination at 532 nm with laser power 0.1 W/cm 2 . In all cases integration time was 30.53 ms. (c) Amplification factor of acceptor emission calculated by eq. 1 for antennas of different size 1-2 acceptor per NP. (d) Representative single particle trace excited at 532 nm with a power density of 1 mW/cm 2 . (e-g) Scheme of experimental setup and obtained results, using excitation by external light source that mimics direct sunlight. (f) Single particle

4 .

 4 For preparation of FRET nanoparticles, different concentrations of DiD (from 0.001 wt% to 0.04 wt % relative to the polymer) were added to the acetonitrile solution of polymer containing desired concentration of R18/F5-TPB, and the particles were prepared as described above. Preparation of NPs of different size was achieved by varying the pH of phosphate buffer at the first dilution. Nanoparticle characterization. Measurements for the determination of the size of nanoparticles were performed on a Zetasizer Nano ZSP (Malvern Instruments S.A.). The mean value of the diameter of the size distribution per volume was used for analysis. Absorption spectra were recorded on a Cary 4000 scan UV-visible spectrophotometer (Varian), excitation and emission spectra were recorded on a FluoroMax-4 spectrofluorometer (Horiba Jobin Yvon) equipped with a thermostated cell compartment. For standard recording of fluorescence spectra, the excitation wavelength was set 530 nm. The fluorescence spectra were corrected for detector response and lamp fluctuations. To calculate FRET efficiency based on fluorescence spectra, a classical equation was used: 𝐸 𝐹𝑅𝐸𝑇 = 1 -𝐼 𝐷-𝐴 𝐼 𝐷

  excitation intensities of donor and acceptor in FRET NPs, respectively; 𝐼 𝐷 𝑒𝑥 and 𝐼 𝐴 𝑒𝑥 are the excitation intensities at the wavelengths of excitation maximum of donor and acceptor in NPs without acceptors, respectively; 𝑓 is the correction factor calculated as the ratio of maximum emission intensity of donor for FRET NPs to that for NPs without acceptor dyes: the antenna effect was also estimated based on the FRET efficiency using the following equation: AE = (nDεDE)/(nAεA), where nD and nA are the numbers of donors and acceptors, respectively, per particle, εD and εA are the extinction coefficients of donors and acceptors, respectively, and E is the FRET efficiency. QYs of NPs were calculated using Rhodamine 101(QY=1) in ethanol as a reference with an absorbance of 0.01 at 530 nm. 2 QY of an acceptor molecule DiD in PMMAMA matrix was calculated for the concentration 0.02 wt% using DiD in methanol (QY=0.33) as a reference. 3Time-resolved anisotropy. For the time-resolved anisotropy measurements, we used an amplified Ti:sapphire laser that produces ultrashort pulses (100 fs) at a repetition rate of 100 kHz. 60 fs linearly polarized pulses centered at 520 nm ( = 12 nm) were obtained by means of an optical parametric amplifier (OPA). The pump's power density was around 120 W cm -2

  fluorescence signal was recorded with an EMCCD (ImagEM Hamamatsu) (0.106 µm pixel size) using an open source Micro-Manager software. The exposure time was set to 30.53 msec per image frame. To enable two channel images W-VIEW GEMINI image splitting optics were used with the following filter set: dichroic 640 nm (Semrock FF640-FDi01-25x36), bandpass filters 593±20 nm(Semrock FF01-593/40-25) and 685±20 nm ( Semrock FF02-685/40-25) )

  mimicking ambient sunlight excitation. The sunlight power density (24 mW cm-2 ) was recorded at midday on 19 October, 2016, Strasbourg region, using Handheld Laser Power Meter, 1917-R and Semrock band-pass filter 527 nm (50 nm bandwidth). The artificial white light mimicking sunlight was provided by a Cold light source from Zeiss, type KL 1500 LCD. The sample was illuminated from the top ~2 cm from the divergent light source output through the same 527-nm filter, which corresponded to 15 mW cm -2 power density at the sample. Single-molecule imaging was done using Nikon Ti-E inverted microscope using CFI Plan Apo 20 air (NA = 0.75) and CFI Plan Apo 60 oil (NA = 1.4) objectives and Hamamatsu Orca Flash 4 camera. Donor channel was recorded through a 600-nm band-pass filter (50 nm bandwidth, Semrock), while the acceptor channel used 647-nm long-pass filter (Semrock). Data were recorded and analyzed using NIS Elements and Fiji software, respectively.

