Halina Abramczyk
email: abramczy@mitr.p.lodz.pl

Beata Brozek-Pluska

Jakub Surmacki

Marc Tondusson

Eric Freysz

Photostability of biological systems-Femtosecond dynamics of zinc tetrasulfonated phthalocyanine at cancerous and noncancerous human Breast tissues

Keywords:

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

Introduction

Biological systems must effectively acclimate to maintain photostability, because there would be no life on earth without it. Thus, molecular biological structures responsible for harvesting solar energy must be resistant to photo induced chemical changes. This feature determines the health disease balance in living creatures. When the photostability protection and reparation mechanisms fail, the processes that convert noncancerous tissue into abnormal tissue are strongly enhanced, leading to disease. Therefore, it is important to characterize the mechanisms governing photostability in living biological systems [START_REF] Abramczyk | Mechanisms of energy dissipation and ultrafast primary events in photostable systems: H-bond, excess electron, biological photoreceptors[END_REF].

To examine photostability new analytical tools, such as Raman imaging and femtosecond spectroscopy, providing high temporal or spatial resolution are required. The high temporal resolution is needed to monitor the primary events occurring upon light excitation, while the high spatial resolution is required to map the localization and distribution of exogenous probes and endogenous cellular components [START_REF] Abramczyk | Oncologic photodynamic diagnosis and therapy: confocal Raman/fluorescence imaging of metal phthalocyanines in human breast cancer tissue in vitro[END_REF][START_REF] Brozek-Pluska | Distribution of phthalocyanines and raman reporters in human cancerous and noncancerous Breast tissue as studied by raman imaging[END_REF].

Raman imaging and femtosecond spectroscopy may open new expanses in cancer biology particularly in metabolic and epigenetic modifications of cancer, and bring revolution in cancer detection and treatment. Limited number of papers has been reported on ultrafast dynamics of biologically important molecules such as: proteins and lipids [4 8]. Despite of several model studies that have been done so far, no ex vivo ultrafast dynamics of human tissue have been reported yet. High spatial [9 14] and temporal resolution [15 20] allows to detecting a single cancerous cell in vivo and monitor molecular events that occur inside and contribute to cancer development.

In the present study, we used sulfonated zinc phthalocyanine as an exogenous probe to examine the processes occurring at the biological interfaces of human breast tissue. Phthalocyanines play an important role as photosensitizers in conventional and targeted photodynamic therapy (PDT) [15 82] and the mechanisms and dynamics of energy transfer are crucial in this therapy. PDT is a three component therapy involving the photosensitizer (phthalo cyanine), the visible light absorbed through the photosensitizer and molecular oxygen. The photosensitizer, accumulated or retained in the target components of human cells, absorbs light and induces a sequence of photophysical events, such as excited singlet state fluorescence emission, intersystem crossing to a triplet state or electron transfer. The energy dissipation depends on many factors, such as phthalocyanine aggregation or photoinduced reactions at photosensitized interfaces. Aggregation also depends on many factors, such as a central metal, substituent type and solvent composition. Dimers and higher ordered aggregates are generally non fluorescent, as these structures have efficient channels for non radiative relaxation. Thus, aggregation decreases the efficiency of the radiative paths that return the photosensitizer to the ground state via fluorescence or long lived phosphores cence. In contrast, the excited monomeric species of the photosensitizer once upon irradiation emits fluorescence [START_REF] Snow | The Porphyrin Handbook[END_REF].

In this study, we used pump probe femtosecond transient absorption spectroscopy to examine the primary processes occurring in sulfonated zinc phthalocyanine (ZnPcS 4 ). However, the early femtosecond and picosecond responses to light irradia tion provide only limited information, as multiple cellular targets make it difficult to distinguish the critical events of PDT that lead to cell death. To understand the molecular basis of cancer cell death through PDT, time resolved spectroscopy methods must be combined with other sensitive techniques that are capable of elucidating the driving forces preceding formation of ROS (reactive oxygen species) from the excited photosensitizer. The sensitive techniques, such Raman and fluorescence imaging, are useful for photosensitizer localization and mapping cellular events during and after photosensitization [START_REF] Abramczyk | Oncologic photodynamic diagnosis and therapy: confocal Raman/fluorescence imaging of metal phthalocyanines in human breast cancer tissue in vitro[END_REF][START_REF] Brozek-Pluska | Distribution of phthalocyanines and raman reporters in human cancerous and noncancerous Breast tissue as studied by raman imaging[END_REF]. It has been shown that the degree of photosensitizer aggregation affects localization and distribution [START_REF] Dumoulin | Synthetic pathways to watersoluble phthalocyanines and close analogs[END_REF][START_REF] Morgan | Mitochondria-based photodynamic anti-cancer therapy[END_REF]. PDT initiates three types of programmed cell death (PCD): apoptosis, necrosis or autophagy. The exact mecha nism underlying the induction of PCD remains unknown, but it seems that ROS generated from the excited photosensitizer are the driving force underlying these events [START_REF] Zawacka-Pankau | The p53-mediated cytotoxicity of photodynamic therapy of cancer[END_REF][START_REF] Piette | Cell death and growth arrest in response to photodynamic therapy with membrane bound photosensitizers[END_REF].

The detailed mechanisms of photosensitizer localization have been previously discussed [START_REF] Abramczyk | Ultrafast dynamics of metal complexes of tetrasulfonated phthalocyanines at biological interfaces: comparison between photochemistry in solutions, films, and noncancerous and cancerous human Breast tissues[END_REF][START_REF] Morgan | Mitochondria-based photodynamic anti-cancer therapy[END_REF][START_REF] Piette | Cell death and growth arrest in response to photodynamic therapy with membrane bound photosensitizers[END_REF]. The initial subcellular localization of each photosensitizer depends on many factors, such as charge, hydrophobicity and plasma protein binding affinity. Based on the charge and hydrophilic or lipophilic properties, the photosensitizer can be localized in the various cytoplasmic membranous structures of certain types of cells or compartments, such as a membrane surfaces, endosomal compartments, organ elles and cytoplasm. Briefly, the charge determines the anionic, cationic, amphiphilic or neutral characteristics of photosensitizers and plays an important role in the cellular uptake and photody namic efficacy of PDT [START_REF] Kobayashi | Cation or solvent-induced supermolecular phthalocyanine formation: crown ether substituted phthalocyanines[END_REF]. One of the most important types of interactions between photosensitizers and membrane lipids are electrostatic interactions. The higher efficiency of binding of water soluble tetrasubstituted cationic aluminum phthalocyanine to phospholipid membranes compared to the anionic tetrasulfonated aluminum and zinc phthalocyanine complexes has been proven [START_REF] Kane | The nuclear magnetic resonance spectra and the electronic spectra of some silicon and germanium phthalocyanines[END_REF]. This higher efficiency can be easily explained by electrostatic interactions of the photosensitizer with negatively charged lipids contained in the membrane [START_REF] Dhami | Photophysical characterization of sulfonated aluminum phthalocyanines in a cationic reversed micellar system[END_REF]. The hydrophilic or lipophilic properties of the photosensitizers are related to their structure, which regulates aggregation and the efficiency of singlet oxygen production [START_REF] Dumoulin | Synthetic pathways to watersoluble phthalocyanines and close analogs[END_REF]. Among the hydrophilic photosensitizers, anionic derivatives with sulfo substituents (Fig. 1), such as ZnPcS 4 or AlPcS 4 are one the best photosensitizers. It has been reported that the high lipophilicity correlates with high cancer affinity of the photosensitizer, while high hydrofility correlates with high phototoxicity of the photosensitizer [82]. Moreover, it has been shown that anionic phthalocyanines have higher selectivity in binding process than cationic phthalocyanines and some cationic compounds can be used in targeted therapy for destroying specific subcellular organelles such as mitochondria [88 92]. Although the cellular mechanisms of the mitochondrial pathway involved in cancer cell elimination through photodynamic therapy remain largely unclear, several studies on the mechanism of PDT induced apoptosis have suggested the involvement of pro and anti apoptotic proteins, e.g., Bcl 2, Bax, Bcl XL, and most importantly p53 [START_REF] Zawacka-Pankau | The p53-mediated cytotoxicity of photodynamic therapy of cancer[END_REF][START_REF] Piette | Cell death and growth arrest in response to photodynamic therapy with membrane bound photosensitizers[END_REF][START_REF] Crescenzi | Photodynamic therapy with indocyanine green complements and enhances low-dose cisplatin cytotoxicity in MCF-7 breast cancer cells[END_REF]. The tumor suppressor protein p53 is critically involved in defense against genome alterations resulting from DNA damage. Although the precise role of p53 remains elusive, it has recently been well documented that p53 confers a crucial barrier for cancer progression, as p53 inactivation during tumorigenesis occurs with high frequency via multiple mechanisms [START_REF] Zawacka-Pankau | The p53-mediated cytotoxicity of photodynamic therapy of cancer[END_REF][START_REF] Piette | Cell death and growth arrest in response to photodynamic therapy with membrane bound photosensitizers[END_REF].

The significant progress made since the mid 1990s in cancer detection and PDT therapy correlates with the development of selective photosensitizers localized to specific sites of cells and tissues such as plasma membrane, nuclei, mitochondria, and lysosomes [START_REF] Ruoslahti | Synthesis of a new water-soluble octa-cationic phthalocyanine derivative for PDT[END_REF][START_REF] Peng | Targeting of bioactive compounds to mitochondria[END_REF]. The high specificity of these new photo sensitizers can be enhanced by using conjugated antibodies that recognise and bind to cancer cells [ 96 98]. The targeting approach combining nanoparticles with laser spectroscopy and Raman imaging can potentially revolutionize cancer diagnosis and therapy [START_REF] Abramczyk | Raman imaging in biochemical and biomedical applications. diagnosis and treatment of Breast cancer[END_REF][START_REF] Peng | Targeting of bioactive compounds to mitochondria[END_REF]. In the present study, we focused on the biological interfaces after suspension and adsorption of photosensitizer in a biological medium of noncancerous and cancerous human breast tissues. Tetrasulfonated zinc phthalocyanine (Fig. 2) was selected as a hydrophilic and anionic photosensitizer to identify the primary processes occurring upon excitation of the chromophore. We examined the vibrational properties and dynamics of sulfonated zinc phthalocyanine at the biological interface of the human breast tissues in a time window ranging from femto seconds to nanoseconds. The results were compared with the dynamics of the photosensitizer in the film on the glass support and in aqueous solutions. Understanding mechanisms of energy dissipation at the biological interface is a key challenge of photochemistry of photosensitizers in vivo. Although photochem ical properties of photosensitizers in liquid solutions has been well documented [49 58]. We have shown [START_REF] Abramczyk | Ultrafast dynamics of metal complexes of tetrasulfonated phthalocyanines at biological interfaces: comparison between photochemistry in solutions, films, and noncancerous and cancerous human Breast tissues[END_REF] that bulk solution properties of photosensitizers at biological interfaces cannot be translated to the solid phase. Therefore, the photochemistry of photosensitizers must be significantly revised when considering biological interfaces. A limited number of papers have reported on the dynamics of the primary events upon light excitation in thin phthalocyanines films [12,15,60,65,73,75,80,99 105].

