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  'archive ouverte pluridisciplinaire HAL, est destinée au dépôt   Distributed under a Creative Commons Attribution| 4.0 International License Permeability and porosity improvement of grouted sand Nadia Saiyouri, Abdelghafour Aït Alaiwa, Pierre-Yves Hicher To cite this version: Nadia Saiyouri, Abdelghafour Aït Alaiwa, Pierre-Yves Hicher. Permeability and porosity improvement of grouted sand. European Journal of Environmental and Civil Engineering, 2011, 15 (3), pp.77-97. 10.3166/EJECE.15.313-333. hal-01007182 is necessary to exploit experimental

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David Lasne

Gerhard A Blab

Stéphane Berciaud

Martin Heine

Laurent Groc

Daniel Choquet

Laurent Cognet

Brahim Lounis
email: b.lounis@cpmoh.u-bordeaux1.fr

Prof B Lounis

Single NanoParticle Photothermal Tracking (SNaPT) of 5 nm gold beads in live cells

à la diffusion de documents scientifiques de niveau recherche, publiés ou non, émanant des établissements d'enseignement et de recherche français ou étrangers, des laboratoires publics ou privés.

Introduction

The movements of molecules in the plasma membrane of living cells are characterized by their diversities, both in the temporal and spatial domain. Membranes exhibit a constitutive complexity, consisting of several different lipids and a great variety of proteins with highly dynamic and compartmentalized spatial distributions. These molecules explore the plasma membrane in various lateral diffusion modes, and frequently interact with each other at specific locations in order to transmit information across the membrane, starting the cascades of specific signaling processes. Those molecular interactions are by nature heterogeneous making ensemble observations of these phenomena rather challenging. Single molecule detection has allowed the elimination of the implicit averaging of conventional optical observations, giving access to heterogeneity, dynamical fluctuations, lateral diffusion, reorientation, colocalization, and conformational changes at the molecular level. Until now, two main approaches have been used to track individual molecules in the plasma membrane of live cells, with distinct advantages and limitations. The first one, Single Particle Tracking (SPT), uses labels large enough to be detectable by conventional microscopes (1,2) through Rayleigh intensity scattering (~ 40 nm gold particles or even larger latex beads). SPT permits to follow the movement of individual molecules for very long times and possibly at very fast imaging rates (3). SPT, for instance, revealed barriers set for diffusion by the cytoskeleton(4), and the diversity of lateral diffusion modes of receptor for neurotransmittors in live neurons (5,6). However the main drawback is the size of the beads which might sterically hinder the interaction between the labeled molecules or alter their movements in confined environments such as synaptic clefts or endocytotic vesicles. Recently, a method which exploits the interference between a background reflection and the scattered field has been page 4 of 23 developed (7). The detection of gold nanoparticles smaller than 20 nm has been achieved in vitro conditions (8,9).

The second widely used technique, Single Molecule Tracking (SMT), uses fluorescent organic dyes (10,11) or autofluorescent proteins (12). As these fluorophores are generally smaller than the target molecules it does not have the drawback of SPT mentioned previously.

Applied to neurosciences, SMT has thus allowed to reveal the lateral diffusion of glutamate receptors (AMPA and NMDA) inside the synapses of live neurons (13,14). The main limitation encountered in SMT studies is photobleaching which severely limits the observation times of a single fluorophore to typically less than one second in live cells.

An experimental technique combining the advantages of SPT and SMT, namely long observation times and small nanometer-sized labels, would thus have great potential. For biological questions, it would allow for recording the full history of proteins in cells including intermediate states even in highly confined regions (e.g. lipid rafts or membrane protein clusters, intracellular vesicles, synapses of neurons….).

For this purpose, semiconductor nanocrystals (such as CdSe/ZnS) have been used recently as fluorescent markers (15,16). They are more photostable than organic dyes and autofluorescent proteins. Quantum dots have proven to be valuable tools for extended observation in living organisms (17,18). However, biocompatible and functionalized nanocrystals are rather bulky labels of few tens of nanometers in diameter and they eventually bleach. In addition, their luminescence is subject to blinking (19) at all time scales (20) which renders observations of changes between different lateral diffusion modes of proteins difficult. page 5 of 23 An alternative approach consists in developing sensitive optical systems for the detection of the absorption of nanoparticles (NPs). We have recently shown that the photothermal methods allow for the detection of individual non-luminescent nano-objects (21)(22)(23). Metal NPs are efficient light absorbers. The luminescence yield of these particles being extremely weak (24), almost all the absorbed energy is converted into heat. The increase of temperature induced by the absorption gives rise to a local variation of the refraction index. A Photothermal Interference Contrast technique was used to detect for the first time individual 5 nm gold NPs embedded in thin polymer films NPs (21) and to demonstrate the detection of single protein labeled with 10 nm gold NPs in fixed cells (22). Because of its limited sensitivity, this technique requires relatively high laser beam intensities restricting its use to fixed biological samples. Photothermal heterodyne imaging (23) is two orders of magnitude more sensitive than earlier methods. It allowed for the unprecedented detection of individual 1.4 nm gold NPs. It uses a combination of a time-modulated heating beam and a non-resonant probe beam. The heating induces a time-modulated variation of the refraction index around the absorbing NP. The interaction of the probe beam with this index profile produces scattered field with sidebands at the modulation frequency. The scattered field is then detected in the forward direction through its beatnote with the transmitted probe field which plays the role of a local oscillator akin a heterodyne technique (25). In the following, "Laser Induced Scattering around a NanoAbsorber" (LISNA) will be used to refer to this detection method.

Results and Discussion

While the sensitivity of LISNA is unparalleled, the imaging rate of this scanning method is a serious limitation. Recording the movement of membrane proteins on live cells requires fast acquisition rates (typically video rate or faster). The acquisition of several µm² images is however not necessary since the localisation of a single NP requires only a limited number of data points taken at well chosen position. For instance, tracking fluorescent objects with confocal microscopes has been proposed by using rotating illuminations (26) . It has been recently implemented in 2-photon microscopes and allowed to perform 3D tracks of 500 nm beads phagocytosed by live fibroblasts at video rate (27).

In order to study the lateral diffusion of membrane proteins in a plane, we present here an algorithm requiring 3 measurement points to localize a NP. The movements of the objects along the axial direction of the microscope are neglected since the cellular material will only consist on one layer of flat cultured cells. However, the algorithm can easily be extended to track 3D movements. We use 5nm gold particles as they can be imaged in live cells with high signal to noise ratios (SNR) at reasonable laser intensities (SNRs >30 for excitation intensities of ~400kW/cm², 5ms integration time per pixel). page 7 of 23

Description of the tracking method

The spatial profile of the LISNA signal from an individual NP is given by the product of the intensity profiles of the heating and probe beams (25). It is well approximated by a Gaussian profile with a constant width, and can be defined by three parameters: the central position (x 0 , y 0 ), and the peak signal S 0 . By measuring the signal at three well defined positions around the tentative location of a NP, both its precise location in space (x, y) and its peak signal S can be unequivocally retrieved.