Figure S2 .Figure S3 .

 S2S3 Figure S2. Poisson distribution of the number of acceptors (0.004 wt %) encapsulated per NP with 0.1-30 wt% of R18/F5-TPB. The horizontal axis corresponds to the number of acceptors per NP. The connecting lines are only guides for the eye.

Figure S6 .

 S6 Figure S6. Photo of experimental setup that uses excitation by a divergent light, mimicking

2. 3 .

 3 FRET to the surface of polymer NPs: Organic nanoparticle antenna for amplified FRET-based biosensing

   227] It was shown that 5 acceptor molecules attached to the proteins on the surface of 3 nm QDs can give ~ 58 % of FRET efficiency.[144]

Figure 2 .

 2 Figure 2.18.a) Fluorescence spectra of PLGA NPs loaded with 5 wt% of R18/F5 with addition of Cy5-biotin. The spectra were obtained at excitation wavelength λex= 530 nm. b) Calculated FRET efficiency.

Figure 2 . 19 .

 219 Figure 2.19. Normalized absorbance (a) and emission spectra (b) of PMMA-SO3 NPs loaded with different amounts of R18/F5-TPB. Emission spectra were recorded using excitation at 530 nm.

150FRET

  to the NPs surface To determine the efficiency of our NPs as energy donor to single acceptors located on their surface we performed the titration of NPs loaded with different amounts of R18/F5-TPB with Cy5-biotin conjugate. During the titration the later adsorbed on the NPs surface and we observed the occurrence of FRET between encapsulated dyes and Cy5-biotin. The addition of increasing concentration Cy5-biotin led to a drop of NPs fluorescence intensity and a rise of fluorescence at the acceptor emission wavelength. Remarkably, these processes were much more prominent at high loading of donor dyes, where already at 1 acceptor per particle, the acceptor emission became higher than the donor one. The acceptor emission reached saturation at about 10 acceptors per NP (100 nM concentration of acceptor), when the self-quenching of acceptors on NPs surface probably started to take place (Figure 2.20). The stability of the signal in time (over 25 min) confirmed that acceptor molecules remained on NPs surface. Control titration of NPs with DMSO, used in the titration, showed no significant change in the spectrum, confirming that observed effects were attributed to Cy5-biotin -NPs interactions.

Figure 2 . 20 .

 220 Figure 2.20. Fluorescence spectra changes of NPs loaded with 1 wt% (a) and 20 wt% (b) of R18/F5 dye

Figure 2 .

 2 Figure 2.21. a) FRET efficiency calculated for NPs with different R18/F5 loading with one acceptor molecule adsorbed on its surface. b) Fluorescence anisotropy value for R18/F5 encapsulated into NPs. c) Amplification of acceptor emission intensity calculated for NPs with one acceptor molecule adsorbed on its surface.

Figure 2 .

 2 Figure 2.22. a) Fluorescence spectra changes of NPs loaded with 20 wt% R18/F5 with adsorbed single molecule of Cy5-biotin on its surface upon addition of streptavidin. b) Changes in IA/ID ratio due to addition of streptavidin. Error bars represent standard error between three experiments (n = 3). c) Fluorescence spectra changes of NPs loaded with 20 wt% R18/F5-TPB with adsorbed single molecule of Cy5-biotin on its surface upon addition of 3nM of streptavidin in the presence of 1µM of biotin. The spectra were obtained at excitation wavelength λex= 530 nm.

10 - 15 )Figure 2 Figure 2 .

 1522 Figure 2.23. a) Fluorescence spectra changes of NPs loaded with 20 wt% R18/F5, with adsorbed single molecule of Cy5-BA on their surface upon addition of hCA. b) Changes in IA/ID ratio due to addition of hCA. c) Fluorescence spectra changes of NPs loaded with 20 wt% R18/F5-TPB, with adsorbed single molecule of Cy5-BA on their surface upon addition of 500 nM of hCA in the presence of 100 µM of EZA inhibitor. The spectra were obtained at excitation wavelength λex= 530 nm.

Finally

  , the addition of 2.5 µM of streptavidin decreased the intensity of the acceptor channel back to the value observed for control NPs without acceptor. These results confirm that NPs could sense proteins at the level of individual particles (Figure2.24 a). Remarkably, one step bleaching event of acceptor emission that was accompanied 154 by appearance of strong emission on the donor channel, confirmed that we really observe single molecule events on the NPs surface (Figure2.24 b).