Although the primary PDT mechanism has been associated with the selective accumulation of photosensitizers in cancer tissue, it remains completely unclear whether the photochemical pathways of energy dissipation upon light excitation are the same in the noncancerous and cancerous tissues. The answer to this long standing question, representing a key challenge in clinical applications, can be elucidated using the time resolved pump probe method to monitor pathways of photodynamic reactions in regions of noncancerous and cancerous cells.

The aim of the present study was to elucidate the processes responsible for the ultrafast dynamics of ZnPcS 4 at biological interfaces of the human noncancerous and cancerous tissues occurring on a time scale from femtoseconds to nanoseconds by using the pump probe transient absorption spectroscopy. The ultrafast dynamics was induced through femtosecond laser pulses centered at 674 and 633 nm corresponding to the absorption maxima of the Q band of the monomer and dimer, respectively.

Experimental section

Phthalocyanine

Zinc phthalocyanine tetrasulfonic acid, tetrasodium salt (ZnPcS 4 ) was prepared using a method similar to that of Griffiths et al. [START_REF] Griffiths | Some observations on the synthesis of polysubstituted zinc phthalocyanine sensitisers for photodynamic therapy[END_REF]. More details about the synthesis can be found elsewhere [START_REF] Abramczyk | Ultrafast dynamics of metal complexes of tetrasulfonated phthalocyanines at biological interfaces: comparison between photochemistry in solutions, films, and noncancerous and cancerous human Breast tissues[END_REF]. Scheme 1 illustrates the method used for the synthesis of ZnPcS 4 .

This method generated a single regioisomer, presented in Fig. 3. Fig. 3 shows the structure of the metal phthalocyanine, zinc (II) phthalocyanine 3,4 0 ,4 00 ,4 000 tetrasulfonic anion.

Steady state UV vis absorption measurements

UV vis absorption electronic spectra were recorded with a Varian Cary 5E spectrophotometer using 2 and 0.10 AE 0.005 mm detachable quartz cells (Hellma). The spectra were recorded at 293 K for the aqueous solutions at concentration of c = 10 3 M, as well as ZnPcS 4 films on BaF 2 supports and the thin sections of 16 mm of cancerous and noncancerous human breast tissue samples stained with ZnPcS 4 .

Fig. 2. Hydrophilic and anionic character of ZnPcS 4 .

Pump probe transient absorption spectroscopy

The source of femtosecond pulses was a mode locked titanium sapphire femtosecond laser (MIRA, Coherent, 800 nm, 76 MHz, 9 nJ, <200 fs) pumped with iode pumped solid state laser (VERDI V5, Coherent, 532 nm). The fundamental beam was amplified with Ti: Sapphire regenerative amplifier (Coherent Legend USP, 800 nm, 1 kHz, 3 mJ, 50 fs). The regenerative amplifier was pumped with diode pumped Nd:YLF laser (JADE, Thales Laser, 527 nm, 1 kHz, 20 mJ, <200 ns). The pulse was split in two and further amplified to a dual single pass amplifier (Coherent Elite Duo, 800 nm, 1 kHz, 2 Â 4.5 mJ, 50 fs). This amplifier was pumped through high power Nd:YLF laser (Evolution, Coherent, 527 nm, 50 mJ, <200 ns). The output of the laser system was split and two 1 mJ laser pulses were used to pump two optical parametric amplifiers (OPA, model TOPAS from Light Conversion). These OPA, combined through frequency conversion modules, generated femtosecond pulses tunable in the 300 and 2600 nm ranges. More details about the femtosecond laser setup can be found in Ref. [START_REF] Abramczyk | Ultrafast dynamics of metal complexes of tetrasulfonated phthalocyanines at biological interfaces: comparison between photochemistry in solutions, films, and noncancerous and cancerous human Breast tissues[END_REF].

Patients and samples

We examined human ductal and lobular carcinoma (in situ and infiltrating) and various benign changes, including benign dysplastic and neoplastic lesions [START_REF]Sigma-Aldrich Mayers Hämatoxylin-Lösung Verfahrens Nr[END_REF][START_REF] Surmacki | The lipid-reactive oxygen species phenotype of breast cancer. Raman spectroscopy and mapping, PCA and PLSDA for invasive ductal carcinoma and invasive lobular carcinoma. Molecular tumorigenic mechanisms beyond Warburg effect[END_REF]. Raman spectroscopy and Raman imaging have been previously used to analyze breast cancer specimens [ 109 111]. The breast tissue samples were obtained during a surgical operation. The research did not affect the course of the operation or treatment of the patients. All procedures were conducted using the protocol approved through the institutional Bioethical Committee at the Medical University of Lodz, Poland (RNN/30/11/KE). The detailed description of the sample preparation procedure can be found in Ref. [START_REF] Abramczyk | Ultrafast dynamics of metal complexes of tetrasulfonated phthalocyanines at biological interfaces: comparison between photochemistry in solutions, films, and noncancerous and cancerous human Breast tissues[END_REF].

Infrared spectroscopy

IR spectra were recorded using a Specord M 80, Germany. Specord M80 is a double beam spectrometer for the measurement of lead 4000 200 cm 1 (2.5 50 mm) with an accuracy ranging from +/ 0.8 cm 1 to +/ 0.3 cm 1 , depending on the spectral Scheme 1. Method used for the synthesis of ZnPcS 4 via the condensation of the sulfonated precursors in the presence of urea, the selected metal salt and a catalyst. range. The spectra were scanned with a 4 cm 1 step and recorded in a 800 4000 cm 1 range at 293 K.

Results and discussion

To learn about the properties of the photosensitizers at the interface of cancerous and noncancerous tissues and the corre sponding biochemical components of the tissue that form biological environment for the photosensitizers we employed a broad range of spectroscopic methods.

In this section, the results for the photosensitizer (ZnPcS 4 ) at the interface of the noncancerous and cancerous human breast tissues using various methods, such as Raman, IR, and UV vis steady state spectroscopy methods and time resolved transient electronic absorption through the pump probe femtosecond spectroscopy will be presented.

Fig. 4 presents the histological images of the tumor mass (cancerous tissue) and tissue from the safety margin (noncancer ous tissue) of the same patient P95. The samples obtained from this patient have also been used to record the Raman and IR spectra to analyze the biochemical composition of cancerous and noncan cerous human breast samples.

The histological images were obtained by staining with hematoxylin and eosin (H&E). H&E staining facilitates the assignment of various structures in tissues and single cells [START_REF]Sigma-Aldrich Mayers Hämatoxylin-Lösung Verfahrens Nr[END_REF]. Hematoxylin stains all basophilic components blue, particularly the nucleus, containing DNA and RNA, and the rough endoplasmic reticulum as a result of the coordinated bonding between aluminum and the phosphor atoms in DNA and RNA [START_REF] Brozek-Pluska | Raman spectroscopy and imaging: applications in human breast cancer diagnosis[END_REF]. Eosin stains cytoplasm, connective tissue and collagen fibers (eosinophilic substances) red due to the ionic bonds between the anionic dye and cationic plasma proteins.

To understand the details of the histological images presented in Fig. 4, we will discuss the basic information on the morphology of the human breast. The human breast consists of 6 10 major duct systems. Each system contains numerous lobules, which terminate ducts. Adipose tissue and supporting stroma surrounds these ducts and lobules. The disease develops in the epithelial cells of the ducts (ductal carcinoma) and lobules (lobular carcinoma). These tissues contain no blood vessels; thus, nourishment is provided via the diffusion of substances from the connective tissue surrounding the epithelial structures [113]. These structures can be easily seen in the histological H&E images presented in Fig. 4. The epithelial cells are surrounded by the stroma, which predominantly, but not exclusively, comprises connective tissue (type I collagen) (Fig. 4a) and adipose tissue (Fig. 4b). The surface of the cancerous tissue is hydrophilic [START_REF] Brozek-Pluska | Phase transitions in oleic acid and in human breast tissue as studied by Raman spectroscopy and Raman imaging[END_REF], and the level of various proteins is markedly elevated during disease development. In contrast, the noncancer ous breast tissue consists of fatty tissue and is hydrophobic, as this tissue primarily comprises glycerol monooleate [START_REF] Brozek-Pluska | Phase transitions in oleic acid and in human breast tissue as studied by Raman spectroscopy and Raman imaging[END_REF].

Steady state Raman scattering

To obtain insight into the vibrational properties of the tissue constituents at biological interfaces at the molecular level and characterize the nature of these interactions, we analyzed the Raman vibrational features in the noncancerous and the cancerous tissues. Fig. 5 shows the typical Raman spectra of the noncancerous and cancerous human breast tissues of patient P95, with the tentative assignments of major vibrational bands.

The results show that the Raman spectra contain multiple contributions from proteins, lipids, and nucleic acids, with contributions from individual RNA and DNA bases (adenine, thymine, guanine, cytosine, and uracil) and the sugar phosphate backbone of DNA. A detailed inspection of Fig. 5a andb revealed that the Raman spectra are characteristic of adipose rich regions, phospholipid rich regions (Table 1), disordered collagen regions (Table 2), collagen poor regions (Table 3).

