FR3038713A1 - SPECTROSCOPY DEVICE, IN PARTICULAR RAMAN SPECTROSCOPY, AND ASSOCIATED METHOD - Google Patents

Abstract

The subject of the invention is a spectroscopy device in which an assembly integrating the sample holder, the source of illumination of the sample and the detector (light sensor) movable with respect to a measurement assembly integrating the elements of excitation of the sample, preferably a laser, and the collection elements of this light ... The invention also relates to the associated method. A particularly targeted use of the invention is the optical analysis by Raman spectroscopy of biopsies from tissue samples, which are fixed on transparent substrates.

Description

SPECTROSCOPY DEVICE, IN PARTICULAR RAMAN SPECTROSCOPY, AND

ASSOCIATED METHOD

TECHNICAL FIELD The invention relates to a spectroscopy device and a method implementing such a spectroscopy device.

Although described with reference to the main application targeted, Raman spectroscopy, the spectroscopy device according to the invention can be implemented to make different spectral analyzes, for example, infrared spectroscopy, fluorescence spectroscopy, etc. .. with the proviso to modify some of the elements of the device, such as the analysis light source, the spectrometer and / or the optical elements.

A particularly targeted use of the invention is the optical analysis by Raman spectroscopy of biopsies from tissue samples, which are fixed on transparent substrates.

Prior art

Raman spectroscopy, also called Raman spectroscopy, and Raman microspectroscopy are non-destructive methods for the analysis of the composition and the molecular chemical structure of a material in order to characterize it.

It exploits the physical phenomenon according to which a medium modifies the frequency of the light circulating there. This frequency offset corresponds to an exchange of energy between the light beam and the medium, and gives information on the sample itself.

Raman spectroscopy consists of sending a monochromatic light onto the sample and analyzing the scattered light.

FIG. 1 illustrates, by way of example, a known device for Raman type 10 spectroscopy. This device 10 essentially comprises a Raman analysis module 12, coupled to a standard microscope 14. This known device is sometimes called "micro-Raman".

On the microscope 14 is mounted a sample holder 16 for carrying the sample to be analyzed.

This sample holder 16 can be moved relative to the microscope 14 to modify the viewing area of the sample.

The microscope 14 is also equipped with a video camera 18 for recording the observations made with the microscope 14. The microscope 14 comprises one or more light sources, so that it is possible to observe in transmission or in reflection according to the nature sample (transparent or not). The microscope 14 further comprises an optical lens system or, better still, several interchangeable optical lens systems coupled to lenses for changing the magnification with which observations of the sample to be analyzed are made.

Moreover, the microscope 14 is optically coupled to the Raman analysis module 12.

The Raman analysis module 12 essentially comprises a light source 20, here a laser 20, for emitting a light beam in order to probe the sample.

The microscope 14 is equipped with means such as a set consisting of a mirror 22, a semi-reflective plate, a notch filter 24, and focusing lenses 26, for example, for directing the light beam emitted by the laser to the sample to be analyzed and to direct the light beam emitted back by the sample to be analyzed to the Raman analysis module 12.

In the notch filter 24, the light beam emitted by the sample is filtered to remove the spectral component of the light beam corresponding to the natural emission frequency of the laser.

The filtered light beam then passes through a slot 26 to focus it, then a diffractive device 28. The diffracted beam is finally captured by means of a light sensor 30, here a CCD (for "Charge-Coupled Device", or "device load transfer ").

In standard analysis procedures, observations of the sample are made: either by microscopic imaging with white light illumination; either by Raman imaging.

Raman imaging necessarily involves switching off the white light, because the excitation beam and the white light illumination use the same optical path, so the use of one excludes the other. , or at worst mutually disturb each other.

The change from one imaging to the other implies a change of focus that can lead to performance failures in the observations made.

With a low magnification, typically 5 times, the observations can make it possible to observe the entire surface of the sample to be analyzed, typically of the order of 1 mm 2, to determine the areas of interest of the sample. However, a low magnification does not achieve sufficient resolution to achieve the details of the sample, such as cells from a tissue sample.

With a larger magnification, typically 50 times, it is possible to visualize the very details of the sample being analyzed, such as single cells of between 10 and 200 μm in size from a tissue sample, and place the laser beam for sample analysis accurately.