Figure 2 . 24 .

 224 Figure 2.24. Single-particle observation of switching of NPs in two channels. a) Wide field image of NPs (upper panel), NPs with adsorbed Cy5-biotn on theirs surface (middle panel) and NPs with Cy5-biotn after the addition of streptavidin (lower panel).The images were obtained by illumination with laser 532nm with power 2 W/cm 2 , integration time for each image was set at 0.1 sec. b) The representative trace of the NPs bearing Cy5-biotin in green and red channels in time under the illumination at 532 nm with laser power 0.2 W/cm 2 .

  Photochromic molecule PCDI was obtained from collaborator Prf. Luca Prodi (University of Bologna, Italy).The synthesis was described earlier[229]

  Dynamic light scattering measurements were performed on a Zetasizer Nano series DTS 1060 (Malvern Instruments S.A.). It uses a laser source of 633 nm, so only NPs without excitation at this wavelength could be used. Absorption and emission spectra were recorded on a Cary 4000 scan UV-visible spectrophotometer (Varian) and a FluoroMax-4 spectrofluorometer (Horiba Jobin Yvon) equipped with a thermostated cell compartment, respectively. For standard recording of fluorescence spectra, the excitation wavelength was chosen to excite the sample optimally. The fluorescence spectra were corrected for detector response and lamp fluctuations. QY was calculated by comparative method, using appropriate reference sample. Steadystate anisotropy was measured on a SLM 8000 spectrofluorometer (Aminco) in a Tmeasurements of NPs loaded with R18/F5-TPB and LR were performed on a two-photon platform including an Olympus IX70 inverted microscope, as described previously. [169] Two-photon excitation at 830 nm (from 1mW to 5mW laser output powers) was provided by a mode-locked Tsunami Ti:sapphire laser pumped by a Millenia V solid state laser (Spectra Physics). The measurements were carried out in a 96-well plate, using a 200-µL volume per well. The focal spot was set about 20 µm above the coverslip. The normalized autocorrelation function, G(τ) was calculated online by an ALV-5000E correlator (ALV, Germany) from the fluorescence fluctuations, σF(t), by G(τ)=<σF(t)σF(t+τ)>/<F(t)> 2 where <F(t)> is the mean fluorescence signal, and τ is the lag time. Assuming that NPs diffuse freely in a Gaussian excitation volume, the correlation function, G(τ), calculated from the fluorescence fluctuations was fitted according toThompson:[230]

2

 2

  humain est composé d'une grande variété de cellules, chacune contenant une quantité importante de biomolécules différentes. Leurs concentrations, leurs modifications et interactions sont généralement contrôlées avec une précision tant spatiale que temporelle élevée. Tout changement dans ces processus complexes peut conduire à des maladies létales. Il est donc essentiel de développer une voie de diagnostic précoce pour toute modification en biochimie humaine afin d'augmenter le taux de survie des patients. Etant donné sa haute sensibilité, sa détection rapide et non-invasive la fluorescence est une technique très prometteuse pour détecter des biomolécules même à faible concentration. [231] En outre, le signal de fluorescence peut être modulé, par transfert d'énergie résonant (FRET), [232] en réponse à la présence de biomolécules cibles. La vitesse, la résolution et la sensibilité de la bioimagerie peuvent être significativement améliorées à l'aide des nanoparticules fluorescentes (NPs) présentant une luminosité supérieures aux sondes organiques et protéines fluorescentes. A l'heure actuelle, les NPs les plus couramment utilisées sont inorganiques comme par exemple les quantum dots (QDs). [41] Si les nanoparticules organiques fluorescentes sont des alternatives intéressantes aux QDs, au vu de leur biodégradabilité potentielle, leur préparation est difficile étant donné le problème d'auto-inhibition des fluorophores encapsulés et, en outre, une photostabilité des fluorophores nettement plus faible que celle des QDs. De plus, leur taille (~10-60 nm) est beaucoup plus grande que le rayon de FRET ce qui présente un obstacle pour une application réussie dans ce domaine. L'objectif de mon projet de doctorat a été de développer des nanoparticules fluorescentes organiques non toxiques et biocompatibles, présentant une petite taille, une luminosité et une photostabilité élevées, capables de changer d'un état éteint à 166 un état allumé (allumage) grâce au FRET et de les appliquer à la détection de biomolécules. Dans ce but, nous nous sommes intéressés aux nanoparticules polymériques dopées en fluorophores préparées par nano-précipitation à partir de polymères biodégradables et biocompatibles approuvés par la FDA (Food and Drug Administration). Les fluorophores pour l'encapsulation doivent satisfaire à un certain nombre de critères: avoir un bon coefficient d'extinction moléculaire, un rendement quantique élevés, être photostables, procurer un bon contraste pour une faible puissance d'excitation (afin d'éviter une phototoxicité pour des expériences prolongées) et être suffisamment hydrophobes pour co-précipiter avec le polymère et ne pas fuir dans le milieu biologique (comme une tumeur ou le cytosol) afin d'assurer un faible bruit de fond de fluorescence. Pour surmonter les problèmes de biodégradabilité, de photostabilité et d'autoinhibition des colorants incorporés, nous avons étudié l'encapsulation de dérivés de pérylène diimide (PDI), qui sont à ce jour les fluorophores les plus photostables connus, [171] dans des nanoparticules polymériques à base d'acide poly(lactique-coglycolique) PLGA. L'un de ces dérivés porte des groupements hydrophobes volumineux dans la région imide (PDI-1), alors que l'autre a été substitué dans les deux régions imide et basse (Lumogen Red) (Figure 1).