The results shown in Fig. 5 highlight differences between the Raman spectra of the noncancerous breast tissue from the safety margin (Fig. 5a) and the cancerous breast tissue from the tumor mass (Fig. 5b). Comparing the spectra in Fig. 5a andb one can notice that the noncancerous human breast tissue contains Raman peaks at 2854, 2888, and 2926 cm 1 typical for glycerol mono oleate derivatives [12 14,120,121]. These peaks are not observed in the Raman spectrum of the cancerous tissue for which a protein peak at 2938 cm 1 is characteristic. One can also see from Fig. 5 that the strong signals at 1158 cm 1 and 1518 cm 1 in the Raman spectrum of the noncancerous tissue are clearly visible. These peaks originate from carotenoids, which are not observed in the cancerous human breast tissue. It indicates that the noncancerous human breast tissue contains a higher amount of glycerol monooleate derivatives typical for adipose tissue, which act as reservoir of carotenoids and fatty acids. The third most important difference between the noncancerous and cancerous human breast tissues is observed in the Raman spectra of the Amide I. The maximum peak position of Amide I has been shifted from 1660 cm 1 in the cancerous tissue to 1654 cm 1 in noncancerous tissue. This effect must be associated with the distinct biological environment, potentially related to the disordered and alpha helical protein structures, respectively. Many distinctions can also be observed in the region of 2800 3000 cm 1 typical for lipid/ protein vibrations, where the contribution from the monounsatu rated fatty acids, common constituents of triglycerides of the adipose tissue, dominates the Raman spectrum of the noncancer ous tissue in contrast to the Raman spectra of the cancerous tissue. We have shown that the Raman spectra of the cancerous tissue demonstrate a protein like profile [12 14,118,121 123]. This finding is consistent with the knowledge that in contrast to noncancerous cells, abnormal cells divide in an uncontrolled process of cell growth and synthesize large amounts of proteins.

Tables 1 3 show the strongest Raman signals for the noncancerous and the cancerous tissues and the tentative assignments. Carotenoids [START_REF] Parker | Applications of Raman and Resonace Raman Spectroscopy in Biochemistry[END_REF] (C C) str.

1655-1680

Proteins Amide I/Unsaturated fatty acids [START_REF] Brozek-Pluska | Raman spectroscopy and imaging: applications in human breast cancer diagnosis[END_REF][START_REF] Verier | In situ monitoring of cell death using Raman microspectroscopy[END_REF] a helix, (C-N) str., (C-H) def./(C C) str. [START_REF] Brozek-Pluska | Raman spectroscopy and imaging: applications in human breast cancer diagnosis[END_REF][START_REF] Verier | In situ monitoring of cell death using Raman microspectroscopy[END_REF] 2854 Fatty acids, triglicerides [113] (C-H 2 ) sym. str. 2888

Lipids [START_REF] Verier | In situ monitoring of cell death using Raman microspectroscopy[END_REF] (C-H 2 ) antisym. str. 2926

Proteins/Lipids [113,[START_REF] Verier | In situ monitoring of cell death using Raman microspectroscopy[END_REF] (CH 3 ) antisym. str. 2935

Proteins/Lipids [START_REF] Verier | In situ monitoring of cell death using Raman microspectroscopy[END_REF] (CH 3 ) sym. str. 3008

Lipids [START_REF] Brozek-Pluska | Raman spectroscopy and imaging: applications in human breast cancer diagnosis[END_REF][START_REF] Verier | In situ monitoring of cell death using Raman microspectroscopy[END_REF] ( CÀ ÀH) str.

Abbreviations: (bend.) bending, (wag.) wagging, (def.) deformation, (tw.) twist, (sym.) symmetric, (antisym.) antisymmetric, and (str.) stretch.

Steady state IR absorption

To obtain complementary information concerning the vibra tional properties of the noncancerous and cancerous tissues, it would be interesting to compare the Raman spectra with IR spectra. Fig. 6 shows the typical IR spectra for the cancerous breast tissue of the same patient (P95) shown in Fig. 5. The results show that the absorbance of various vibrations differs markedly, particularly in the region of Amide I and fatty acids (1642, 1651, 1670, and 1686 cm 1 ).

Table 4 presents the tentative assignments of major vibrational bands of IR spectra for the cancerous (infiltrating ductal carcinoma G2) and the noncancerous breast tissues of patient P95.

A detailed analysis of the IR and Raman spectra of the human breast tissue is beyond the scope of this paper, and additional report directly related to this issue will be prepared in future studies.

Steady state electronic absorption measurements

To obtain information concerning the electronic features of the photosensitizer in the noncancerous and cancerous tissues we have compared the electronic spectra of ZnPcS 4 in various environments.

Fig. 7a shows the steady state electronic absorption spectra of ZnPcS 4 in water, in film on a glass support, and at the biological interface of noncancerous and cancerous human breast tissues. The results show that sulfonated zinc phthalocyanine absorbs in the 600 680 nm region, known as the Q band, corresponding to a p p* ring transition [START_REF] Leznoff | Phthalocyanines[END_REF]. Another intensity band, due to electronic transitions, consistent with the B (or Soret band), is observed at 335 nm. A maximum at around 679 nm (Fig. 7a) has been attributed to the Q band ((a 2u ) ! (e g ) transition) of ZnPcS 4 monomers in aqueous solution. Recently we have shown [5] that the absorption band shape and the intensity change in the concentration range of 10 6 10 2 M, indicating that aggregation of ZnPcS 4 molecules in aqueous solution occurs. The band at 633 nm in Fig. 7a has been assigned to a dimer or higher aggregates. As for most metallophthalocyanines, the ZnPcS 4 dimers with a cofacial dimer geometry [125 127] show absorption that is blue shifted relative to the monomer absorption band at 679 nm. The face to face aggregation, reflecting the extensive interaction between the Proteins Amide I/Unsaturated fatty acids [START_REF] Brozek-Pluska | Raman spectroscopy and imaging: applications in human breast cancer diagnosis[END_REF][START_REF] Verier | In situ monitoring of cell death using Raman microspectroscopy[END_REF] a helix, (C-N) str., (C-H) def./C C str.

Abbreviations: (wag.) wagging, (tw.) twist, (sym.) symmetric, (antisym.) antisymmetric, and (str.) stretch.

Fig. 6. Comparison between the IR spectra of the cancerous (infiltrating ductal carcinoma G2) and noncancerous breast tissues of patient P95 at 0% humidity. p systems of adjacent rings [START_REF] Kasha | Energy transfer mechanisms and the molecular exciton model for molecular aggregates[END_REF], is stabilized through hydrogen bonding in tetrasulfonated ZnPcS 4 [START_REF] Barbosa | mora, Identification of mutations in two major mRNA isoforms of the Chediak-Higashi syndrome gene in human and mouse[END_REF].

In contrast to the solutions, the absorption spectra at the biological interface of the tissues and in the film presented in Fig. 7a exhibit broad, structureless bands characteristic for the absorption of monomers, dimers, and higher order aggregates.

Assuming the thickness of the ZnPcS 4 layer for about 100 mm we have estimated the concentration of the photosensitizer in the tissues to be around 10 2 M.

For comparison, in Fig. 7b we showed the steady state absorption spectra of AlPcS 4 in water solution, in a film, and at the biological interface of noncancerous and cancerous human breast tissues. AlPcS 4 does not to aggregate in either aqueous or organic solvents [START_REF] Renoit | Q-band splitting and relaxation of aluminum phthalocyanine tetrasulfonate[END_REF]. The sharp band at around 679 nm has been assigned to monomeric species, and the weak band at 602 nm to the vibronic band.

Excited state dynamics of ZnPcS4 at biological interfaces of the human cancerous and noncancerous breast tissues, films and in aqueous solutions. S0 ! S1 transition (Q band transition).

Upon absorbing a photon, a molecule is promoted to higher energy states and very quickly relaxes back to ground state through radiative or non radiative (heat) pathways. All these pathways can be depicted in the Jablonski diagram [START_REF] Lakowicz | Principles of Fluorescence Spectroscopy[END_REF].

When radiative path is chosen, profound chemical rearrange ments occur leading to dangerous photoreactions in living cells, thereby reducing photostability protection. In contrast in fast non radiative processes no profound chemical or structural rearrange ments can occur [START_REF] Abramczyk | Mechanisms of energy dissipation and ultrafast primary events in photostable systems: H-bond, excess electron, biological photoreceptors[END_REF]. This pathway of ultrafast nonradiative decay can be observed in many biological systems [START_REF] Abramczyk | Mechanisms of energy dissipation and ultrafast primary events in photostable systems: H-bond, excess electron, biological photoreceptors[END_REF][START_REF] Satzger | Reassignment of the low lying cationic states in gas phase adenine and 9methyl adenine[END_REF][START_REF] Rode | Photophysics of inter-and intra-molecularly hydrogen-bonded systems: computational studies on the pyrrole-pyridine complex and 2 (2 0 -pyridyl) pyrrole[END_REF].

To elucidate additional electronic dynamics from the signals of the transient absorption DA(t), we need to understand the details of the electronic transitions in zinc phthalocyanines, which are induced and probed through the laser pulses in the pump probe experiments.

To monitor dynamics upon phthalocyanine excitation the time evolution of transient absorption signals were monitored with a time resolution of 50 fs.

To obtain insight into the mechanisms of energy dissipation, we monitored the dynamics of ZnPcS 4 at the interfacial region of human breast tissues upon excitation of the S 0 ! S 1 transition in the Q band at 674 nm (maximum absorption of monomer) and at 633 nm (maximum absorption of dimer) and probing at different wavelengths (570 nm, 633 nm, 674 nm) corresponding to the excited state, dimer, and monomer absorption spectra, respective ly. Fig. 8 shows the transient absorption signals DA(t) of ZnPcS 4 at the interface of the cancerous tissue, noncancerous tissue, in the film, and in solution as a function of the time delay in the pump probe experiments when pumped with 674 nm and probed with 674 nm. The results are presented in the full time window up to 10 ps (Fig. 8a) and in the narrower windows up to 100 ps (Fig. 8b) and up to 1 ns (Fig. 8c).

