FR2936064A1 - Surface-enhanced Raman spectroscopy imaging device for acquisition of in-vivo images of biological sample, has bundle of optical fibers with nanostructured distal face that is in form of network of conical points formed by cores of fibers - Google Patents

Abstract

The device has a bundle (1) of optical fibers (10) comprising a proximal face (150) and a nanostructured distal face (100), where the distal face has sub-micrometric scale, and is coated with a thin metallic layer (110). The distal face is presented in a form of a network of conical points (13), where each point is formed by a core (11) of optical fibers. The cores of the fibers have radial concentration gradient of chemical doping species so that refractive index of the cores decreases from a center towards periphery. The layer has thickness ranging between 1 and 50 micrometers. An independent claim is also included for a method for manufacturing a surface-enhanced Raman spectroscopy imaging device.

Description

The invention relates to a Raman spectroscopy imaging device with surface enhancement, as well as to methods for using and manufacturing such a device. BACKGROUND OF THE INVENTION The device of the invention is particularly suitable for biological and medical applications, and in particular for producing images in vivo with low invasiveness.

Raman spectroscopy is a powerful technique for studying a wide variety of chemical and biological systems. It provides both qualitative and quantitative information and with high spatial resolution from samples such as biomolecules, viruses, cells, tissues, thus providing molecular information on the components of these complex systems . The main limitation of this technique is its low sensitivity, due to the smallness of the Raman cross section (typically of the order of 1030 ± 10-25 cm 2 / molecule), which is well below the absorption cross-sectional area. intrinsic or extrinsic fluorescence (of the order of 10-15 cm 2 / molecule).

The Raman signal can, however, be very strongly exalted by a factor of up to 1014 by exploiting an interaction between the analyzed molecules and a nanostructured metal surface. This phenomenon is known as surface-enhanced Raman Spectroscopy (SERS) spectroscopy. The article by K. Kneipp, H. Kneipp, I. Itzkan, R. R. Dasari, and S. Feld Ultrasensitive Chemical Analysis by Raman Spectroscopy Chem. Rev. 1999, 99, pages 2957-2976 provides an introduction to the SERS technique and its applications. A more in-depth theoretical description of the exaltation effect of the Raman signal induced by a nanostructured metal surface can be found in the article by Yuan Vo-Dinh "Surface-

Enhanced Raman Spectroscopy Using Metallic Nanostructures ", TrAC Trends in Analytical Chemistry 1998, 17, pp. 557-582. Thirty years after the discovery of this effect, SERS substrates are beginning to be commercially available. The substrate consists of a silicon wafer with a gold coating and a regular network of nanoscale holes, a promising way to reduce the cost of SERS substrates. to improve their spatial homogeneity and to increase their reproducibility is to manufacture them from bundles of optical fibers.The following articles may be mentioned in this connection: - DJ White, PR Stoddart, Nanostructured optical fiber with surface-enhanced Raman scattering functionalii : y Opt.Lat 2005, 30, 598-600 - DJ White, AP Mazzolini, PR Stoddart Manufacturing of a range of SERS subst Spectroscopy on nanostructures multicore optical fibers, Journal of Raman Spectroscopy 2007, 38, 377-382 - Hankus ME, Li H., GJ Gibson, BM Cullum, Surface-Enhanced Raman Scattering-Based Nanoprobe for High-Resolution, Non-Scanning Chemical Imaging Anal. Chem. 2006, 78, 7535-7546. V. Guieu, F. Magugné-Labarthet, L. Servant, D. Talaga, N. Sojic Ultrasharp Optical-Fiber Nanoprobe Array for Local Raman-Enhancement Imaging Small 2008, 4, No. 1, 96 - 98. In all As mentioned above, a SERS substrate is made from an optical fiber bundle or, what is equivalent, a multi-core optical fiber. Such a device, also known as fiber for imaging, consists of a plu'alité of identical optical fibers extending in the same direction, not twisted and whose sheaths are melted together or, more generally, connected together.