In practice, after the acquisition of a white light and/or an epifluorescence image, a LISNA image is recorded in order to check gold-labeling density and specificity. Then a region of interest is chosen in which LISNA signals are taken at random positions. When a signal above a predefined threshold is obtained, three data point are taken around this position at the apices of an equilateral triangle. A first set of NP coordinates and peak signal is then calculated (x t , y t , S t ). If S t and the three measured signals are above a second predefined threshold, this procedure is repeated iteratively by recentering the equilateral triangle on (x t ,y t ) for the next three measurements which will give (x t+Δt , y t+Δt , S t+Δt ), and thus the track of the moving particle.

Theoretical capabilities

The maximum speed for the tracking of an individual NP is determined by the time needed to perform the three measurements. We use integration times of 5 ms per point separated by 6 ms waiting times to ensure stability of the piezoscanner and data transfer.

Consequently, the tracking rate of the position of nanoparticles is ~30 Hz. The maximum speed of a moving object that can be tracked is thus limited to that of NP which can not page 8 of 23 escape from the triangle during the 3 measurements. Using a triangulation radius of 180 nm (~1.5 × the width of a single NP LISNA profile(25)), objects moving with diffusion speeds up to ~0.2 µm²/s will be tracked.

Tests on simulated trajectories.

We first tested SNaPT by generating 2D Brownian movements of individual NPs embedded in thin polyvinyl alcohol (PVA) films with the piezoscanner stage (see Fig. 1). The tracking scheme was then used to recover the simulated (and known) trajectories of the NPs as a function of the input diffusion constant. The tests were conducted for different detection SNRs (30 to 100). From the recovered trajectories, three main parameters were calculated: the ratio of successful tracks, defined as tracks which were not lost before a given time limit (200 data points, 7.5 s), the instantaneous diffusion constant D (see methods) and the deviation from the simulated trajectory. For SNRs ~ 30, diffraction limited beams and a triangulation radius of 180 nm, a cut-off in the diffusion constants is found around 0.15 µm²/s, above which only few simulated trajectories could be retrieved (Fig 2). Below this value, a very good agreement between the generated and measured diffusions is obtained over more than 2 orders of magnitude of D and the standard deviation between the measured and the simulated trajectories gives the pointing accuracy of the method: 20 nm for SNR~30 or 7 nm for SNR~100. Only few trajectories with high diffusion constants (0.15-0.3 µm²/s) could also be retrieved. Interestingly, by enlarging the heating beam size and the triangulation radius by 50%, the cut-off in the diffusion constants could be increased by ~30% (not shown) but at the price of a 10% loss of the pointing accuracy. Measurements of very slow diffusion constants are limited by the pointing accuracy and the stability of the setup. The minimum detectable page 9 of 23 diffusion constant D min was experimentally determined with SNR~30 to be equal to D min =1.4×10 -3 µm 2 /s (Fig. 2). We will now address the important issue of the possible effect of the laser intensities used in SNaPT (200-400 kW/cm²). First, the local temperature rise due to laser absorption in the vicinity of the NP can be estimated. Since the thermal conductivity of metals is much higher than that of the surrounding medium, the temperature inside a spherical NP can be considered uniform and equal to the temperature at its surface. It writes:

Tracking on live cells

For applications on live cells, we validate the

SNaPT on live neurons

For future applications, we further tested SNaPT by recording long trajectories of diffusing AMPA receptors (AMPARs) in the membrane of live neurons. The first evidence for lateral motion of AMPARs came from SPT studies where AMPARs labeled with latex bead (~0.5 µm diameter) were tracked at the surface of cultured hippocampal neurons (6).

Using SMT experiments and thus reduced label sizes, Tardin et al (13) showed that AMPARs can also be mobile inside synapses. Altogether these studies established that AMPARs alternate between different membrane compartments through lateral diffusion.

The measure of the dynamics of these exchanges is important for the understanding of the synaptic physiology (30). As SMT is limited to short acquisitions times, the need for methods like SNaPT to follow nanometer sized labels at video rate for long times is required.

We thus labeled a small proportion of surface expressed native AMPARs containing the

Conclusion

We have developed a new tracking method which combines the advantages of small marker size afforded by SMT and the unlimited observation time afforded by SPT with stable signals.

In this work, we have only considered tracking of nanoparticles in the two dimensions of a planar membrane. The method itself, however, can be easily extended to full 3D tracking by simply adding a fourth measurement point to the presented scheme (27). We have demonstrated that SNaPT allows long recordings of membrane protein lateral diffusion with signal stabilities not yet achievable with fluorescent labels even by using semi-conductor page 13 of 23 nanocrystals. Further use of optimized bioconjugation strategies to bind the 5nm gold nanoparticles to the target protein with minimal ligand sizes (31), will make SNaPT a powerful approach for the study of individual proteins in vitro or in live cells.

Methods

LISNA setup

We built a microscopy setup to perform SNaPT and wide-field SMT as well as white light imaging of cells (Figure 1). A non-resonant probe beam (HeNe, 632. recorded by the same CCD camera using a standard condenser for illumination.

Cell culture, transfection of COS7 cells and staining.

COS7 cells were cultured in DMEM medium supplemented with streptomycin (100 µg/ml), penicillin (100 U/ml), and 10% bovine serum in a humidified atmosphere (95%) at 5% CO 2 and 37 o C. Cells were used for 12-14 passages and were transferred every 4 days. For transfection the cells were plated onto 15 mm #1 glass plates to a confluence of ~ 30% and incubated with 1 µl FUGENE and 0.5 µg DNA coding for a metabotropic receptor for glutamate containing of myc-tag at the extracellular N-terminus (mGluR5a-myc( 28)).

Transfection efficiency was on the order of 40%. After 12h, immunostaining was performed using antimyc antibodies tagged with Cy5 dyes (herein named amyc-Cy5, 3 min at room temperature, 20 µg/ml, 0.3% BSA). After two rinses in PBS, a secondary immunostaining by antiIgG-10 nm or antiIgG-5 nm gold (goat anti-mouse, BBInternational, to label the amyc-Cy5, Auroprobes Amersham, 3 min at room temperature, 0.3% BSA) was performed at a antibody concentration of 300 ng/ml, followed by 3 rinses in medium. The coverslips were then mounted in a custom chamber with culture medium supplemented with 20 mM Hepes.

All data were taken at room temperature within 20 minutes after a last rinse.

Neuron culture, GluR2 staining

Hippocampal neurons from 18 days old rat embryos were cultured on glass coverslips as previously (6). For SNaPT experiments, 7-10 DIV neurons were incubated 3 min at room temperature with 10 µg/ml anti-GluR2. After two rinses in culture medium, a secondary immunostaining by F(ab)-5nm gold conjugates (5nm gold conjugate goat F(ab')2 anti-mouse page 15 of 23 IgG, BBInternational, to label the anti-GluR2, Auroprobes Amersham, 3 min at room temperature, 0.3% BSA) was performed at a concentration of 300 ng/ml followed by 3 rinses in culture medium. After fast rinses, the coverslips were mounted in a custom chamber with culture medium supplemented with 20 mM Hepes. All data were taken at room temperature within 20 minutes after the last rinse.