Figure 1 .

 1 Figure 1. Structure chimique de PDI-1, des fluorophores LR et du polymère PLGA + schéma d'une nanoparticule de PLGA dopée en colorant.

Figure 2 .

 2 Figure 2. a) Représentation 3D de l'intensité de fluorescence de nanoparticules individuelles chargées avec 1 % en poids de Lumogen Red et de quantum dots QD585, mesurée par microscopie TIRF, avec excitation de laser de 532 nm. b) Imagerie, après 2h d'incubation, de nanoparticules chargées avec 1 wt% de Lumogen Red dans des cellules HeLa (l'agglutinine-Alexa488 a été utilisée pour marquer la membrane cellulaire).

Figure 3 .

 3 Figure 3. a) Structures chimiques du donneur octadecyl ester rhodamine B (R18) et de son contre-ion tétrakis(pentafluorophényl)borate (F5-TPB) et de l'accepteur cyanine 5 DiD + ordre à courte échelle de cations R18 (vert) et de leurs contre-ions F5-TPB (bleu) dans la matrice PMMA-MA, qui empêche l'agrégation du colorant et conduit à une courte distance inter-fluorophore et un EET ultra-rapide et un FRET résultant sur une molécule d'accepteur unique (rouge). b) Schéma conceptuel d'une nanoantenne collectrice géante à l'intérieur d'une nanoparticule polymérique.

Figure 4 .

 4 Figure 4. a) Observation en particule unique de sondes sur deux canaux spectraux (vert pour l'émission du donneur, rouge pour celle de l'accepteur. b) Représentation 3D de l'intensité de fluorescence de nanoparticules individuelles chargées avec 30 % en poids de R18/F5-TPB et de ~1.5 Cy5 per nanoparticule de quantum dots QD655, mesurée par microscopie TIRF avec excitation de laser de 532 nm c)Traces représentatives d'une particule unique excitée à 532 nm à une puissance de 1mW/cm 2 .

Figure 5 .

 5 Figure 5. a) Structure chimique de l'accepteur photochrome de la famille des diaryléthènes en forme fermée. b) Spectre d'absorption dans le dioxane comparé aux spectres d'absorption et d'émission de R18/F5-TPB dans des NPs de PLGA (chargées à 1 wt%). c) Schéma du concept photocommutation à l'intérieur d'une nanoparticule. d) Variation d'intensité relative, sous irradiation alternée, de nanoparticules de PLGA dopées en flurophore Rhodamine R18 avec différents contre-ions (1 wt%) et en fluorophore photochrome PCD1 (0.3 wt%).

Figure 6 .

 6 Figure 6. a) Modification du spectre d'émission des NPs dopées avec 20 wt% de R18/F5 et avec une molécule de Cy5-biotine absorbée à la surface lors de la détection de la streptavidine. b) Le changement du IA/ID lors de la détection de la streptavidine.

Figure 7 .

 7 Figure 7. a) Observation en particule unique de sondes sur deux canaux spectraux (vert pour l'émission du donneur, rouge pour celle de l'accepteur) avant et après l'addition de streptavidine. b) Traces représentatives en particule unique en excitation 532 nm pour des nanoparticules avec l'accepteur adsorbé.