Thus, the use of a known imaging system, integrating the device 10, requires an operator to constantly change the magnification mode to find areas of interest at low magnification and then place the laser beam precisely with the image obtained in high magnification. This back and forth between low and high magnifications requires the operator to mentally match the two images. This work is tedious and can cause observation biases, that is, observation errors.

In addition to the disadvantage of requiring an operator to constantly switch from low magnification to high magnification, with the inherent risk of observation errors, the Raman spectroscopy device 10 illustrated in FIG. 1 is cumbersome and also not simple to use. use due to the preliminary optical adjustments necessary to implement the actual analysis, these settings consisting for example of alignments, the search for the optimal focal position which is not the same between a good focus for microscopic visualization and a good focus for Raman analysis.

There is therefore a need to improve spectroscopy devices, particularly those employing Raman spectroscopy, to overcome at least some of the aforementioned drawbacks.

The object of the invention is to respond at least in part to this need.

Presentation of the invention

To do this, the invention proposes a spectroscopy device comprising: a first set comprising: a sample holder, preferably a plane, for receiving the sample to be analyzed by spectroscopy, the sample holder being at least partially translucent, A light source for illuminating the sample, able to illuminate the sample; a first light sensor for receiving the light emitted by a light source of illumination of the sample and passing through the sample and the holder; sample, the first light sensor being preferably associated with a display device of the image picked up by the first light sensor, - a second set comprising: a light source for analyzing the sample, and / or a second light sensor adapted to capture the light transmitted through or reflected by the sample to be analyzed, • light collecting means diffused by the sample to be analyzed, excited by the light emitted by the analysis light source, associated with a spectrometer for analyzing the light scattered by the sample to be analyzed.

The elements of the first set are mounted integral in translation in at least one direction, preferably in two directions in a plane parallel to the sample holder, preferably in three directions, two of which are included in a plane parallel to the sample holder and the third is perpendicular to the other two,

The elements of the second set are mounted integral in translation.

According to the invention, the second set is independent in relative translation of the first set in at least one direction, preferably at least two directions lying in a plane parallel to the sample holder, preferably in three directions, two of which are included in a plane parallel to the sample holder and the third is perpendicular to the other two.

Thus, the invention essentially consists in making the assembly integrating the sample holder, the source of illumination of the sample and the detector (light sensor) mobile with respect to the measurement assembly integrating the excitation elements. of the sample, preferably a laser, and the collection elements of this light.

In other words, according to the invention, the displacement of the excitation part of the sample relative to that of the imaging part proper in a spectroscopy device is decoupled.

Advantageously and as will be clear from reading the description which follows, the spectroscopy device according to the invention greatly facilitates the use because, unlike with the devices according to the state of the art, an operator can view a sample simultaneously at low magnification and at high magnification.

Furthermore, the device according to the invention has a smaller footprint than the spectroscopy devices of the prior art, particularly in the direction perpendicular to the sample holder.

In addition, such a system is simple both in terms of implementation and use, and robust.

Preferably, the device according to the invention has one or more of the following characteristics, taken alone or in combination: the lighting light source of the sample is able to illuminate the sample with a grazing incidence light, in particular with an angle of incidence between 0 ° and 30 °, - the light source of the sample is integrated in the sample holder. the light source of illumination of the sample comprises one or more diodes located on the same side of the plane of the sample holder, preferably fixed on the sample holder, preferably on either side of an area adapted to receive the sample to be analyzed.

Advantageously, the first set further comprises a first optical lens system disposed between the sample holder and the first light sensor. This first lens system preferably has a magnification of between 1 and 20, in particular equal to 10.

According to an advantageous embodiment, the second light sensor is associated with a second optical lens system disposed between the sample holder and the second light sensor, the second optical lens system preferably having a different magnification, especially greater, magnification of the first optical lens system. This second lens system preferably has a magnification of between 20 and 100, in particular equal to 50.

Preferably, the light source for analyzing the sample is a laser, preferably associated with a collimator of the beam emitted by the laser, arranged between the laser and the sample holder.

The second optical lens system is advantageously arranged between the laser preferably between the collimator and the sample holder.

According to an advantageous variant embodiment, the collection means comprise at least one of a beam splitter from the sample to be analyzed, a notch filter and a collimating lens.

Preferably at least one, more preferably both light sensors are CMOS sensors, more preferably connected to display screens.