Data analysis

In the case of SMT, trajectories are recorded following the procedure described in (13). Only

  LISNA images are obtained by raster scanning of the samples by means of a piezoscanner stage (see fig1). In this work, we used LISNA to perform Single nanoParticles Tracking (SNaPT) in live cells at video rate. For this purpose, we have developed a tracking strategy to record the page 6 of 23 2D trajectories of individual moving 5 nm gold nanoparticules. The results obtained by SNaPT are compared to those obtained by SMT in a study of the lateral diffusion of individual metabotropic glutamate receptors on the plasma membrane of live COS7 cells. Furthermore, long trajectories (several minutes) of individual glutamate receptors (AMPA) recorded on the plasma membrane of live neurons are shown.

  SNaPT technique by measurements of the well characterized lateral diffusion of mGlurR5a (a metabotropic glutamate receptor, member of the super-family of G-Protein coupled receptors) in the plasma membrane of live COS7 cells (Fig 3 (a))(28). The cells were transfected and expressed a mGlurR5a-myc cDNA fusion construct consisting of the mGluR5a receptor sequence and a N-terminal extracellular myc tag. Those proteins were first sparsely labeled with fluorescent antibodies (anti-myc-Cy5) in order to perform SMT control experiments. For SNaPT, a secondary labeling stage, with either antiIgG-10nm or antiIgG-5nm gold antibodies was used (see methods). LISNA images of non-labeled COS7 cells show intrinsic signal only at regions close to the nucleus(29). Apart from these regions which will not be considered in this work, a 2 fold increase of the noise is found and no signal is detected. Transfected cells are easily discriminated from untransfected ones in fluorescence images (not shown). Specificity of the gold labeling was ensured as LISNA images of stained non-transfected cells are similar to that of non-labeled cells. LISNA images of cells with labeled receptors reveal the presence of point-like signals which correspond to immobile (or slowly moving) mGluR5-linked NPs. But most NPs move during the raster scan of the sample, which produces characteristic stripe signals (see fig 3(c)). Fig 3(c-d) further shows an example of 2 SNaPT trajectories (20s acquisition time each, see supplementary materials for trajectories of several minutes). Rapid changes in diffusion page 10 of 23regimes are clearly visible as reported previously on PtK2 cells and neurons(28). Although the signal exhibits increased fluctuations when the NP is in a fast diffusive state, it remains relatively stable during the overall trajectory (Fig.3e) allowing a precise assignment of the number of tracked NPs. Fig 3(b) presents the histogram of the signals measured during 48 SNaPT recordings of mGlurR5a receptors labeled with 5nm gold NPs. The distribution contains two main peaks corresponding to the detection of one NP in the first peak and two NPs in the second(22).

Fig. 4 (

 4 Fig.4(a-c) shows the distributions of D measured on the first 750ms of trajectories acquired

  abs σ the NP absorption cross section, κ the thermal conductivity of the medium (water) and a the radius of the NP. For 5 nm gold NPs in aqueous medium, and I = 400 kW/cm 2 one finds a rather low NP temperature rise of ~ 1.5 K. Furthermore it decreases as the inverse of the distance from the NP surface. Second on short time scales, SMT and SNaPT give the same results despite different excitation intensities (<10 kW/cm² for SMT). Third, we investigated longer time scales by recording 115 trajectories (600 data points each, page 11 of 23 15 cells) and calculating D over a sliding window (25 data points) along these trajectories (Fig.4(d)). No evolution of the mean and standard deviation of the distributions of D is noticed and the proportion of immobile NPs do not change. Finally, 2×2 µm² regions of cells were exposed during 30 minutes to 400 kW/cm 2 continuous laser illumination. Comparison of images taken before and after the illuminations revealed no discernible adverse effect on the cell morphology as compared to control samples (Fig.4(e) (f)). Altogether these data indicate that SNaPT should allow single molecule experiments on live cells when long recordings and nanometer-sized labels are needed.

GluR2 subunit in live

  Fig 5(a)). Intrinsic signals originating from some portions of neurites were also observed(29).

  8 nm) and an absorbed heating beam (532 nm, frequency doubled Nd:YAG laser) are overlaid and focused on the sample by means of a high NA microscope objective (100×, NA=1.4). The intensity of the heating beam is modulated at a frequency Ω (typically Ω/2π = 700 kHz) by an acoustooptic modulator. A second microscope objective (80×, NA=0.8) collects the interfering probetransmitted and forward-scattered fields. The intensity of the heating beam sent on the samples was 400 kW/cm 2 for 5 nm gold NPs and 200 kW/cm 2 for 10 nm gold NP. The forward interfering fields are collected on a fast photodiode and fed into a lock-in amplifier in order to extract the beat signal at Ω. Integration times of 5 ms are used. Images and tracking are performed by moving the sample over the fixed laser spots by means of a 2D piezoscanner.Fluorescence and white light imaging.Fluorescence images are recorded using the probe beam in a wide-field excitation configuration by inserting an additional lens to focus the beam in the back aperture of the objective lens. Samples were illuminated for 10 ms at a rate of 30 Hz on a surface of 400 µm 2 with an intensity of 7±1 kW/cm 2 . The fluorescence from single Cy5 fluorophores is collected in epi and imaged on a back-illuminated thinned CCD camera. White light images are page 14 of 23

  SMT trajectories containing at least 25 data points were analyzed. For each track (recorded by SNaPT or SMT) , the Mean Square Displacements (MSDs) are calculated on 25 consecutive data points (~1 s) along the trajectory and the instantaneous diffusion constant D is deduced as a function of time, from the initial slope of each MSD curve (fitted on the first 7 points).

Figures

  Figures

Figure 1 :

 1 Figure 1: Schematic of the experimental setup. Inset: LISNA image of 5 nm NPs embedded in

Figure 2 :

 2 Figure 2: Tracking capabilities: ratio of successful tracking (open circles) and measured

Figure 3 :

 3 Figure 3: (a) Biological construct. (b) Histogram of the signals for 5 nm gold NPs detected on

Figure 4 :

 4 Figure 4: (a) to (c) Distributions of the instantaneous diffusion constants D of mGluR5a on

Figure 5 :

 5 Figure 5: White light image (a, left) and LISNA image (a, right) of a live neuron labelled with

Figure 1 page 20 of 23

 123 Figure 1

Figure 2 page 21 of 23

 223 Figure 2

Figure 3 page 22 of 23

 323 Figure 3

Figure 4 page 23 of 23

 423 Figure 4

Figure 5

 5 Figure 5

Acknowledgements

We thank Pierre Gonzales for COS7 cell cultures, Christelle Breillat and Delphine Bouchet-Teissier for neuron cultures. G.A. Blab acknowledges financial support by "Fonds zur Förderung der wissenschaftlichen Forschung" (FWF, Austria, Schrödinger-Stipendium) and the "Fondation pour la Recherche Médicale" (FRM, France). This research was funded by CNRS (ACI Nanoscience and DRAB), Région Aquitaine and the French Ministry for Education and Research (MENRT).