to form a compact beam. A beam of a few hundred micrometers in diameter may contain several thousand individual optical fibers. In the articles by White et al. (2005 and 2007) and Hankus et al. it is suggested to stretch the bundles of fibers to reduce their diameter and thus increase the spatial resolution of the SERS substrate. An optical fiber bundle has two opposite end faces; one of these faces-said distal face, the opposite face being called proximal-is immersed in a chemical bath wet etching. When tapered beams obtained by stretching are used, the distal face corresponds to the thinnest end of the bundle. The core and the cladding of the optical fibers are made of silica glass and contain different dopants in order to modify their refractive index. Typically, the hearts may be doped with germanium oxide and the sheaths contain fluorides. Incidentally, doping modifies the chemical properties of glass, and allows differential etching of the core and sheaths. This effect allows the nanostructuration of the distal face of the fiber bundle. In all the known embodiments of the prior art, the etching conditions (chemical composition of the bath, immersion time) are chosen so as to cause preferential erosion of the cores of the optical fibers, which thus form cavities. The sheaths surrounding the hearts form ridges that culminate in ultra-sharp nano-points, arranged in a regular pattern. The distal surface is then covered with a thin layer of gold or silver having a thickness of a few tens of nanometers. The nanostructured and conductive surface thus obtained constitutes an excellent SERS substrate. In the experiments conducted by White et al. and by Guieu et al. (see above), the SERS substrate is illuminated from the outside and the Raman-scattered back light is harvested using a microscope objective. In other words, the nanostructured surfaces

manufactured from optical fiber bundles are used as SERS substrates of conventional type. Hankus et al., On the other hand, collect Raman light that has been guided by the hearts of individual optical fibers to the proximal end of the beam. On the other hand, the lighting is made from the outside of the fibers, as in the other cases. The lighting conditions of the SERS substrates known from the prior art do not make it possible to acquire Raman images in vivo, in particular for diagnostic purposes, in a manner as minimally invasive as a conventional fibroscopy or endoscopy examination. An object of the invention is precisely to provide such a device. Unlike the prior art, in a device of the invention the excitation light is injected into the heart of the optical fibers at the proximal face of the beam to propagate in a guided manner to the nanostructured distal face; and the Raman light generated at said distal surface propagates along said fibers to be collected from the proximal surface. According to the invention, this object can be achieved by means of a Raman surface-elevation spectroscopy imaging device comprising a bundle of optical fibers having a first end, said proximal, and a second end, said distal, said distal end having a structured surface on a sub-micrometric (ie nanometric) scale and coated with a thin metallic layer, characterized in that said surface structured on a sub-micrometric scale is in the form of a network of tips each formed by a core of an optical fiber beam. According to preferred embodiments of the invention: the cores of the optical fibers may have a radial concentration gradient of a doping chemical species, because of which the refractive index of said cores decreases from the center to the periphery.

The tips of the grating formed on said structured surface may have apexes having a radius of curvature of between 20 nm and 100 nm. The thin metal layer may be made of a metal chosen from: Cu, Ag, Au, Li, Na, K, In, Pt and Rh. The metal thin film may have a thickness of between 10 and 200 nm. The average distance between two points of said network may be between 200 nm and 500 μm, and preferably between 1 and 50 μm. Similarly, the diameters of the cores of said optical fibers may be between 200 nm and 500 μm and preferably between 1 and 50 μm. - The device may also comprise means for injecting into the hearts of said optical fibers, at said proximal end, an excitation light propagating from said distal end; and means for collecting, at said proximal end, a light generated at said structured surface and propagating in a guided manner within the cores of said optical fibers. The device may also comprise spectral analysis means of the light generated at said structured surface and collected at said proximal end. Said excitation light may be substantially monochromatic. Another object of the invention is a method of using such a device for image acquisition by Raman spectroscopy with surface acquisition. Yet another object of the invention is a method of manufacturing such a device comprising:

a step of structuring an end face of an optical fiber bundle by wet etching; and a step of depositing a thin metallic layer on the structured face.

Advantageously, said wet-phase etching structuring step may include immersing an end of said bundle of optical fibers in an aqueous solution comprising a strong acid and a salt of said acid, the chemical composition of the acid and of the salt, their concentrations and the immersion time being chosen so as to obtain a preferential etching of the sheaths of said optical fibers relative to the cores. In particular, said aqueous solution may contain hydrofluoric acid and ammonium fluoride, the coricentration of ammonium fluoride being greater than that of hydrofluoric acid.

Other characteristics, details and advantages of the invention will emerge on reading the description given with reference to the appended drawings given by way of example and which represent, respectively: FIG. 1, in a schematic way, the structure a device according to the invention; FIGS. 2A, 2B and 2C, electron microscopy images of the nanostructured distal surface of such a device; FIG. 3, for comparison, an electron microscopy image of the nanostructured distal surface of a device according to the prior art (Guieu et al.); and FIG. 4, a Raman scattering spectrum obtained using the device of the invention. In FIG. 1, the reference 1 indicates a bundle constituted by a plurality of individual optical fibers 10. By way of example, the bundle 1 may be of the IGN-035/06 type manufactured by Sumitomo Electric, having a diameter of 350 μm. and consisting of 6000 individual fibers. Good

that the beam 1 is shown in a rectilinear configuration, it is flexible and can be folded with a radius of curvature as small as 15 mm. Each individual fiber 10 consists of a core 11 of silica glass (SiO 2) doped with GeO 2, surrounded by a sheath 12 also made of silica glass but doped with fluoride (F). In a conventional manner, the dopings are chosen such that the refractive index of the core is higher than that of the sheath, so as to ensure the guiding of the light. The diameter of each core is of the order of 3 μm and the cores of different individual fibers are spaced about 4 μm apart.