The results shown in Fig. 8 indicate that directly upon excitation at 674 nm, a negative transient absorption DA(t) signal at 674 nm was recorded. The negative signal was assigned to the bleaching of the ground state due to the HOMO(a 1u ) ! LUMO(e g ) transition in the Q band for monomer ZnPcS 4 molecules. The bleaching is instantaneous and limited by the laser pulse width. The recovery at 674 nm was three exponential and fitted with time constants of 1.7 AE 0.4 ps, 24.4 AE 3.1 ps and 59.3 AE 4.4 ps for the noncancerous tissue and 8.3 AE 1.2 ps, 17.8 AE 1.7 ps, and 42.2 AE 1.6 ps for the cancerous tissue. The recovery in the ZnPcS 4 film was also three exponential, with the time constants 1.8 AE 0.2 ps, 13.1 AE 0.8 ps, and 28.4 AE 1.8 ps. These results indicate that the dynamics of the ground state recovery are different for the noncancerous and cancerous tissues, particularly the shortest time constants (1.7 ps vs 8.3 ps).

The recovery of ZnPcS 4 bleaching in solution at 674 nm achieved the best fit, showing three exponential functions with time constants of 11.1 AE 0.9 ps, 25.9 AE 0.5 ps, and 307 AE 79 ps. It is important to stress that the dynamics in the solution contains a slow component of 307 ps that has not been observed neither in the tissues nor in the film. Fig. 9 shows the transient absorption signal DA(t) of ZnPcS 4 in the noncancerous and cancerous human breast tissues, ZnPcS 4 film and aqueous solution as a function of the time delay when pumped at 633 nm and probed at 633 nm.

Fig. 9 shows that directly upon excitation with the laser pump pulse at 633 nm, a negative DA(t) signal at 633 nm was recorded.

The negative signal was assigned to the bleaching of the ground state due to the HOMO(a 1u ) ! LUMO(e g ) transition in the Q band for ZnPcS 4 dimer. The bleaching was instantaneous and limited by the laser pulse width. The recovery of the bleaching at 633 nm was three exponential and fitted with time constants of 1. Lipids C O str. [113,[START_REF] Notingher | Raman spectroscopy cell-based biosensors[END_REF][START_REF] Verier | In situ monitoring of cell death using Raman microspectroscopy[END_REF][START_REF] Lasch | Imaging of colorectal adenocarcinoma using FT-IR microspectroscopy and cluster analysis[END_REF] Abbreviations: (bend) bending, (wag) wagging, (def.) deformation, (tw) twist, (sym) symmetric, (antisym) antisymmetric, and (str) stretch.

The recovery in the ZnPcS 4 film was also three exponential, with the time constants 1.5 AE 0.1 fs, 9.5 AE 0.6 ps, and 19.8 AE 1.1 ps. These results indicate that the dynamics of the ground state S 0 recovery is different in the noncancerous and the cancerous tissues. The recovery of ZnPcS 4 bleaching in solution at 633 nm when pumped at 633 nm was three exponential, with the time constants 12.1 AE 0.7 fs, 72.8 AE 14.1 ps, and 116 AE 27 ps. Tables 5 and6 summarize the characteristic time constants for the dynamics of the ground state by monitoring the DA(t) signal of bleaching recovery for the monomeric species at 674 nm and the dimer species at 633 nm. The results presented in Figs. 8 and9 clearly demonstrate that the photochemical properties of photosensitizers in solutions cannot be directly translated to the biological interfaces. The comparison of the time constants shown in Tables 5 and6 demonstrates that the mechanisms of energy dissipation for phthalocyanines in bulk solutions and at biological interfaces are dramatically different. First, the dynamics of ZnPcS 4 molecules at the biological interface of noncancerous tissue contains a very fast component of around 1 ps, which does not exist in bulk solutions.

Second, the dynamics of ZnPcS 4 in solution contains the slow component of hundreds of picoseconds, which is not observed at biological interfaces of the tissues. The time constants in solution are consistent with those obtained by Howe et al. [START_REF] Howe | Ultrafast studies of excited-state dynamics of phthalocyanine and zinc phthalocyanine tetrasulfonate in solution[END_REF] for PcS 4 and ZnPcS 4 in DMSO probed at 720, 790, and 820 nm, but the very fast component of around 1 ps at biological interfaces is a new feature suggesting a different channel of energy dissipation.

To gain further insight into the dynamics of ZnPcS 4 we monitored the time evolution of the decay signal in the region of the excited state absorption (ESA) from S 1 to higher S n states. Fig. 10 shows the ESA transient absorption signal DA(t) of ZnPcS 4 in noncancerous and cancerous human breast tissues, in film and in aqueous solution (concentration 10 3 M) as a function of the time delay in the full time window up to 10 ps (10a), 100 ps (10b) and 1 ns (10c), pumped at 674 nm and probed at 570 nm. Fig. 10 shows that the instantaneous bleaching of the negative signal of ZnPcS 4 in the tissues, film, and water solution at 674 nm presented in Figs. 8 and9 is accompanied by a sudden rise at 570 nm, followed by decay. This decay is three exponential and was fitted with time constants of 0.98 AE 0.1 ps, 1.6 AE 0.2 ps, It is obvious that the distinct dynamics of ZnPcS 4 in the noncancerous and the cancerous tissues must be associated with distinct biological environment.

It is worth emphasizing that the dynamics of ZnPcS 4 in the ground and the excited state at biological interfaces reveals three different time scales: a) relatively fast component ranging around 1 ps, b) a component of a few picoseconds (8 19) and c) relatively slow component of a few tens of picoseconds (21 72 ps). In contrast, the dynamics of ZnPcS 4 in solutions exhibits a very slow component in the range of a few hundred picoseconds (108 307 ps) which is not observed at the biological interface of the tissue. The slow component was previously reported for many phthalocyanines in solutions (232 ps [START_REF] Jarota | Ultrafast dynamics of metal complexes of tetrasulphonated phthalocyanines[END_REF], 2.9 ns [START_REF] Savolainen | Characterizing the functional dynamics of zinc phthalocyanine from femtoseconds to nanoseconds[END_REF], 460 [START_REF] Howe | Ultrafast studies of excited-state dynamics of phthalocyanine and zinc phthalocyanine tetrasulfonate in solution[END_REF]).

The longest time constants observed in ZnPcS 4 solutions in the range of 108 307 ps were assigned to the radiative decay via fluorescence from S 1 to the ground state.

The most important question that arises after detailed analysis of the energy dissipation channels is why dynamics of photosen sitizer differs so dramatically when studied in solutions and at biological interfaces. One must be noticed that the interactions between the photosensitizer and the tissues seem to be evidently governed by the hydrophobic or hydrophilic properties of the biological interfaces. Fig. 4a shows that the noncancerous human breast tissue is dominated by adipose tissue, embedded ducts and blood vessels. Adipose tissue is hydrophobic as it contains glycerol derivatives (monooleate derivatives [START_REF] Brozek-Pluska | Phase transitions in oleic acid and in human breast tissue as studied by Raman spectroscopy and Raman imaging[END_REF]). In contrast, the cancerous human breast tissue is dominated by epithelial cells of the ducts, where the cancer occurs (Fig. 4a) The surface of the this type of tissue is hydrophilic [START_REF] Brozek-Pluska | Phase transitions in oleic acid and in human breast tissue as studied by Raman spectroscopy and Raman imaging[END_REF] and contains a high amount of various proteins. Recently we have also proved that water amount confined in cancerous tissue is markedly higher than that in noncancerous tissue [START_REF] Brozek-Pluska | Phase transitions in oleic acid and in human breast tissue as studied by Raman spectroscopy and Raman imaging[END_REF]. These features of the tissues can explain why the hydrophilic zinc phthalocyanine (ZnPcS 4 ) interacts with the hydrophilic environment of the cancerous breast tissue and exhibits dynamics more similar to the dynamics in aqueous solution than that at the of hydrophobic interface of the noncancerous breast tissue.

Conclusions

The paper focuses on issues that may open up new horizons and opportunities for understanding the complex spatio temporal interactions of molecules at biological interfaces in realistically crowded environment of the human cells and tissues. Despite of several model studies that have been published so far, no ultrafast dynamics of human tissue has been reported yet. We have found spectacular differences in the dynamics of photosensitizers in the noncancerous and cancerous breast human tissues. So far, the cancer pathology has been derived from the static properties of tissues and the dynamic behavior has not been used to discriminate between the noncancerous and cancerous tissues. In this paper we examined the ground and the excited state dynamics of a photosensitizer (ZnPcS 4 ) at the biological interface of the human breast tissue by the pump probe transient absorption femtosecond spectroscopy to obtain information concerning the events occurring in a time scale ranging from femtoseconds to nanoseconds.

First, the results showed that the dynamics of the photosensi tizer was markedly faster in the interfacial regions of the biological tissue than in solutions. Second, the photosensitizer localized in noncancerous tissue dissipates the energy through different pathways than that in cancerous breast tissue. We have shown that the lifetimes characterizing both the ground state S 0 and the first excited state S 1 in the interfacial regions of noncancerous tissue are markedly shorter than those in cancerous tissue.

Summarizing our study proved that femtosecond spectroscopy open new capabilities in cancer biology and perfectly fit into the research on functions of biologically active molecules and understanding of light energy dissipation. Our studies have shown that for phthalocyanines as for photosynthesis, the reactions of the photoreceptors rhodopsin, bacteriorhodopsin, photoactive yellow protein, phytochrome, and flavin chromophores femtosecond spectroscopy is a unique technique to study ultrafast processes activated by light collection and molecular mechanisms responsi ble for harvesting the light energy can be used for practical applications in cancer diagnostics.

Fig. 1 .

 1 Fig.1. Anionic and cationic zinc photosensitizers.

Fig. 3 .Fig. 4 .

 34 Fig. 3. Structure of the metal phthalocyanine, zinc(II)phthalocyanine-3,4 0 ,4 00 ,4 000tetrasulfonic anion.

Fig. 5 .

 5 Fig. 5. Raman spectra of the noncancerous (a) and cancerous (infiltrating ductal carcinoma G2) (b) breast tissues from patient P95, using a 16-mm layer of tissue on BaF 2 window.

Fig. 7 .

 7 Fig. 7. a) Absorption spectra of ZnPcS 4 in noncancerous tissue (a), cancerous tissue (b), film (c) and aqueous solution c = 10 3 M (d); b) Absorption spectra of AlPcS 4 in noncancerous tissue (a), cancerous tissue (b), film (c) and aqueous solution c = 10 3 M (d) 18.

Fig. 8 .