Stéphane Berciaud

Laurent Cognet

Brahim Lounis
email: b.lounis@cpmoh.u-bordeaux1.fr

Photothermal Absorption Spectroscopy of Individual Semiconductor Nanocrystals

Photothermal Heterodyne Detection is used to record the first room temperature absorption spectra of single CdSe/ZnS semiconductor nanocrystals. These spectra are recorded in the high cw excitation regime and the observed bands are assigned to transitions involving biexciton and trion states. Comparison with the single nanocrystals photoluminescence spectra leads to the measurement of spectral Stokes shifts free from ensemble averaging.

Core shell CdSe/ZnS semiconductor nanocrystals exhibit unique size-dependent optical properties 1 which make them ideal candidates for applications in various fields such as the design of new optical devices 1-3 or bio-labeling 4 . For full exploitation, understanding of their optical properties is however crucial. Ensemble photoluminescence and linear absorption measurements provided a comprehensive description of the excitonic levels structure of CdSe/ZnS nanocrystals 5 and allowed for a precise assignment of the optical transitions 6 . Individual nanocrystals are now commonly detected using standard fluorescence microscopes with low excitation intensities due to the high radiative quantum yield of their band edge exciton state X (1S e , 1S h,3/2 ) (see reference 6 for spectroscopic notations). Their luminescence displays a blinking 7 behavior with intensity dependent on-times distributions and strong photon anti-bunching 8, 9 .

CdSe/ZnS nanocrystals have relatively high absorption cross-sections 8, 10 (typically 2 15

~cm -

) and when excited with sufficiently high intensities, excitons are created at average rates N abs significantly higher than their radiative recombination rates Γ rad 8, 11 . In this regime, efficient non-radiative Auger recombinations of the prepared multiexcitons take place [START_REF] Efros | Semiconductor Nanocrystals[END_REF] . In CdSe/ZnS nanocrystals, these recombinations occur in the picoseconds range as measured in time resolved experiments on ensemble samples [START_REF] Klimov | Quantization of multiparticle auger rates in semiconductor quantum dots[END_REF] . Individual nanocrystals could thus be detected through their absorption using the Photothermal Heterodyne method [START_REF] Berciaud | Photothermal Heterodyne Imaging of Individual Nonfluorescent Nanoclusters and Nanocrystals[END_REF] .

Although recording the luminescence spectra from single nanocrystals has become routine, a measurement of the absorption spectra of individual nanocrystals has however never been reported due to a lack of sensitive enough methods. In this letter, we present the first room temperature photothermal absorption spectra of individual nanocrystals.

In the photothermal techniques [START_REF] Berciaud | Photothermal Heterodyne Imaging of Individual Nonfluorescent Nanoclusters and Nanocrystals[END_REF][START_REF] Boyer | Photothermal Imaging of Nanometer-Sized Metal Particles Among Scatterers[END_REF] the signal is proportional to the amount of laser power absorbed by a single nanoparticle converted into heat by non-radiative relaxation processes. This allowed to detect individual tiny metal nanoparticles and to perform spectroscopy of their Plasmon resonance [START_REF] Berciaud | Observation of Intinsic size effects in the optical response of individual gold nanoparticles[END_REF] . For nanocrystals the signal has its origin from two possible non-radiative relaxation pathways. First, at the high cw excitation rates required in the photothermal method (typically nanocrystals [START_REF] Klimov | Quantization of multiparticle auger rates in semiconductor quantum dots[END_REF] ) and is followed by a non radiative relaxation of the remaining exciton to the band-edge state X. Since N abs is much higher than Γ rad ( 1 20 1 ns ~), multiexciton creation efficiently prevents radiative recombination of the monoexcitons. Thus, the first contribution to the signal arises from continuous cycling of the nanocrystal between the band-edge X and biexciton XX states (see Fig. 1).

The second mechanisms contributing to the signal come into play when the nanocrystal is in a charged state. It is known that continuous excitation of a CdSe nanocrystal can lead to a spontaneous or Auger type ejection of charges to the shell or to the local environment of the nanocrystal [START_REF] Nirmal | Fluorescence intermittency in single cadmium selenide nanocrystals[END_REF][START_REF] Efros | Random Telegraph Signal in the Photoluminescence Intensity of a Single Quantum Dot[END_REF][START_REF] Krauss | Charge and Photoionization Properties of Single Semiconductor Nanocrystals[END_REF] . In this case, absorption leads to the formation of trions and the photothermal signal arises from fast Auger nonradiative recombination of X* state to the ground charge state 0* ( ).

The absorption spectroscopy setup (Fig. 2) using Photothermal Heterodyne signals was described elsewhere [START_REF] Berciaud | Observation of Intinsic size effects in the optical response of individual gold nanoparticles[END_REF] . In short, it consists in a heating beam (tunable cw dye laser, Coherent 599, tuning range 530-590 nm corresponding to photon energies of 2.10-2.34 eV) overlaid with a probe beam (HeNe laser, 633nm wavelength , well below the band-edge absorption of the nanocrystals). The heating beam is circularly polarized with an intensity of As expected, we observe a large heterogeneity of luminescence spots heights due to diversity in nanocrystals structures, surface states and orientations with the respect to the polarization of the excitation light. As can be seen on Fig. 3 (b) the photothermal signal levels recorded from individual nanocrystals are also heterogeneous. However, we cannot establish a clear correlation between the heterogeneities of the luminescence and photothermal signal levels. We also notice that some nonluminescent nanocrystals (which do not appear in Fig. 3 (a)) are observed on the photothermal image.

Furthermore, the photothermal signal scales linearly with the heating intensity (Fig. ).

We acquired the photothermal absorption spectra for the nanocrystals which colocalize in the luminescence and photothermal images. Both of them contain a first absorption peak at respectively 2.160 eV and 2.170 eV, blue shifted from the emission lines. Similarly to the emission spectrum, the width of the single nanocrystal absorption peak is narrower than that of the ensemble. The two peaks of the ensemble absorption spectrum, recorded in the weak excitation regime, are assigned to the band-edge X state and to the higher energy (1S e , 2S h,3/2 ) state [START_REF] Norris | Measurement and assignment of the size-dependent optical spectrum in CdSe quantum dots[END_REF][START_REF] Ekimov | Absorption and intensity-dependent photoluminescence measurements on CdSe quantum dots: assignment of the first electronic transitions[END_REF] (Fig. 4 (bottom, solid line)). The non-resonant Stokes shift ΔE S is defined as the energy difference between the first absorption peak and the luminescence maximum energy. We found ΔE S ~ 70 meV in agreement with previous ensemble studies [START_REF] Kuno | The band edge luminescence of surface modified CdSe nanocrytallites: Probing the luminescing state[END_REF] ,

The photothermal absorption spectrum presented on Fig . 4(top, solid line) exhibits one clear band and a rising feature at higher energies which is not fully scanned due to insufficient laser tuning range. We assign the low energy band to the two unresolved XX X → and * X * 0 → transitions. Indeed, Coulomb interactions induce comparable red-shifts of the transitions [START_REF] Patton | Trion, biexciton, and exciton dynamics in single selfassembled CdSe quantum dots[END_REF] with respect to E X , the energy of the X 0 → transition (see Fig. 1). The rising feature in the spectrum may involve X X X ′ → and * X 0 ′ → transitions where X′ denotes a high energy exciton states (such as (1S e , 2S h,3/2 ) or (1P e , 1P h,3/2 )).