For reasons which will become clear later, it is advantageous that the doping of the cores of the optical fibers 10 is not uniform, but gradually decreases (for example linearly) from the center to the periphery. The optical fiber bundle 1 has two opposite end faces, called the proximal (reference 150) and distal (reference 100) faces. The distal face 100 of the beam 1 is structured on a sub-nanometric scale. More specifically, this face is in the form of a network of conical tips 13 each formed by a core 11 of an individual optical fiber 10. The height of each spawn 13 is about 5-7 pm and the radius of curvature at the apex of the tips is between 20 and 100 nm. In addition, the distal surface 100 is covered with a thin metal layer 110, generally made of gold or silver, of a thickness of a few tens of nanometers (for example, 10-200 nm) which can be produced in particular by sputtering. . FIGS. 2A to 2C show images of the distal face of a device according to the invention, made by scanning electron microscopy using different magnifications. FIG. 2B, in particular, shows the relatively regular arrangement of the tips 13. FIG. 3 shows, for comparison, the active surface of a device

SERS optical fiber beam known from the prior art, and precisely the aforementioned article by V. Guieu et al. The values which have been indicated for the dimensions of the tips, the thickness of the metal coating 110 and its chemical composition have been given only by way of example. Those skilled in the art are able to determine the appropriate values to produce an exaltation effect of Raman scattering. The distal surface 150 of the beam 1 may be provided with connection means making it possible to connect it functionally to a Raman spectrometer such as a Labram HR spectrometer from Horiba-Jobin-Yvon. Such a spectrometer comprises a source 2 of excitation light, in particular a laser; a device 3 for spectral analysis of light, such as a diffraction grating; and a light detector 4. The light source (for example, an Ar-Kr laser emitting at 647 nm) injects light radiation into the cores 11 of the fibers 10 of the beam 1. It is understood that the fibers must to be able to guide this radiation, whether it is in single mode or multimodal mode. After having spread along the fibers 10, the light radiation emitted by the source 2 (called excitation radiation) interacts by Raman diffusion with an analyte adsorbed by the metal coating 110 of the nanopoints 13. In a manner known per se the analyte absorbs the excitation light and re-emits, by Raman scattering, a radiation having a frequency shift characteristic of its vibrational levels and allowing its identification. Raman light is in turn guided by the hearts of the optical fibers to the distal surface 150, where it is decomposed into its spectral components and analyzed in frequency. FIG. 4 shows a Raman spectrum obtained using the device of the fiber 1, on the active face 100 from which crystal violet (methyl-violet) has been adsorbed. The peak P1 corresponds to the oxide doped with oxide

of germanium, and can be observed even in the absence of the nanopoints 13. The peaks P2, P3 and P4, on the contrary, characterize the aralyte (that is, in the example, the crystal violet), and not are not detected when the distal face 100 of the device is not structured. In the graph of FIG. 4, the abscissa axis represents the Raman shift (expressed in cm) and the ordinate axis the signal intensity (in arbitrary units). Since the Raman scattered light propagates in a guided manner along the cores of the optical fibers of the beam, the spatial information is preserved. It is therefore possible to determine the spatial distribution, on the active face 100, of the analytes identified by Raman spectroscopy. As shown in FIG. 3, the devices known from the prior art, the nanopoints making it possible to exalt the Raman effect, are not produced in correspondence of the fiber cores, but on the contrary between said cores. Therefore, it is not possible to illuminate the analyte molecules located at said points (and therefore likely to produce an exalted Raman signal) by light radiation guided along the cores. Hence the need to illuminate the nanostructured surface from the outside.

The same goes for the acquisition of the Raman signal: only the use of a structure according to the invention allows an efficient coupling of this signal with the optical fiber cores (although Hankus and collaborator claim to have obtained such a coupling using a structure in which the nanopoints are made from the sheaths).