 8 Fig. 8. Transient absorption signal DA(t) of ZnPcS 4 in noncancerous (~), cancerous (&) human breast tissues, film (*) and aqueous solution (concentration 10 3 M) (^) as a function of the time delay in the full time window up to 10 ps (a), 100 ps (b) and 1 ns (c), pumped at 674 nm and .probed at 674 nm.

Fig. 9 .

 9 Fig. 9. Transient absorption signal DA(t) of ZnPcS 4 in noncancerous (~), cancerous (&) human breast tissues, film (*) and aqueous solution (concentration 10 3 M) (^) as a function of the time delay in the full time window up to 10 ps (a), 100 ps (b) and 1 ns (c), pumped at 633 nm and probed at 633 nm.

Fig. 10 .

 10 Fig. 10. Transient absorption signal DA(t) of ZnPcS 4 in noncancerous (~), cancerous (&) human breast tissues, film (*) and aqueous solution (concentration 10 3 M) (^) as a function of the time delay in the full time window up to 10 ps (a), 100 ps (b) and 1 ns (c), pumped at 674 nm and probed at 570 nm.

Table 1

 1 Characteristic Raman peaks for noncancerous and cancerous breast tissues of adipose-rich regions, phospholipid-rich regions.

	Wavenumber/cm 1	Tentative assignments
	729	Nucleic Acids, Phospholipid (choline) [112-114]
	1004	Carotenoids/Phenylalanine [115]
		(C-C) str.
	1064	Lipids/Collagen [112,113]
		(C-C) str.
	1158	Carotenoids (C-C str.)/Proteins (C-C/C-N str.) [112-116]
	1220-1285	Nucleic Acids (T, A)/Proteins (Amide III), Lipid, phospholipid CÀ ÀH bend [112,113]
	1304	Lipids, phospholipids [112]
		(C-H 2 ) tw
	1444	Lipids/Proteins [115,117]
		(C-H) wag.
	1528

Table 3

 3 Characteristic Raman peaks for noncancerous and cancerous breast tissues of collagen-poor regions.

	Wavenumber/cm 1	Tentative assignments
	838	Nucleic acids, Proteins [113]

(O-P-O) antisym. str./ring br. Tyrosine 1076 Proteins/Lipids [117] (C-N) str./chain (C-C) str. 1127 Proteins/Lipids/Carbohydrates [117] (C-N) str./chain (C-C) str./(C-O) str. 1304 Lipids, phospholipids [112] (C-H 2 ) tw. 1444 Lipids/Proteins [115,117] (C-H) wag. 1655-1680

Table 2

 2 Characteristic Raman peaks for noncancerous and cancerous breast tissues of disordered collagen regions.

	Wavenumber/cm 1	Tentative assignments
	814	Collagen/RNA [112,117]
		(C-C)/(O-P-O) str.
	852	Collagen, Tyrosine [121,113]
	880	Lipids/Carbohydrates/Collagen [112]
		(C-C-N + ), (C-O-C) ring, (C-C)
	935	Hydroxyproline/Collagen backbone [113,114]
		(C-C)
	1018	Collagen [113]
		(C-N)
	1031	Collagen [113]
		(C-C)
	1125	Carbohydrates (C-O)/Proteins (C-N str.) [112,113]
	1220-1285	Nucleic Acids (T, A)/Proteins (Amide III)/Lipid, phospholipid ( CÀ ÀH bend.) [112,113]
	1444	Lipids/Proteins [115,117]
		(C-H) wag.
	1655-1680	Proteins Amide I/Unsaturated fatty acids [112,117]
		a helix, (C-N) str., (C-H) def./C C str.
	3311	Water [113]
		(OH) str.
	3060	Nucleic acids/Proteins [117]
		(C-H) aromatic

Abbreviations: (A) adenine, (T) thymine, (bend) bending, (wag.) wagging, (def.) deformation, (tw.) twist, (sym.) symmetric, (antisym.) antisymmetric, and (str.) stretch.

Table 4

 4 The tentative assignments of major vibrational bands of IR spectra for the cancerous (infiltrating ductal carcinoma G2) and noncancerous breast tissues of patient P95.

	Wavenumber/cm 1	Tentative assignments
	1096	Phospholipids, O-P-O sym. str. [113]
	1160	Carotenoids CÀ ÀC str. [115]
	1199	CÀ ÀC 6 H 5 Phe, Trp [112]
	1238	Phospholipid, O-P-O asym. str. [113]
	1220-1285	Nucleic Acids (T, A)/Proteins (Amide III), Lipid, phospholipid ( CÀ ÀH bend.) [112,113]
	1299	Lipids, phospholipids [112]
		CH 2 tw.
	1383	Lipids CH 3 sym. bend., lipids [113]
	1402	Amine CÀ ÀN str. [113]
		Lipids N + (CH 3 ) 3 sym. bend.
	1461	Phospholipids (CH 2 ) scissoring [113]
	1551	Amide II, proteins [113]
	1580	Nucleic acids [112,113]
	1642	Water OH bend [113]
	1655-1680	Proteins Amide I [112,117]
		a helix, CÀ ÀN stretch, CH deformation, Unsaturated fatty acids
		C C str. [112,117]
	1738	Lipids C O str. [113,115,117,119]
	1742

Table 5

 5 Comparison of time constants for ZnPcS 4 at the biological interfaces of noncancerous and cancerous tissues, and in the film and aqueous solution pumped at 674 nm and probed at 674 nm.

		Time constants and amplitudes
		Time constant	Amplitude	Time constant	Amplitude	Time constant	Amplitude	Time constant	Amplitude
	Noncancerous tissue	1.7 AE 0.4	À0.04 AE 0.008	24.4 AE 3.1	À0.1 AE 0.01	59.3 AE 4.4	À0.1 AE 0.006
	Cancerous tissue	8.3 AE 1.2	À0.04 AE 0.01	17.8 AE 1.7	À0.11 AE 0.1	42.2 AE 1.6	À0.15 AE 0.1
	Film	1.8 AE 0.2	À0.005 AE 2.8E-4	13.1 AE 0.8	À0.005 AE 6.7E-4	28.4 AE 1.8	À0.01 AE 5.6E-4
	Aqueous solution			11.1 AE 0.9	À0.07 AE 0.005	25.9 AE 0.5	À0.27 AE 0.01	307 AE 79	À2.92 AE 0.01

Table 6

 6 Comparison of time constants for ZnPcS 4 at the biological interfaces of noncancerous and cancerous tissues and in the film and aqueous solution pumped at 633 nm and probed at 633 nm.

		Time constants and amplitudes
		Time constant	Amplitude	Time constant	Amplitude	Time constant	Amplitude	Time constant	Amplitude
	Noncancerous tissue	1.1 AE 0.1		8.3 AE 0.9		51.2 AE 2.6
	Cancerous tissue	5.6 AE 0.4	À0.8 AE 0.2	16.1 AE 0.5	À4.4 AE 0.1	51.7 AE 3.8	À7.4 AE 2.4
	Film	1.5 AE 0.1	À0.06 AE 0.001	9.5 AE 0.6	À0.3 AE 0.003	19.8 AE 1.1	À0.5 AE 0.01
	Aqueous solution			12.1 AE 0.7	À1.4 AE 0.1	72.8 AE 14.1	À84.1 AE 0.2	116 AE 27	À0.05 AE 0.01

Table 7

 7 Comparison of the time constants of ZnPcS 4 at the biological interfaces of noncancerous and cancerous tissues and in film and solution pumped at 674 nm and probed at 570 nm. AE 0.9 ps for the noncancerous tissue and 2.7 AE 0.2 ps, 11.6 AE 0.5 ps, 37.0 AE 0.6 ps for the cancerous tissue. The results in Fig. 10 clearly demonstrate that the dynamics of ZnPcS 4 in the noncancerous tissue are markedly faster than those in the cancerous tissue. The decay of the signal in the ZnPcS 4 film was also three exponential, with the time constants 1.3 AE 0.1 ps, 11.7 AE 1.5 ps, and 49.2 AE 2.5 ps. The decay of the signal in the ZnPcS 4 aqueous solution was also three exponential, with the time constants 1.3 AE 0.3 ps, 21.6 AE 1.2 ps, and 108 AE 16 ps. Table7compares the time constants characterizing the dynamics of the excited state S 1 decay of ZnPcS 4 at biological interfaces of noncancerous and cancerous tissues and in film and aqueous solution when the monomeric species are excited at 674 nm. The results from Table7show that the dynamics of the excited state S 1 decay for the noncancerous tissue is much faster than that of the cancerous tissue. The dynamics of the noncancer ous tissue is described with three time constants (0.98 AE 0.1 ps, 1.6 AE 0.2 ps, 8.9 AE 0.9 ps) and does not contain the slow component observed in the cancerous tissue(37.0 AE 0.6 ps), film (49.2 AE 2.5 ps.) and solution (21.6 AE 1.2 ps, and 108 AE 16 ps).

		Time constants and amplitudes
		Time constant	Amplitude	Time constant	Amplitude	Time constant	Amplitude	Time constant	Amplitude
	Noncancerous tissue	0.98 AE 0.1	0.04 AE 0.009	1.6 AE 0.2	0.03 AE 0.01	8.9 AE 0.9	0.01 AE 0.003
	Cancerous tissue	2.7 AE 0.2	0.005 AE 0.001	11.6 AE 0.5	0.03 AE 0.002	37.0 AE 0.6	0.07 AE 0.001
	Film	1.3 AE 0.1	0.01 AE 6.4E-4	11.7 AE 1.5	0.005 AE 0.001	49.2 AE 2.5	0.006 AE 9.1E-5
	Aqueous solution	1.3 AE 0.3	0.03 AE 0.14			21.6 AE 1.2	0.09 AE 0.001	108 AE 16	0.001 AE 0.001

Acknowledgment

The project was funded through The National Science Centre Poland Grants: UMO 2012/07/B/ST4/01588 and UMO 2015/19/B/ ST4/01878.