We performed the same measurements for 23 individual nanocrystals and observed qualitatively comparable spectra. For each of them, we extracted the energy separation ΔE between the position of the low energy absorption band and the luminescence peak. The histogram of the values of ΔE is shown on Fig. 4 (inset). It is well fitted by a Gaussian distribution with a FWHM of meV 12 and a mean value of ~50 meV smaller by ~20 meV than ΔE S . This deviation is in agreement with the values of biexciton and trion binding energies deduced from femtosecond photoluminescence up-conversion [START_REF] Achermann | Multiexcitons confined within a subexcitonic volume: Spectroscopic and dynamical signatures of neutral and charged biexcitons in ultrasmall semiconductor nanocrystals[END_REF] , transient photoluminescence [START_REF] Caruge | Transient photoluminescence and simultaneous amplified spontaneous emission from multiexciton states in CdSe quantum dots[END_REF] studies (~20-30 meV for 2nm nanocrystal) as well as from the emission properties of single colloidal nanocrystals [START_REF] Shimizu | Surface-enhanced emission from single semiconductor nanocrystals[END_REF] or self-assembled quantum dots 22 in charged states.

In conclusion we have demonstrated the applicability of the Photothermal Heterodyne method to room temperature detection and absorption spectroscopy of individual CdSe/ZnS semiconductor nanocrystals in the multi-excitonic regime. We foresee promising spectroscopic studies of nanocrystals using this method with samples at cryogenic temperatures.

1 -

 1 are focused on the sample using a high NA objective. A fluorescence confocal microscopy scheme is also implemented on this setup. It is used to image individual luminescent nanocrystals and to record their luminescence spectra. For these measurements, the heating laser is used for the excitation with an attenuated intensity ( 2 1 ~cm kW ) and a fixed energy (2.30 eV). The collected luminescence photons are split towards an avalanche photodiode and a spectrometer. The samples were prepared by spin-coating dilute solutions of nanocrystals (average radius: in 1% mass polyvinyl-alcohol aqueous solution) onto clean microscope coverslips. The dilution and spinning rates were chosen such that the final density of nanoparticles in the samples is less than 2 µm . A drop of viscous silicon oil was added on top of the samples to ensure homogeneity of heat diffusion. Individual fluorescent nanocrystals were first located by recording a the sample (Fig.3 (a)). For each spot, a luminescence time trace and an emission spectrum were recorded simultaneously. The blinking behavior, a signature of single nanocrystal emission, is clearly visible in the time trace shown on Fig.3 (c). We then recorded a photothermal image of the same sample area (Fig.3 (b)). Cleary the luminescence and photothermal images correlate well ( % 80 > of the luminescence spots correlate with a photothermal spot), proving that the spots in the photothermal image stem from individual nanocrystals. Contrary to luminescence, the photothermal signals do not show any blinking behavior (Fig.3 (d)). They remain stable during time scales larger than those necessary to record an absorption spectrum.

Figure 4 (

 4 top) presents the results obtained from the individual nanocrystal highlighted on Figure 3. For the absorption spectrum, the laser frequency was scanned with steps of 4 meV and ms 200 integration times per point. The signals are corrected for the wavelength dependence of the diffraction-limited laser spot size and the heating power at the sample was measured during the acquisitions for normalization. Contrary to the ensemble luminescence spectrum, which has an inhomogeneously broadened Gaussian shape (open circles of Fig. 4 (bottom) with peak energy at eV 100 on Fig. 4 (top)). The absorption spectrum of the same nanocrystal (measured with the photothermal method) and that of an ensemble of nanocrystals (measured with a spectrophotometer) are also shown on Fig. 4 (solid lines).

Figure 1 .

 1 Figure 1. Energy diagram of the excitonic states involved in the photothermal signal from nanocrystals in the neutral and charged state. For notations see details in the text.

Figure 2 .

 2 Figure 2. Schematic of the experimental setup.

Figure 3 . 2 .

 32 Figure 3. (a) Comparison of photoluminescence (b) and Photothermal Heterodyne images of the same area

Figure 4 .

 4 Figure 4. Top: Photoluminescence (open circles) and photothermal absorption (solid lines) spectra recorded for the individual nanocrystal highlighted in Fig. 3. For clarity a smoothing of the absorption spectra is shown. Bottom: Photoluminescence (open circles) and linear absorption spectra (solid line) of an ensemble of nanocrystals. Inset: Histogram of the values of ΔE obtained from photoluminescence and photothermal absorption spectra of 23 individual nanocrystals. The distribution is fitted by a Gaussian curve (black line).

Figure 1 12 Figure 2 13 Figure 3 14Figure 4

 1221334 Figure 1

ACKNOWLEDGMENTS. We wish to thank O. Labeau, P. Tamarat and G. Blab for helpful discussions and A. L. Efros for the reading of the manuscript. This research was funded by CNRS (ACI Nanoscience and DRAB), Région Aquitaine and the French Ministry for Education and Research (MENRT).


L Cognet

C Tardin

D Boyer

D Choquet

P Tamarat

B Lounis
email: b.lounis@cpmoh.u-bordeaux1.fr

Prof B Lounis

Single metallic nanoparticle imaging for protein detection in cells

We performed the first visualization of membrane proteins labeled with 10nm gold nanoparticles in cells, using an all optical method based on photothermal interference contrast (PIC). The high sensitivity of the method and the stability of the signals allows 3-dimension imaging of individual nanoparticles without the drawbacks of photobleaching and blinking inherent to fluorescent markers. A simple analytical model is derived in order to account for the measurements of the signal amplitude and the spatial resolution. The PIC method provides an efficient, reproducible and promising way to visualize low amounts of proteins in cells by optical means.

An important development in modern optical microscopy is the direct observation of single molecules(1), in media as varied and complex as molten polymers (2) or living cells (3)(4)(5)(6)(7).

Selecting a single molecule at a time allows the elimination of all the implicit averages of conventional optical observations, gaining access to heterogeneity, dynamical fluctuations (8,9), diffusion (10), reorientation (11), colocalization (12), and conformational changes (13) at the molecular level. Single molecule methods have changed the way we think and carry out experiments on complex molecular systems. Yet, the optical detection of a single molecule remains difficult, because the spatial resolution is limited by the wavelength of light, so that the signal must be extracted from the background arising from billions of other molecules in the focal spot of the microscope. An elegant way to find the needle in the haystack is to attach a "label" to the molecule of interest. The ideal label must fulfill contradictory requirements. It should generate an intense optical signal, but at the same time be as small as possible, not to perturb the observed molecule too severely.