In addition, in the case of drawn optical fiber bundles, the core diameters are too small to provide guided propagation of excitation radiation and Raman radiation. It follows that the stretching of the fibers can actually improve the spatial resolution of the measurements, but only on the condition of illuminating the sample from the outside and to acquire the Raman signal also from the outside, by renouncing to exploit the guiding properties of the optical fibers. 2936064 'o

Therefore, only the structure of the invention makes it possible to perform remote SERS imaging by making full use of the optical fiber guiding properties. In particular, only this structure seems to allow Raman endomicroscopy. Moreover, tests have shown that the nanopoints are very mechanically resistant, and are not likely to be damaged by contact with a biological sample. The use of index gradient fibers (ie having a dopant concentration in the core which decreases from the center to the periphery) is advantageous because the radial variation of the refractive index tends to concentrate the light in the center of the heart, and therefore at the apex of the tip. This results in greater efficiency of Raman scattering. As in the case of the prior art, the nanostructuration of the active face 100 of the optical fiber bundle 1 can be obtained by wet etching. However, unlike the prior art, the etching conditions must be chosen so as to ensure preferential erosion of the sheaths with respect to the cores. Under these etching conditions, the germanium oxide acts as a protection agent for the hearts against etching. It is understood that the use of a radially variable doping, with a higher concentration of dopant in the center of the cores than at their periphery, facilitates the obtaining of very sharp points, rather than rod-shaped protuberances. The use of index gradient fibers is therefore also advantageous for this reason of a strictly technological nature. Wet-phase etching involves immersing the distal end of a bundle of optical fibers in a corrosive chemical bath. The immersion time and the composition of the bath should be chosen so as to ensure preferential erosion of the sheath of the individual fibers with respect to the core. The structures reproduced in FIGS. 2A and 2C were obtained by immersing the face for 90 minutes.

end of a Sumitomo beam IGN-035/06, previously polished, in a mixture consisting of: 5 parts of a 40% aqueous solution of NH4F; 1 part of a 48% aqueous solution of HF; and 1 part of deionized water. The exact proportions of the components may vary, provided that the concentration of ammonium fluoride remains higher than that of hydrofluoric acid. Other strong acid / salt pairs can also be used, depending on the chemical composition of the optical fibers (and in particular the nature of the dopants used in the cores and sheaths, respectively). Other etching techniques may also be used, such as ion beam etching; but these techniques are substantially more complex and more expensive to implement than chemical etching which is the preferred embodiment of the invention.

Claims (14)

REVENDICATIONS1. A surface exaltation Raman spectroscopy imaging device comprising a bundle (1) of optical fibers (10) having a first, so-called proximal end (100), and a second, so-called distal end (150), said distal end having a surface structured on a sub-micrometric scale and coated with a thin metal layer (110), characterized in that said surface structured on a sub-micrometric scale is in the form of a network of tips (13) each formed by a core (11) of an optical fiber (10) of the beam. 2. Device according to claim 1, wherein the cores of the optical fibers of the beam have a radial concentration gradient of a doping chemical species, because of which the refractive index of said cores decreases from the center to the periphery. 3. Device according to one of the preceding claims, wherein the points of the network formed on said structured surface have apex having a radius of curvature of between 20 nm and 200 nm. 4. Device according to one of the preceding claims, wherein the thin metal layer is made of a metal selected from: Cu, Ag, Au, Li, Na, K, ln, Pt and Rh. 5. Device according to one of the preceding claims, wherein the thin metal layer has a thickness between 10 and 200 nm and preferably between 1 and 50 pm. 6. Device according to one of the preceding claims, wherein the average distance between two points of said network is between 200 nm and 500 pm and preferably between 1 and 50 pm. 7. Device according to one of the preceding claims, wherein the diameters of the cores of said optical fibers are between 200 nm and 500 pm.30. 8. Device according to one of the preceding claims, also comprising means (2) for injecting into the hearts of said optical fibers, at said proximal end, an excitation light propagating from said distal end; and means (4) for collecting, at said proximal end, a light generated at said structured surface and propagating in a guided manner within the cores of said optical fibers. 9. Device according to claim 8, also comprising means (3) for spectral analysis of the light generated at said structured surface and collected at said proximal end. 10. Device according to one of claims 8 or 9, wherein said excitation light is substantially monochromatic. 11. Use of a device according to one of the preceding claims for the acquisition of images by surface acquisition Raman spectroscopy. 12. A method of manufacturing a device according to one of claims 1 to 10 comprising: a step of structuring an end face of an optical fiber bundle by wet etching; and a step of depositing a thin metallic layer on the structured face. The method of claim 12 wherein said wet etching structuring step comprises immersing an end of said optical fiber bundle in an aqueous solution comprising a strong acid and a salt of said acid, the chemical composition of the acid and salt, their concentrations and the immersion time being chosen so as to obtain a preferential etching of the sheaths of said optical fibers with respect to the cores. 14. The method of claim 13 wherein said aqueous solution contains hydrofluoric acid and ammonium fluoride, the concentration of ammonium fluoride being greater than that of hydrofluoric acid.