A Bal Čytis

D Hakobyan

M Gabalis

A Žukauskas

D Urbonas

M Malinauskas

R Petruškevi Čius

E Brasselet
email: etienne.brasselet@u-bordeaux.fr

S Juodkazis

Hybrid curved nano-structured micro-optical elements

Keywords:

Tailoring the spatial degree of freedom of light is an essential step towards the realization of advanced optical manipulation tools. A topical challenge consists of device miniaturization for improved performance and enhanced functionality at the micron scale. We demonstrate a novel approach that combines the additive three-dimensional (3D) structuring capability of laser polymerization and the subtractive subwavelength resolution patterning of focused ion beam lithography. As a case in point hybrid (dielectric/metallic) micro-optical elements that deliver a well-defined topological shaping of light are produced. Here we report on hybrid 3D binary spiral zone plates with unit and double topological charge. Their optical performances are compared to corresponding 2D counterparts both numerically and experimentally. Cooperative refractive capabilities without compromising topological beam shaping are shown. Realization of advanced designs where the dielectric architecture itself is endowed with singular properties is also discussed.

Introduction

Advanced optical manipulation of matter at the microscopic scale implies the development of optical elements enabling on-demand structuring of electromagnetic fields [START_REF] Padgett | Tweezers with a twist[END_REF][START_REF] Dholakia | Shaping the future of manipulation[END_REF][START_REF] Woerdemann | Advanced optical trapping by complex beam shaping[END_REF]. Significant progress in the miniaturization of optical systems has been made by combining multiple optical functions within a single structure. An example of the trend towards miniaturization and hybridization is the lithographic fabrication of micro-lens arrays for multi-spot illumination or multi-aperture detectors. Photoresist reflow method is employed to create micro-lenses with a subsequent laser writing step used to inscribe a di↵ractive phase correction element [START_REF] Dannberg | Wafer-level hybrid integration of complex micro-optical modules[END_REF]. Such hybridization is pursued either to circumvent limitations in fabrication method, as in the aforementioned example, or when additional flexibility is required for specific fields. Fabrication methods need to be even more adaptive for applications such as optical sorting in microfluidics. For such purposes laser photopolymerization allows to depart form the 2D geometry imposed by lithographic approaches and create complex 3D optics [START_REF] Malinauskas | Femtosecond laser polymerization of hybrid/integrated micro-optical elements and their characterization[END_REF]. Since this method is inherently capable of additively producing arbitrary refractive dielectric structures, further hybridization requires an approach with complimentary capabilities. In contrast, subtractive focused ion beam (FIB) milling of metals is capable of creating complex di↵ractive elements and metasurfaces with unmatched precision [START_REF] Yu | Flat optics with designer metasurfaces[END_REF]. Since in such elements metal films and shells are sub-wavelength in thickness as well as in their patterning resolution, they provide a way to complement laser polymerized bulk refractive structures.

One of the areas where new methods to compound optical functionality and enact miniaturization are in high demand is light structuring. Structured light, also named complex light, refers to optical fields that possess non-trivial spatial distribution of one or several characteristics such as intensity, phase or polarization. Its description and main applications have been already extensively reviewed within the past decade, see for instance refs. [START_REF] Andrews | Structured Light and Its Applications: an Introduction to Phase-Structured Beams and Nanoscale Optical Forces[END_REF][START_REF] Yao | Orbital angular momentum: origins, behavior and applications[END_REF], and it still attracts growing attention from the optics and photonics community. A few paradigmatic examples are worth mentioning to appreciate the impact of structured light, especially when optical phase structuring is considered. In optical trapping, it is known since almost two decades that light beams endowed with optical phase singularities (i.e. vortex beams), which correspond to a local electric field amplitude proportional to exp(i` ) with the azimuthal angle and `the integer topological charge, allow enhancement of the axial trapping e ciency [START_REF] Simpson | Optical tweezers with increased axial trapping e ciency[END_REF]. Nevertheless, phase structuring continues to amaze as demonstrated just recently with the drastic increase of transverse trapping e ciency [START_REF] Taylor | Enhanced optical trapping via structured scattering[END_REF]. Sharper focus has been achieved for radially polarized light beams that consist of a superposition of contra-circularly polarized vortex beams with opposite unit topological charges [START_REF] Dorn | Sharper focus for a radially polarized light beam[END_REF]. Vortex beams also provide a spatial (orbital) degree of freedom that adds to the one associated with the polarization state (spin) of light. The fact that the orbital degree of freedom has an unbound set of eigenstates formally has demonstrated great promise in optical communications [START_REF] Wang | Terabit free-space data transmission employing orbital angular momentum multiplexing[END_REF][START_REF] Bozinovic | Terabit-scale orbital angular momentum mode division multiplexing in fibers[END_REF]. This also provides with enhanced optomechanical capabilities since a vortex beam with topological charge `carries `~angular momentum per photon [START_REF] Allen | Orbital angular momentum of light and the transformation of laguerre-gaussian laser modes[END_REF] whereas the spin contribution is limited to ±~angular momentum per photon. Optical imaging is another topic that has been deeply impacted by phase structuring. At the microscopic scale, this has been celebrated in 2014 with Nobel Prize in Chemistry for the development of super-resolved fluorescence microscopy. On the other hand, astronomical observations now benefit from optical vortex coronagraphy developed ten years ago [START_REF] Foo | Optical vortex coronagraph[END_REF]. As such, it seems not exaggerated to say that engineering of light beams has revolutionized our ways of taming light and current technological developments are likely to bring further unexpected surprises in the future.

Conventional methods to generate vortex beams involve the use of spatial light modulators, forked di↵raction gratings, phase plates or mode converters [START_REF] Yao | Orbital angular momentum: origins, behavior and applications[END_REF]. However, recent e↵orts to achieve optical trapping and manipulation at the microscale resulted in forgoing bulky options in favor of miniaturized structures, such as spiral zone plates (SZP) of both di↵ractive [START_REF] Liu | Twisted focusing of optical vortices with broadband flat spiral zone plates[END_REF] and plasmonic [START_REF] Gorodetski | Generating far-field orbital angular momenta from near-field optical chirality[END_REF] varieties, topological nanoslits [START_REF] Brasselet | Topological shaping of light by closed-path nanoslits[END_REF], photopolymerized structures [START_REF] Brasselet | Photo-polymerized microscopic vortex beam generators : precise delivery of optical orbital angular momentum[END_REF], metasurface-based devices [START_REF] Yu | Flat optics with designer metasurfaces[END_REF][START_REF] Shalaev | High-e ciency all-dielectric metasurfaces for ultracompact beam manipulation in transmission mode[END_REF], or self-engineered birefringent objects [START_REF] Brasselet | Optical vortices from liquid crystal droplets[END_REF][START_REF] Loussert | Manipulating the orbital angular momentum of light at the micron scale with nematic disclinations in a liquid crystal film[END_REF]. On the other hand, ultrafast laser 3D nanolithography o↵ers unmatched structuring flexibility on non-flat surfaces such as optical fiber tips [START_REF] Malinauskas | Femtosecond laser polymerization of hybrid/integrated micro-optical elements and their characterization[END_REF] or inside buried microfluidic channels [START_REF] Wu | In-channel integration of designable microoptical devices using flat sca↵old-supported femtosecond-laser microfabrication for coupling-free optofluidic cell counting[END_REF]. It makes it a practical choice for custom-on-demand 3D printing of microoptics [START_REF] Gissibl | Spatial beam intensity shaping using phase masks on single-mode optical fibers fabricated by femtosecond direct laser writing[END_REF].

Although optical manipulation setups realized at the microscale provide for added versatility and integration, new fabrication challenges also arise when multi-functional behavior is needed. In that case, multiplexing structures achieving independent optical operation into a single multi- functional architecture is an option. One can mention the monolithic integration of dielectric spiral phase plates and axicons fabricated by 3D photopolymerization into helical axicons in order to generate higher-order Bessel beams [START_REF] Žukauskas | Monolithic generators of pseudo-nondi↵racting optical vortex beams at the microscale[END_REF]. Also noteworthy is the very recent realization of multiplexed 2D metallic metasurface holograms [START_REF] Wen | Helicity multiplexed broadband metasurface holograms[END_REF][START_REF] Huang | Broadband hybrid holographic multiplexing with geometric metasurfaces[END_REF]. Here we propose to combine the additive 3D structuring capability of laser photopolymerization and the subtractive subwavelength resolution patterning of focused ion beam lithography to produce hybrid (dielectric/metallic) micro-optical elements for the purpose of topological shaping of light. Our demonstration relies on the fabrication of hybrid 3D binary SZPs enabling the generation of single-charge and double-charge optical vortices. We show, both numerically and experimentally, the cooperative refractive interaction between the individual 2D metallic and 3D dielectric sub-parts without compromising topological beam shaping from the SZP. Also, we discuss advanced 3D designs where the dielectric architecture itself is endowed with singular properties and report on their fabrication. Our results suggest that additive manufacturing methods such as 3D direct laser writing, once combined with state-of-the-art nanofabrication technologies, pave the way towards the realization of 3D hybrid metasurfaces of arbitrary complexity.

Design and fabrication of hybrid 3D singular micro-optics

The design of a SZP combines the radial Hilbert transform operation with Fresnel zone plate focusing, which gives rise to a focused vortex beam in transmission. It is described by a 2D binary transmittance function t of the form [START_REF] Wei | Fractional spiral zone plates[END_REF]:

t(r , ') = { 0 if (2k 1)⇡  `' ⇡r 2 f  2k⇡1 if 2k⇡  `' ⇡r 2 f  (2k + 1)⇡, (1)
where (r , ') refer to the polar coordinates, is the wavelength, f is the e↵ective focal length of the SZP and index k = (0, 1, 2, ...) is the spiral zone number. Designed SZP were chosen to have a diameter of 2R = 30 µm which gives up to 10 zones. Experimentally, 3D SZPs designed with focal length f = 18 µm and `= (1, 2) following Eq. ( 1), are fabricated by means of FIB lithography milling through an optically opaque layer of Au. The large depth-of-focus of the FIB allows to pattern metal layers overlaying various 3D structures. In the present work milling was done through Au coatings on both flat disk and axicon pedestals, each created by direct laser writing using femtosecond laser nano-polymerization. Axicons were used in this first study to evaluate whether focused ion beam lithography is viable for 3D fabrication of complex patterns without changing the focusing parameters. Similarly, flat disk pedestals represent a planar reference.