Table 1 .1. Effect

 1 of energy transfer on the fluorescence characteristics of the donor in the case

Table 1 . 2 .

 12 Comparison of commonly used techniques for NPs characterization.

	Technique	Measures	Sample	Sensitivity	Notes
					Determine
	DLS	average size and size distribution	dilute suspension	1nm -10μm	only hydrodynamic size of NPs a
	FCS	average size and size distribution, concentration and brightness b	dilute suspension of fluorescent sample	down to 1nm	Two -photon excitation or pinhole system required
		brightness b and
		brightness	Fluorescent,		Visible "size"
	TIRF	distribution, photostability,	immobilized on a glass	No direct access to size	reflects both real size and
		fluorescence	surface		brightness
		behavior
			< 1μg has to		Sample
			be prepared		preparation
			as a thin film		can be time
	TEM	size and characterization	and be stable under an	down to 1nm	consuming, phase-contrast
			electron beam		results could
			and a high		be difficult to
			vacuum		interpret,
	SEM	size and characterization	conductive or sputter-coated easier to prepare than TEM sample	down to 1nm	Sample preparation can be time consuming
			must be		slow scan
	AFM	size and characterization	adhered to a surface, be rigid and	1nm -8μm	time, which can lead to thermal drift of
			dispersed		the sample

Table 2 . 1 .

 21 Mean size from TEM measurements, estimated number of dyes per NP, QY, and calculated particle brightness.

	wt%	PLGA				PMMA				PCL
	dye
		Size	N	QY	Brightne	Size	N	QY	Brightne	N	QY	Brightne
		(nm)			ss	(nm)			ss			ss
					(M -1 cm -				(M -1 cm -			(M -1 cm -
					1 )				1 )			1 )
	0.2	27.6	10	0.75 7.7 x10 5 25.8	8	1.02 8.5 x10 5 8	1.02 8.4 x10 5
	1	30.0	62	0.55 3.6 x10 6 29.8	61	0.87 5.6 x10 6 51	0.79 4.3 x10 6
	5	32.5	392	0.41 1.7 x10 7 33.8	444	0.72 3.4 x10 7 331	0.65 2.3 x10 7
	10	34.1	906	0.35 3.4 x10 7 34.4	933	0.67 6.7 x10 7 777	0.57 4.7 x10 7
	20	34.7	1911 0.22 4.5 x10 7 33.8	1771 0.41 7.7 x10 7 1566 0.37 6.1 x10 7
	30	35.3	3020 0.10 3.3 x10 7 33.2	2518 0.31 8.2 x10 7 2368 0.34 8.6 x10 7

Table 2 .

 2 Hydrodynamic diameter and spectroscopic properties of PMMA-MA NPs encapsulating 30 wt% of R18/F5-TPB prepared at varied pH. After preparation, NPs were diluted in pH 7.4 buffer. b Statistics by volume was used in DLS measurements (error is s.e.m., n  3). PDI is polydispersity index. c Error is the standard deviation based on analysis of 80 -500 NPs. d Quantum yield of NPs without acceptor molecules (error is s.e.m., n  3). e Estimation is based on NPs size measured by TEM. Normalized single particle brightness of NPs under illumination of a 532 nm laser with power 0.1 W/cm 2 .

	Sample	pH a Size,	PDI Size,	QY d	Anisotro	Donors/NPs e SPB f	SPB g
		DLS	TEM		py		estim.	exp.
		(nm) b	(nm) c
	NPs30	9.0 36  1 0.11 309	0.410.04 0.001	1900	1	1
	NPs45	7.4 65  4 0.11 458	0.390.03 0.0028 6300	3	5
	NPs60	6.7 105  5 0.11 6022 0.380.02 0.0025 15000	8	12
	NPs67	6.5 139  8 0.13 6727 0.360.03 0.0024 21000	11	20
	NPs107 5.8 201 11 0.17 10743 0.350.03 0.0029 85000	45	31

a f Estimated changes in single molecule brightness based on QY and size of NPs measured by 129 TEM. g

Table S2 .

 S2 Dependence of Förster distance for homo FRET on dye loading

	NPs	loading,	Homo	FRET	Average distance
	wt%		distance R0, nm	between
					encapsulated
					R18/F5-TPB
					dyes, nm a
	0.1		6.6		7.3
	1		6.2		3.4
	5		5.6		2.0
	10		5.3		1.6
	20		4.8		1.2
	30		4.6		1.0

Table S3 .