Several kinds of optical labels have been developed and utilized in recent years. The most commonly used labels are fluorophores, usually organic dyes that can be chemically grafted to the molecule under study, but also autofluorescent proteins genetically fused to the protein of interest. Because of the wavelength change it involves (Stocke's-shift), fluorescence can be separated from background. Usual dye fluorophores are so small that they hardly hamper the diffusion of their host proteins or lipids, nor do they affect their functions or interactions with partners, at least if they are not attached to sensitive areas. The main drawbacks of organic fluorophores are the photobleaching and the blinking, i.e. the processes by which photochemical reactions transform the excited fluorophore into a non-fluorescent product. A fluorophore's lifetime is severely limited by the heavy laser irradiations used in single molecule detection.

Photobleaching can be reduced by removing free oxygen, but is never eliminated completely, and it is the most serious obstacle to many experiments such as in living cells due to the toxicity of the oxygen scavengers. Nanocrystals of II-VI semiconductors (such as CdSe) have recently been used as fluorescent markers (14)(15)(16)(17). Nanocrystals of different sizes can be excited by the same laser, and still be distinguished by their size-dependent luminescence spectrum. They must be passivated, protected and functionalized by surface layers, because of their high chemical reactivity, and in order to prevent luminescence quenching by acceptor surface states. Therefore, a passivated nanocrystal is a rather bulky label of several nanometers in diameter. Although they resist bleaching longer than organic dyes, their luminescence is subject to blinking (18), and they also bleach eventually.

Metal particles are commonly used for single-particle or single-molecule tracking (19) and in immunocytochemistry (20,21). They are often colloids, with diameters ranging between a micron and a few nanometers, or chemically synthesized clusters (22,23) with well-defined numbers of atoms and chemical structures. Metal particles are very appealing labels because they do not photobleach, and do not optically saturate at reasonable exciting intensities. Single large metal particles can be imaged in optical microscopy by means of various methods, such as imaging at the plasmon frequency with dark field illumination (24), with differential interference contrast (DIC) and video enhancement (25), or with total internal reflection (26). All these methods are based on the intense Rayleigh scattering of sub-micron particles, down to a diameter of about 40 nm. However, they do not apply to very small metal particles, because Rayleigh scattering decreases like the sixth power of diameter. Moreover, the scattering signal must be discriminated from a strong background, specially if the particles are to be detected in cells or tissues. Recently, we developed a photothermal interference contrast (PIC) technique to optically detect absorbing nanoparticles (27). The principle of the PIC method relies on the strong optical absorption of a small metal particle at their plasma resonance which gives rise to a photothermal effect, i.e. a change in temperature around the particle when it is illuminated by a laser light (heating laser). This temperature change leads to a variation of the local index of refraction which can be optically detected when combining high-frequency modulation and polarization interference contrast using a second laser beam. We could previously image very small gold colloids down to 5 nm or 2.5 nm in diameter embedded in thin polymer films, with a signal-tonoise ratio better than 10 or of the order of 2 respectively. We also showed that, in addition to its intrinsic sensitivity, the photothermal image is remarkably insensitive to scattering background, even when arising from such strong scatterers as 300 nm latex beads.

In the present work, we show that it is possible to image gold colloids in thick samples (e.g. cells) with a modified PIC setup. We demonstrate imaging of receptor proteins stained with individual 10 nanometer size gold particles in the plasma membrane of COS7 cells. The resolution in 3D of the PIC method is also derived and confronted to measurements.

METHODS

PIC imaging

The setup is derived from(27) (Fig. 1A). The samples were mounted onto an inverted microscope equipped with a 100x oil immersion objective (NA=1.4 ) and a 3D piezoscanner. An horizontally polarized beam of He-Ne laser (633nm wavelength) is split into two beams (herein called probe and reference beams) by a Wollaston prism and sent to the microscope objective through a telecentric lens system. The transmitted probe and reference beams are collected by a microscope objective (NA=0.8) and imaged on a mirror (at normal incidence) by a 100mm focal length lens, forming an afocal system. The retroreflected beams having the same optical path as the incident ones, recombine in a Wollaston prism. The vertically polarized recombination reflected by the polarizing cube is sent to a fast photodiode. Heating of the nanoparticles is performed by a 514 nm wavelength beam from an Argon laser which intensity is modulated at high frequency (900kHz) by an acousto-optical modulator. We performed resolutions studies in two different heating beam configurations: a focused illumination where the green beam is superimposed with the probe beam and an epi-illumination where the green beam is defocused at the "entrance" of the objective and a wide region of the sample is illuminated.

The heating beam induces a periodic phase difference between the probe and reference beams when the red probe beam is positioned on a heated nanoparticle. This gives rise to a modulation of the red intensity which is filtered from green stray light of the heating beam and detected with a fast photodiode. The photodiode signal is sent to a lock-in amplifier to detect the phase difference between the probe and reference beams at the same frequency as the heating beam intensity modulation. A lock-in amplifier is used to detect the phase difference at the modulation frequency with an integration time of 10 ms. Microscopic images are obtained by scanning the sample with a XYZ piezo-electric translation stage.

Cell culture, transfection of COS7 cells and immunostaining. COS7 cells were cultured plated on #1 glass slides in DMEM medium supplemented with streptomycin (100 µg/ml), penicillin (100 U/ml), and 10 % bovine serum in a humidified atmosphere (95 %) at 5 % CO 2 and 37 o . Cells were used for 12-14 passages and were transferred every 4 days. Transfection was performed using FUGEN. Cells exhibiting confluence of ~ 30% were used for transfection with 10 µg for 1 ml volume of cDNA coding for a metabotropic receptor for glutamate containing of myc-tag at the extracellular N-terminus (mGluR5-myc(28)). Transfection efficiency was on the order of 50%. After 12h, the cells were fixed using the following protocol: 10min in 4% paraformaldehyde / sucrose, 15min in phosphate saline buffer (PBS) with 50nM NH4Cl, then 3 rinses in PBS with 0.3% Bovine Serum Albumine (BSA). Then, a first immunostaining was performed using antimyc antibodies tagged with alexa568 dyes (herein named amyc-Alexa568, 20min at room temperature 10µg/ml, 0.3% BSA). After two rinses in PBS, a secondary immunostaining by antiIgG-10nm gold (goat anti-mouse to label the amyc-Alexa568, Auroprobes Amersham, 20min at room temperature, 0.3 % BSA) was performed for different concentrations (10µg/ml to 0.1µg/ml) followed by 3 rinses in PBS.

Fluorescence and scattering imaging.

Fluorescence images are recorded using the focused green heating beam for excitation. The power was reduced to ~ 10µW. The fluorescence is collected with the high NA objective, filtered with a high pass filter (OG550) and imaged in a confocal configuration onto an avalanche photodiode. Scattering images are recorded with the modulated heating beam in absence of the red beams. The scattered green light is detected by the fast photodiode (red-pass filter removed) and demodulated with the lock-in amplifier.