The first stage of fabrication involves photopolymerization of the disk or cone shaped pedestal by direct laser writing. A hybrid organic-inorganic photopolymer SZ2080 doped with Irgacure 369 photoinitiator at 2 wt.% [START_REF] Žukauskas | Characterization of photopolymers used in laser 3D micro/nanolithography by means of laser-induced damage threshold (LIDT)[END_REF] is chosen as the material for the pedestal. This polymer is particularly suitable for fashioning various micro-optical structures. Indeed, it is known to possess the required mechanical robustness as well as a refractive index closely matched to that of glass in the visible spectral region (n SZ2080 = 1.504, n Glass = 1.52) [START_REF] Ovsianikov | Ultra-low shrinkage hybrid photosensitive material for two-photon polymerization microfabrication[END_REF]. Prior to laser exposure the photoresist is prepared by drop-casting SZ2080 onto a cover-glass substrate that defines the (x, y) plane followed by pre-baking on a hotplate for 60 min at 90 C. Direct-write fabrication is conducted using a Yb:KGW (PHAROS, Light Conversion Ltd.) laser with 300 fs optical pulse duration at second harmonic ( = 515 nm), 200 kHz repetition rate, under tight focusing conditions using a 63⇥, N A = 1.4 microscope objective (Carl Zeiss). The 15 µm radius 3D flat disk pedestal structures are produced under 70 µW average power with corresponding pulse energy of 0.35 nJ and 0.988 TW/cm 2 intensity per pulse at focus, using a 0.5 mm/s scanning velocity along raster trajectories with 50 nm voxel overlap. Conversely, the axicon structures with a full apex angle of 165 are fabricated using 27.5 µW average power (0.14 nJ, 0.388 TW/cm 2 ) at 0.1 mm/s scanning velocity along spiral trajectories with 50 nm voxel overlap. Photopolymer development is performed by immersing it into pure 4-methyl-2-pentanone for 1 h to remove any unexposed residue.

The resulting photopolymerized structure is then coated with a 200 nm thick Au film using magnetron sputtering (AXXIS, JKLesker). Binary patterns given by Eq. ( 1) are transferred onto the Au sputtered polymerized structures by means of FIB milling, using a Raith IonLINE ion beam lithography tool. An interferometrically controlled piezoelectric stage as well as FIB imaging capability allow for precise control over the positioning of the pedestal structures for spiral pattern transfer. After alignment a 35 kV Ga + ion beam is focused down to a 25 nm diameter spot and set to scan along the desired pattern until the 200 nm Au layer was selectively stripped over 10 4 sequential patterning loops. The binary transmission patterns are thus reproduced with high fidelity, closely approximating the geometric parameters deduced from numerical simulations. This is illustrated in Fig. 1 where SEM images of the structures before and after FIB milling are shown in panels (a) and (d), and in panels (b), (c), (e) and (f), respectively. These images reveal the presence of residual gold islands in the milled areas that most likely result from impurities on the surface of the polymerized pedestal acting as crystallization sites for the sputtered gold. Such nanoscale islands are more resilient to ion bombardment than the rest of the structure.

Numerical simulations and experimental results

The optical behavior of the fabricated 3D SZPs is simulated numerically by using a commercially available FDTD software package (FDTD Solutions, Lumerical Solutions, Inc.). The simulation structure for the hybrid micro-optical elements is defined by applying a gold layer with 200 nm thickness (using the refractive index n metal = 0.5106 + i 2.2495) and spiral patterns defined by Eq. ( 1) onto a dielectric (using the refractive index n dielectric = 1.504) axicon and a flat pedestal for reference. Two simulations are performed for each structure, namely by using x-polarized and y-polarized plane waves at wavelength = 532 nm impinging at normal incidence (along the z axis) from the substrate side. By doing so, the response to an arbitrary polarized illumination can be deduced from the linear superposition principle by using appropriate complex weighting factors. In addition, the simulation box x ⇥ y ⇥ z dimensions are 44 µm ⇥ 44 µm ⇥ 34 µm and its surface boundaries are ended by perfectly matched layer boundaries to prevent from spurious e↵ects of reflections from the finite-size calculation volume. A non-uniform meshing algorithm was used. The mesh grid sizes varied from 10 nm close to the metal surface to 60 nm at remote regions of the simulation, with the average cell size being at around 30 nm.

Simulation results are compared with experimental optical characterization of the fabricated structures, which involves the propagation features of the transmitted light field. In practice, the structures are illuminated at normal incidence from substrate side by a 532 nm wavelength Gaussian laser beam, focused using a low numerical aperture lens, the sample being placed in the focal plane of the lens. Considering that the gold layer is thick enough to act as an almost opaque layer, we choose a beam waist radius w 0 twice larger than the radius R of the structure, which thus mimics plane wave illumination. If the metal layer was not opaque enough, a significant portion of laser radiation could be transmitted through the flat gold film surrounding the 3D polymerized structures. As this light would have a phasefront that is di↵erent than the one shaped by the refractive pedestals it could distort the optical characterization results. The issue regarding the opacity of the gold layer is checked in simulations by replacing gold by a so-called perfect electric conductor (PEC) material with infinite conductivity ! 1 that expectedly mimics perfect binary amplitude mask given by Eq. ( 1) better than the metallic mask does. Numerically, in Lumerical FDTD software simulations, PEC was set as a material with refractive index n PEC = 710 + i 710, whereas for analytical calculations ! 1 enters as the imaginary part of permittivity. Identical results are found regardless of whether Au or PEC values are used. In practice, the optical intensity profiles at a distance z from the plane of the structure are recorded by means of a microscope objective (100 ⇥ , N A = 0.8) and a CCD camera. On the other hand, information on the phase profiles are unveiled by recording interference patterns that result from the superposition of the transmitted field with a collinear Gaussian reference arm. Finally, we note that we are purposely restricting our investigations to the particular case of incident circular polarization for both simulations and experiments. Indeed, present use of micro-optical structures leads to transmitted light whose non-paraxial features cannot be ignored. Hence it is fitting to use the circular polarization basis as it allows to address the ensuing coupling between the polarization state and spatial degrees of freedom (i.e. the so-called spin-orbit interaction of light) with ease.

Two-dimensional spiral zone plates

In the case of flat SZP structures, see Figs. 1(b) and 1(c), numerical FDTD simulations of the transmittance of left-handed circularly polarized light (see appendix for used field representation convention) predict strong focusing behavior, as expected from the designed focal length f = 18 µm recalling that R = 15 µm. Calculations give slightly lower focal distance, f = 16.4 µm with no noticeable e↵ect of the helicity = ±1 of the incident circularly polarized light. This is illustrated in Figs. 2(a) and 2(b) that show total intensity cross-sections in the meridional plane (x, z) for `= (1, 2). These intensity patterns emphasize similar focusing distance whatever `that can be associated to a nominal numerical aperture of the elements, N A = sin ✓ ⇡ 0.67, based on the "viewing angle" of the structure ✓ = arctan(R/f ). For comparison, we find f = 19 µm in the case of a flat pedestal with `= 0 whatever the value of . The latter discrepancies between the designed focal distance and the calculated ones might be explained by residual form birefringence arising from metallic layer structuring (though not subwavelength).

In the focal plane of the SZP, doughnut-like intensity pattern is predicted both for `= (1, 2). This is reminiscent of the generated optical vortices whose respective unit and double topological charge are clearly exhibited by one-arm and two-arm spiraling interference patterns obtained from the coherent superposition with a co-axial Gaussian beam, see Figs. 2(c) and 2(d). In addition, we note the increase of the radius of the doughnut shaped intensity pattern with |`| as expected from the well-known p `-behavior of Laguerre-Gaussian-like beams. Indeed, quantitatively, intensity ring diameters are ⇠ 1.4 µm and ⇠ 2.1 µm for `= 1 and `= 2, respectively. Such a behavior is actually retrieved experimentally, as shown in Figs. 2(e) and 2(f) that nicely matched with their simulation counterparts. In particular, we find intensity ring diameters ⇠ 1.3 µm and ⇠ 2.0 µm for `= 1 and `= 2, respectively. Also we note that the handedness of the interference spiraling pattern does not depend on incident field polarization as expected from the scalar nature of the SZP design.

Noticeably, experimentally observed broken axisymmetry of the intensity pattern in the focal plane (see top panels of Figs. 2(e) and 2(f)) is not a spurious manifestation of instrumental limitations. Indeed, 1-fold and 2-fold rotational symmetry is found in simulations as shown in top panels of Figs. 2(c) and 2(d). Its origin is attributed to the finite size of the SZPs whose `-arm design is unavoidably associated with `abrupt ends at the periphery of the structure, as one can see from SEM images in Fig. 1. This is supported by further simulations made on structures with larger sizes. Indeed, broken axisymmetry of the total intensity patterns in the (x, y) plane tends to vanish as the radius R of the SZP increases, which implies a practical trade-o↵ between axisymmetry and fabrication time. However, experimental results show a somewhat lower axisymmetry than predicted by simulations. This is in part due to the Gaussian beam used in experiment, which is only an approximation of the plane wave used in simulations. Fabrication imperfections are likely to also have an e↵ect, especially at the structure edges where the narrowest groves are defined. Finally, beam alignment in experiment produces additional sources of error the FDTD simulations are not susceptible to. Remarkably, such SZPs behave as high-N A micro-optical elements whose N A increases with wavelength as expected from the f factor in Eq. ( 1). As such, the transmitted light has nonparaxial features that are easily deciphered by FDTD simulations. This is illustrated in Fig. 3, where the full vectorial nature of light is analyzed in the focal plane of flat SZP structures with `= 1 [columns (a) and (b)] and `= 2 [columns (c) and (d)] for both right-handed ( = 1) and left-handed ( = +1) circularly polarized incident field. For `= (1, 2), nonparaxial features can be straightforwardly grasped by mere inspection of the doughnut-like total intensity pattern by noting that the on-axis intensity is nonzero or zero depending on the incident circular polarization helicity = ±1, see upper row of Fig. 3. This is reminiscent to the fact that both the contracircularly polarized (with respect to the input polarization state) and the longitudinal components of the output field are nonzero and may exhibit on-axis intensity. This is detailed in the three rows of Fig. 3 labeled 'R', 'L' and 'z', which respectively refer to right-handed circular, lefthanded circular and longitudinal contributions to the total electric field. As a rule of thumb, the longitudinal field component carries an optical phase singularity of topological charge `+ while the spatial distribution of the phase of the contra-circular component is associated with a topological charge `+ 2 , as one can ascertain from the phase profiles in Fig. 3. This is a consequence of the spin-orbit interaction of light resulting from the scattering of light [START_REF] Bliokh | Spin-to-orbital angular momentum conversion in focusing, scattering, and imaging systems[END_REF] by -5 -10 0 SZPs and is reminiscent of total angular momentum conservation.