 S3 Comparison of the theoretical FRET efficiency inside NPs calculated in the absence of homo-FRET (EET) with the experimental FRET value.

	NPs			𝐽(𝜆),M -		QD b Number	R0 c ,	Theoretical	Experimental
	loading,	1 cm -1 nm 4 a		of	Å	calculation d	value
	wt%					acceptors		𝐸 𝐹𝑅𝐸𝑇		𝐸 𝐹𝑅𝐸𝑇
							per NP
	0.1			9.7310 15	0.99	1.6	70	6%		9%
	1			1.1310 16	0.83	2.0	69	6%		10%
	5			1.2910 16	0.74	2.3	70	6%		21%
	10			1.3310 16	0.55	2.7	67	5%		27%
	20			1.3810 16	0.38	3	63	4%		28%
	30			1.4110 16	0.36	4.3	61	4%		34%
	a Overlap integral. b Quantum yield of donor dyes. c R0 is the Förster distance for FRET pair
	R18 and DiD, which depends on the donor loading. d Theoretical FRET efficiency calculated
	in the absence of exciton diffusion.
	a	Normalized excitation emission (a.u)	450 0.0 0.2 0.4 0.6 0.8 1.0	500	550	600 Donor dye loading: 650 0.1 % wt 1 % wt 5 % wt 10 % wt 20 % wt 30% wt	0.00 0.01 b	630	640	650	660	670
						Wavlength (nm)				Wavlength (nm)

Table 2 . 2 .

 22 Size, concentration and two-photon brightness calculated from FCS measurements (830 nm, 2 mW) of NPs loaded with different concentrations of the R18/F5-TPB, their QY and calculated number of encapsulated fluorophores.

	Sample	D, nm	C, nM Brightness/TMR	QY a	#R18/F5-
						TPB
	0.2 wt%	20	11.7	7	0.98±0.08 4
	1 wt%	20	12.0	25	0.88±0.08 18
	5 wt%	25	13.0	150	0.73±0.04 180
	10 wt%	21	15.4	215	0.59±0.05 210
	20 wt%	25	19.8	280	0.50±0.08 720
	a Rhodamine 101 in ethanol was taken as a reference.

The full description of the work can be found in the article enclosed below

Acknowledgements

This work was supported by the European Research Council ERC Consolidator grant BrightSens 648528. C. Ruhlmann for help with electron microscopy. K.T. was supported by a fellowship from the Ministre de la Recherche (France).

My sincere thanks also goes to Prof. Yves MELY for accepting me as a Master student to the Lab four years ago.

Last but not the least, I want to acknowledge Ecole doctorale des Science de

Author contributions

A.S.K. proposed the concept; A.S.K. and K.T. designed the experiments; K.T. performed most of the experiments and data analysis. A.R. helped with data analysis and electron microscopy. P.D. and F.F. performed time-resolved anisotropy measurements, Y.M. and P.G. participated in discussions and manuscript correction. A.S.K., K.T. and A.R. wrote the manuscript.

Competing financial interests

The authors declare no competing financial interests.

Additional information

Supplementary information is available in the online version of the paper. Reprints and permissions information is available online at www.nature.com/reprints. Correspondence and requests for materials should be addressed to A.S.K.

Supporting Information

Giant light harvesting nano-antenna for singlemolecule detection at ambient light Kateryna Trofymchuk, Andreas Reisch, Pascal Didier, François Fras, Pierre Gilliot, Yves Mely, and Andrey S. Klymchenko Zoomed region of the excitation spectra focused on the excitation of the acceptor.

Emission was detected at 700 nm.

Detection of proteins at single molecule level

The

TEM imaging

Five microliters of the particles solution were deposited onto carbon-coated copper-rhodium electron microscopy grids that were used either as obtained or following an air or amylamine glow-discharge. The grids were then treated for 1 min with a 2%uranyl acetate solution for staining. They were then observed with a Philips CM120 transmission electron microscope equipped with a LaB6 filament and operating at 100 kV. Areas covered with nanoparticles of interest were recorded at different magnifications on a Peltier cooled CCD camera (Model 794, Gatan, Pleasanton, CA). Image analysis was performed using the Fiji software.

Controlled switching of fluorescent organic nanoparticles through energy transfer for bioimaging applications

Résumé