RESULTS AND DISCUSSION

Signal calculation, resolution

In this section, we derive an analytical form of the modulated signal detected by the photodiode.

We chose a coordinate system with an origin at the focal point of the heating beam which coincides with the one of the two red beams, the probe beam (Fig 1B). Since we use small absorbing nanoparticles, we can make the assumption of a punctual heat source with a modulated induced by this punctual heat source in a homogeneous medium is derived from the equation of heat conduction [START_REF] Carslaw | Conduction of heat in solids[END_REF] and is given by ( )

            - - + - × = - ∆ - - R r r heat e R r r t r r r I t r r r T 0 0 0 0 0 0 cos 1 4 ) , , ( ω πκ σ . C R ω κ 2 =
is the characteristic length for heat diffusion at ω, where κ is the thermal conductivity of the medium (0.19 W.K -1 .m -1 for water) and C its heat capacity for a unit volume.

At the working modulation frequency, R is on the order of the probe beam transverse focal spot size (27). The temperature rise induces a change in the refractive index medium at position r :

(

0 0 0 0 t r r f r I t r r r T T n t r r r n heat - ⋅ = - ∆ ⋅ ∂ ∂ = - ∆ T n ∂ ∂ ) ( ). , ) , , ( ) , , ( 0 0
is the variation of the refractive index per unit of temperature (~10 -4 K -1 for water at room temperature [START_REF] Tilton | [END_REF]). The heat diffusion characteristic length R being on the order of the optical wavelength, diffraction effects play important roles. The exact expression of the emerging probe beam can in principle be derived using light scattering theory [START_REF] Chu | Laser Light Scattering[END_REF]. The solution is complicated, and is out of the scope of this paper.

To have a simple qualitative physical insight we shall consider now the case were the geometrical optics is valid. For weak refractive index changes, we use the thin-phase grating approximation by assuming that the phase of the optical field can be calculated along the optical rays as they would be in the absence of phase-shifting object. The global phase-shift is thus obtained by accumulating along unperturbed optical rays the variation of refractive index through the entire object. When the object is located in regions where the probe wave front is plan, the signal of the interference between the probe and the beams, detected at the modulation frequency ω, writes: where η is the conversion factor of the detection chain and l is a distance on which the variations of refractive index are significant (l>R). The index ω in f ω refers to the component of f at frequency ω detected by the lock-in amplifier. We assume that the probe and reference beams have the same intensity profiles so that only probe I appears in the equations.

( ) ( ) ( ) ( )  ⌡ ⌠  ⌡ ⌠ - ⋅ =  ⌡ ⌠  ⌡ ⌠ - ∆ ⋅ = ∞ ∞ ∫ ∫ l probe heat l probe r r f dz z y x I dy dx z y x I r r r n dz z y x I dy dx z y x S 0 0 0 0 0 0 0 0 0 0 0 ) , , ( , , 4 , 2 ) , , ( 2 , ,
This latter formulae gives the origin of the resolution of the PIC method when we scan the sample i.e. the particle position 0 r . In the transverse plane (xOy), it is given by the product of the heating profile with the convolution between the probe beam and the phase shifting object profiles. However, the axial resolution in the plane probe wave-front approximation is given by the product of the heating profile by the probe beam profile.

In order to estimate the resolution of the PIC method and the probe-reference optical phase shift, the modulated refractive index variations ) , ( 0 r r n ω ∆ are modeled by a "phase-sphere" of radius Th ρ centred on the heated particle with a uniform modulated refractive index (

( ) 0 r n ω ∆
). We ). In the case where the probe beam profile is nearly uniform over the "phasesphere", this phase shift is given by:

∂ ∂ ≈ ∆ = ∆ ∫ ∫ ( ) ( ) ( ) ( ) ∫∫ ∞ - - - - ∆ = 2 0 2 0 2 0 0 0 0 0 ) , , ( 4 ,

Resolution and Phase-shift measurements

The transverse and axial resolutions of the PIC method were studied on samples of 10nm gold colloidal nanoparticles embedded in a PVA film (few tens of nm thick) spin-coated on glass slide. We used a heating beam which fulfilled the back aperture of the microscope objective such that its transverse dimension was nm 224 2 1.22 heat = ΝΑ / λ (FWHM) and its axial dimension

1.0 = ΝΑ / µm 2 heat
1.22 λ π (FWHM). However the probe and reference beams under-filled the aperture of the objective. Its transverse size at the focal plane was 550±50 nm which leads to a longitudinal size 5.0±0.9µm. As mentioned above, the theoretical transverse resolution of the PIC method involves the profiles of the heating and probe beams.

For simplification, assuming Gaussian beams at the objective focal plane and a uniform phase profile in a sphere of nm Th 370 2 = ρ diameter, equation (1) leads to a theoretical transverse (resp. axial) resolution of 209±2 nm (resp. 0.85±0.05 µm) in the well focussed heating configuration and 530±70nm (resp. 5.0±0.9µm) in the uniform heating one.

In the focused heating beam configuration, we measured a transverse resolution of 215±5 nm (Fig. 2A&B) and an axial resolution of 1.2±0.2 µm (Fig. 2A&C). And as expected, the resolution degraded in the uniform heating configuration and is 460±25 nm (Fig 2D ) in the transverse plane and is 6.0±0.5 µm for the axial direction (Fig 2E).

In both configurations the measurements are in fairly good agreement with what expected from our simple theoretical analysis.

The pointing accuracy at which single nanogold particles can be localized with the PIC method is potentially very high. Since photothermal signals of metal particles are stable and do not saturate, single nanoparticles can be imaged with very high signal-to-noise ratio (SNR). In a typical situation, 10 nm gold particles absorbing 300nW heating power are detected with a peak SNR greater than 30 when the integration time is 10 ms per pixel. Taking the image pixel size equal to the standard deviation of the PIC resolution [START_REF] Thompson | [END_REF], the expected pointing accuracy is then 10 nm in the transverse directions, far below the optical diffraction limit [START_REF] Thompson | [END_REF](33)(34). This pointing accuracy can be dramatically improved with higher SNRs(32-34), using for example larger heating intensities or longer integration times. This very high precision localization of nanoparticles could be of considerable interest for the precise localisation of proteins in cells.

In order to measure the phase-shift φ ∆ induced by heating a single particle we insert an electrooptic modulator (EOM) between the two lenses of the telecentric system. The axis of the EOM are set parallel to the respective polarisations of the reference and the probe beams. Applying a modulated voltage to the EOM induces a phase-shift which mimics the phase-shift produced by a heated particle. φ ∆ is thus deduced from the amplitude of the modulated voltage obtained when the demodulated signals equal that of the heated single particles.