165 ᵒ (c) Simulation Experiment f =12.3 (µm) f =16.4 (µm) f =12.3 (µm) = 1 = 1 = 2 1 μm 1 μm 1 μm 1 μm 1 μm 1 μm

Three-dimensional spiral zone plates

Analysis of 3D SZPs is performed similarly to above 2D case. A comparative study is presented in Fig. 4 in the case `= 1, as shown in panels (a) and (b). The 3 µm height flat pedestal does not appreciably change the focusing behaviour of the SZP element. FDTD simulations show that the amount of bulk material in the pedestal does not a↵ect the focusing performance of the device, as it only imparts some collective phase o↵set. However, di↵erences in optical path length due to the curved surface of the axicon induces changes. The qualitative di↵erence between these two cases is the prediction of a substantial decrease of the focal length from f ⇡ 16.4 µm down to f ⇡ 12.6 µm, taking the top surface of the 2D SZP and the tip of the 3D SZP as the origin from which is evaluated the focal length. Such a trend is retrieved experimentally, indeed we observe a decrease of the focal length from f = 20 ± 2 µm down to f = 15 ± 2 µm (precision of the used translation stage prevents claiming a better accuracy). Interestingly, the latter decrease of focal length can be also assessed without need of numerics by mere evaluation of the combined optical powers of the 3D refractive dielectric and the 2D di↵ractive metallic elements taken separately. Namely, f 1 hybrid = f 1 dielectric + f 1 metallic where f i refers to the focal length of the element "i". On the one hand, we recall that the focal distance of the 2D SZP defined by Eq. ( 1) is designed for f metallic = 18 µm. On the other hand, the focal for an axicon with an apex angle ↵ and radius R can be approximated as half of the typical elongated 1). However, as the focal distance is decreased the SZP pattern needs to be scaled down accordingly, along with corresponding increases to FIB patterning fidelity requirements. Therefore, a refractive subcomponent is especially useful in creating high-N A structures, where the hyperbolic dependence on focal distance f could create challenges for planar FIB fabrication. Finally, intensity and phase characteristics in the focal plane of 3D SZPs with `= 1 and `= 2 are also analyzed experimentally and compared with numerical simulations, as shown in the upper row of Fig. 4. Both experimental and simulation results hint at axicon pedestals slightly shrinking down the annular focal spot. Observations suggest that the hybrid axicon/SZP structure is a way to create â ȂIJperfect optical vorticesâ Ȃİ whose dark hollow radius does not depend on the topological charge [START_REF] Ostrovsky | Generation of the â ȂIJperfectâ Ȃİ optical vortex using a liquid-crystal spatial light modulator[END_REF] by appropriately selecting the full apex angle of the axicon. However, the e↵ect is not very prominent in this first study, therefore, further work is forthcoming.

The transmittance of the spiral zone structure has been evaluated based on numerical simulations. The total transmittance through the 30 µm diameter structures is T ⇡ 22.5% for flat pedestal structures and T ⇡ 25% for patterned cones. This is in part due to the 50% opacity of the metal pattern, the suppression of transmittance through sub-wavelength (aspect ratio of 1) slits as well as the di↵ractive nature of the pattern itself. However, if the Poynting vector is integrated only in the R ⇡ 2 µm focal spot region, transmittance values become T ⇡ 5.1% for flat pedestal structures and T ⇡ 5% for patterned cones. The influence of the photopolymerized pedestals on the total losses is only marginal.

In addition to the aforementioned refractive modifications, the proposed hybrid polymerisation and FIB milling design is useful for applications in microscopy and microfluidics where only an optical vortex beam should be generated with blocked light which does not pass through the binary transmission mask. This creates beneficial dark-background imaging in microscopy setting or a well-defined focused optical vortex in microfluidic channel for trapping/sorting applications. The flat pedestal design is appealing for easier integration into practical microfluidic chip with polymerised functional structures and for creating specific phase o↵sets.

Above demonstration of principle that 3D SZPs of good quality can be fabricated by combining 3D laser polymerization to focused ion beam lithography of ultrathin metallic films sets the basis for the development of arbitrary hybrid curved metasurfaces. In particular, one can consider the realization of advanced designs where the dielectric architecture itself is endowed with singular features. An example is provided in Fig. 5 that shows the design and SEM images of a cuspy pyramid, where the basis corresponds to a continuous deformation of the circle with six-fold rotation symmetry that belongs to the family of hypocycloid curves. This example thus corresponds to a 3D design endowed with cusp singularities that possesses a convex counterpart generated from epicycloid curves. FIB milling can be used to further add sub-wavelength size incisions (either through a metal mask or directly into the polymer) with presission beyond that of laser photopolymerization to further complement its functionality. From previous works dealing with cuspy 2D designs [START_REF] Brasselet | Topological shaping of light by closed-path nanoslits[END_REF][START_REF] Brasselet | Optical vortices from closed-loop subwavelength slits[END_REF] that showed intriguing capabilities regarding the generation of optical phase singularities, 3D singular designs are likely to bring a new twist to beam engineering.

Conclusion

We have reported on the realization and analysis of hybrid 3D micro-optical elements enabling optical phase engineering by combining 3D dielectric structures obtained from laser polymerization to subwavelength-thick metallic film patterned by focus ion beam milling. As such, this represent an original attempt to produce hybrid curved metasurfaces. Demonstration has been carried out in the particular case of 3D binary spiral zone plates with unit and double topological charge, whose optical performances are compared to their 2D counterparts both numerically and experimentally. This work illustrates how fabrication techniques combining refractive and di↵ractive optical approaches can jointly lead to the elaboration of multifunctional of optical elements at the microscale, possibly endowed with topological shaping capabilities. State of the art nanofabrication tools also allow realizing more complex designs where both the dielectric and metallic sub-parts can possess singular features, which may open enhanced engineering of optical singularities and ensuing development of optical techniques based on singular optics.

Here we demonstrated the unique merit of FIB beam milling due to long depth-of-focus by patterning on a 3D surface of an axicon. Fast and accurate FIB milling becomes feasible at high fabrication throughput by mitigation of surface charging [START_REF] Gervinskas | Control of surface charge for high-fidelity nanostructuring of materials[END_REF]. Combination of the direct laser and ion-beam writing methods is promising for novel applications in 3D micro-and nano-photonics [START_REF] Malinauskas | Ultrafast laser processing of materials: from science to industry[END_REF].

2R 2 2RFig. 1 .

 21 Fig. 1. SEM images of photopolymerized flat disk pedestal (a) an axicon with 165 full-apex angle (d) structures of diameter 2R = 30 µm after sputter-coating with 200 nm of Au. SEM images of the same structures after FIB milling of SZP patterns with vortex orders `= 1 (b,e) and `= 2 (c,f) in the Au layer.

Fig. 2 .

 2 Fig. 2. (a,b) FDTD simulated total intensity cross-sections in the meridional plane (x, z) for `= 1 (a) and `= 2 (b) in the case of left-handed circularly polarized incident light on flat SZPs. (c,d) Total intensity (x, y) cross-section in the focal plane (top panel) and corresponding interference pattern that results from the coherent superposition with a coaxial Gaussian beam (bottom panel) `= 1 (c) and `= 2 (d). (e,f) Experimental counterpart of panels (a) and (b), respectively.

Fig. 3 .

 3 Fig. 3. FDTD simulations of the vectorial content of light in the focal plane of flat SZP structures with `= 1 [columns (a) and (b)] and `= 2 [columns (c) and (d)] for both righthanded ( = 1) and left-handed ( = +1) incident circular polarization. Both intensity (left part of each column) and phase (right part of each column) of total ('tot'), right-handed circular ('R'), left-handed circular ('L'), and longitudinal ('z') contributions to the total electric field are shown. Bottom-right colormap refers to phase from 0 to 2⇡.

Fig. 4 .

 4 Fig. 4. FDTD simulated total intensity cross-sections in the meridional plane (x, z) in the case of left-handed circularly polarized incident light on flat pedestal for `= 1 (a), and axicon pedestal with 165 full apex angle for `= 1 (b) and `= 2 (c). Top panels refer to the confrontation between simulation and experiment for both total intensity (x, y) cross-section in the focal plane (left side) and corresponding interference pattern that results from the coherent superposition with a co-axial Gaussian beam (right side). Bottom line: sketches of the side view of the 2D and 3D SZPs.

Fig. 5 .

 5 Fig. 5. (a) Three-dimensional illustration of the design of a topological pyramid of height H, where the basis corresponds to a sixth-order hypocycloid curve inscribed in a circle of radius R. Side-view and top-view SEM images of such a pyramid fabricated by laser photopolymerization for R = H = 50 µm are shown in panels (b) and (c), respectively.

Acknowledgments

This work was partially supported by the Australian Research Council Discovery grant DP130101205 and the Linkage grant LP120100161 with Raith-Asia. DH and EB acknowledge support by the French State in the frame of the "Investments for the future" Programme IdEx Bordeaux (reference ANR-10-IDEX-03-02). AZ and MM acknowledge funding by the Research Council of Lithuania under the Lithuanian-French Programme "Gilibert" (No. TAP LZ-4/2015). SJ acknowledges the startup funding of the Nanotechnology facility by strategic funding from Swinburne University. FDTD simulation work was performed on the swinSTAR supercomputer at Swinburne University of Technology.

Appendix

We used the following convention for the complex representation of the optical field. Namely, a fully polarized plane wave propagating towards z > 0 is expressed as E = E 0 e i(kz !t ) e, where k is the wavevector, ! is the angular frequency and e is the unit vector that defines the polarization state. In the present work, we consider both left-handed and right-handed circular polarization states that corresponds to e = (x + iy)/ p 2 with = ±1, respectively. In addition, a beam carrying an optical phase singularity of topological charge `corresponds to a field amplitude proportional to the pure phase factor exp(i` ) where is the polar angle in the plane (x , y). Ensuing angular momentum characteristics (within the paraxial approximation) are ~spin angular momentum per photon carried by e -polarized light and `~orbital angular momentum per photon carried by light field with amplitude proportional to exp(i` ).