For 10 nm gold spheres absorbing an average heating power of 300nW modulated at a frequency of 1 MHz, we measured rad

Immunohistochemistry assay

We performed an immunohistochemistry assay to demonstrate the capabilities of the PIC method to image low amounts of immunogold labeled proteins in cells. Protein conjugated colloid gold particles can be obtained commercially in sizes ranging from one nanometer to hundreds of nanometers. Electron microscopy prefers to use metal particles smaller than 10 nm because of their better penetration in cellular organelles [START_REF] Hayat | Colloidal Gold: Principles, Methods and Applications[END_REF]. We used COS7 cells transfected with membrane protein mGlurR5-myc (see methods). The membrane proteins were primarily labeled with Anti-myc antibodies tagged with alexa568 dyes. As a secondary labeling stage, we used antiIgG-10nm gold antibodies (see methods). Fluorescence images easily discriminate cells that contained fluorescence-labeled receptors from untransfected ones (Fig. 3B,E&H).

Specificity of the gold labeling was subsequently ensured as no signal was "usually" detected by the PIC method on untransfected cells as shown on Fig. 3A-C (less than 4.10 -3 particles per focal spot area ~0.07µm 2 for untransfected cell (N=10 cells), compared to much more than one particle per 0.07µm 2 in transfected cell (N=28 cells) for the same labeling conditions). Photothermal images of transfected cells are shown on Fig. 3 for two concentrations of antiIgG-10nm gold (10µg/ml in Fig. 3D-F and 0.5µg/ml in Fig. 3G-I). AntiIgG-10nm gold labeling is clearly observed on the cell membrane. The images are identical when the same regions are recorded successively due to the non-photbleaching feature of the labels. Three dimensional pictures of the membrane protein labeling can thus be obtained by changing the axial position of the samples (supplementary materials). Resolved discrete spots which may be attributed to single nanoparticles can be observed in cell regions with low density of labels as in the inset of Fig. 3I, (recorded with a high scan resolution, 40nm). We further decreased the labeling concentration of antiIgG-10nm gold to 0.1µg/ml in order to obtain a sparse labeling on the cell membranes (Fig. 4B-C). Several arguments indicate that we achieved single 10nm gold particle detection in cells.

First, when increasing the intensity of the heating laser by 5 folds, no weaker spot appears in the image (see Figure 4B-C). This indicates that we detect the smallest absorbing objects present on the plasma membrane of the cells. Second, we constructed and analysed the histograms of the signal heights measured on three different samples: bare 10 nm gold colloids in a spin-coated PVA film (Fig4D), antiIgG-10nm gold antibodies also embedded in a PVA (Fig4E) and membrane receptors labeled with a dilute solution (0.1µg/ml) of antiIgG-10nm gold (Fig4F).

The fairly narrow unimodal distribution obtained in Fig4D confirms that the spots stem from individual nanospheres (27). The width of the distribution is equal to 0.3 (center normalized to 1).

It arises from the dispersion of the particle sizes (10% given by the manufacturer) which translates to 30% dispersion in the signal heights since absorption cross-section scales as the volume of the nanosphere. Interestingly, the distribution obtained from spin coated antiIgG-10nm gold antibodies is bimodal. It is well fitted by two Gaussian curves centred at signal heights 1 (normalisation value) and 2.0±0.1, with respective widths δ=0.32±0.03 and 0.4±0.1 ≈ √2×δ. The relative positions and widths of the two peaks indicate that the spots arise from either one (63±10%) or two metal particles. Assuming a Poisson distribution, the mean number of nanoparticles per antibody is thus equal to one. However, we would expect to detect 13% of antibodies labeled with more than 2 nanoparticles which is not the case as stronger signals that could have been attributed to more than two particles were extremely rare. Steric limitation can account for this as the typical size of a IgG is of the order of the size of the nanoparticles.

The histogram constructed from PIC images of cells (Fig4F) is well fitted by four Gaussian curves. The widths of the Gaussian curves were set to SQRT(N)×δ, N being the number of the peak and δ a free fit parameter [START_REF] Schmidt | [END_REF]. The average signal height of the peaks scales linearly with the peak number (inset on Fig4G). By comparison with the results obtained from spin-coated antiIgG-10nm gold antibodies one can conclude that single antiIgG-10nm gold antibodies are detected. The first peak corresponds to single IgG antibodies labeled with only one nanoparticle.

Altogether the data presented on Fig4 indicates that individual 10 nm nanoparticles can be detected on the plasma membrane of cells. The SNR at which the individual 10 nm nanoparticles are detected depends on the heating beam intensity. For this study (Fig. 3 and4), the intensity was of the order of 3MW/cm 2 which corresponds to a power of ~150nW absorbed by a nanoparticle (in an aqueous environment). The SNR was then better than 20

CONCLUSION

We demonstrated in this work that 10nm particles commonly used to label proteins in cells can be imaged using an all optical method. We measured the transverse and axial resolution of the PIC method and compared the measurements to a simple model. For live biological samples, the SNRs might however be limited by the admissible temperature rise in the sample. For particles detected on the cell membranes with a SNR of 10, we estimated a temperature increase of 15K on the surface of the 10 nm gold particles (27). This temperature rise decreases as the reciprocal distance from the particle center.

The high sensitivity of the method allowed precise quantification of the extremely low number (1 or 2) of 10 nm gold colloids present on commercially available IgG antibodies. This could be of great interest when low expression proteins are aimed to be detected. The method does not require amplification to achieve high sensitivity, giving an unbiased picture of the expression pattern of the cell. More generally, the PIC method is accurate and robust enough to perform stoechiometry of gold nanoparticles as commonly used in protein and DNA chips. Fluorescent labels excited by the heating beam can also be detected on the same setup using the Single Photon Counting avalanche photodiode. (B) Coordinate system with an origin at the focal point of the heating beam which coincides with the one of the two red beams, the probe beam.

  σ is the absorption cross section of the nanoparticle and ( ) 0 r I the intensity of the green beam at its position (figure 1.b). The temperature rise ∆T at a

  nm particle absorbing an average heating power of 300nW modulated at a frequency of 1 MHz, we find rad

  The advantages of the PIC method over fluorescent methods for single molecule detection arise from the absence of saturation of the signals, from the absence of the autofluorescence or scattering due to the environment -or to the cells themselves-and from the absence of blinking and photobleaching of the labels. The latter limitation is very restrictive to perform 3D localization of the single fluorescent molecules as multiple records of the same molecule are hardly feasible. On the contrary, the stability of the PIC signals provides a way to localize in 3D single particles with a number of recordings and signal levels which can be arbitrary high. Single nanoparticles can thus be localized in the scattering environment formed by a cell with very high pointing accuracy. Our work show that it is in principle possible to multiplex fluorescence and PIC images. Spots from a PIC image can be accurately located with respect to specific cellular organelles fluorescently labeled. Furthermore, nanoparticles with a shifted plasmon resonance (of different shape or composition) could be used to perform multiple color PIC imaging.

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We thank Dr. Dionysia Theodosis for the gift of the antiIgG-10nm gold antibodies and Pr. Michel Orrit for stimulating discussions. This work was supported by grants from the CNRS, the Conseil Régional d'Aquitaine and the ministère de la recherche.

The authors declare that they have no competing interests.