According to another aspect, the invention relates to a method of spectroscopy including Raman spectroscopy method, implemented by means of a spectroscopy device described above, comprising the steps of: (i) exciting a sample to be analyzed at means of a beam emitted by the analysis light source, (ii) collecting the light scattered by the sample to be analyzed following the excitation by the beam emitted by the analysis light source, and (iii) analyzing the light collected using the spectroscope.

Preferably, the method comprises two steps prior to step (i) and consisting of: a) determining an area of interest of the illuminated sample to be analyzed using the sample light source , in particular from the image displayed by the display device of the image picked up by the first light sensor, and b) turn off the illumination light source of the sample.

The advantages of the device according to the invention are numerous. In particular, the device according to the mode integrating the second light sensor, the second system of optical lenses at higher magnification, the magnification of the first optical lens system has many advantages among which: the possibility of simultaneously viewing the laser beam , the sample and this with two different magnifications, which has never been achieved for a Raman spectroscopy device, or even for the single imaging system; a display at low magnification with illumination, advantageously in grazing light, of the sample, which is independent of the displacements along the three directions X, Y, Z. This configuration is therefore robust because it does not require any verification of alignment of optics. Such a system has never been realized in a Raman spectroscopy device according to the state of the art, in which in contrast the field of view changes with the displacement in X and Y and the focal length changes with the displacement in Z ; a display in high magnification, advantageously in grazing light of the sample, which makes it possible to follow the displacements in two X and Y directions on the sample while being dependent on the position along the remaining direction Z. This high magnification visualization associated with a large field image avoids a ceaseless coming and going between the two magnifications as on devices according to the state of the art; a possible Raman analysis by choosing a region of interest either from the low-magnification image or from the high-magnification image. By choosing the low magnification image, it is possible to choose analysis regions without being limited by the field sized by the choice of magnification via the chosen objective and still having the region of interest included in the image. This possibility can not be envisaged with the devices according to the state of the art in which the Raman analysis is made exclusively from a high magnification image sized by the choice of the objective used in fine for the analysis. Raman.

An important application of the device according to this embodiment of the invention is the analysis of tissue slide. In fact, an image in a large field makes it easier and faster to locate the tumor zones or not on the slide, to define regions of investigation, using the image in high magnification if necessary, to position precisely the excitation laser beam on the areas of interest in order to analyze them by Raman spectroscopy.

The device according to the invention is advantageously compact, robust, easy to use and automated so that it can be installed as an analysis tool in hospitals, analytical laboratories and private medical practices.

DETAILED DESCRIPTION The invention will be better understood on reading the following description, in support of the accompanying drawings, in which: - Figure 1 shows schematically in partially cutaway view a known spectroscopy device; FIG. 2 diagrammatically represents an example of a spectroscopy device according to the invention; - Figure 3 schematically shows a sample holder with a system for integrated grazing lighting, which can be implemented in the spectroscopy device of Figure 2; FIGS. 4 to 7 illustrate observations made on biopsy tissue samples by means of the spectroscopy device of FIG. 2; FIG. 8 represents an example of Raman spectrum acquired with the spectroscopy device of FIG. 2, from the observation of FIG. 6.

Figure 1 has already been described in the preamble, so it is not detailed below.

In FIG. 2, there is shown a spectroscopy device 100 according to the invention. This spectroscopy device essentially comprises an imaging system 102 and a spectrometer 104 optically connected to the imaging system 102, for example by means of an optical fiber 106. The optical fiber 106 is for example a fiber marketed under the name " Thorlabs M18L01, 105μηι 0.22NA ", while the spectrometer is for example a spectrometer" Tomado Spectral System HyperFlux U1 ".

As illustrated, the imaging system 102 first includes a sample holder 108 for receiving a sample to be analyzed. It should be noted here that the sample to be analyzed is preferably a biopsy, that is to say obtained by sampling, the spectroscopy device of Figure 2 does not aim a priori in vivo analysis directly on patient. Typically, the sample may have a thickness of between 5 and 200 μm, corresponding to the thickness of the biopsy.

The sample holder 108 may take the form of a blade, translucent or, better, transparent, at least in places, especially at locations where it receives the sample to be analyzed. In particular, the sample-carrying blade 108 may be translucent or transparent in a central portion, for example at a central disk, the rest of the sample-carrying blade 108 being itself opaque. The sample plate 108 may be provided with means for fixing, at least temporarily, the sample to be analyzed in position on the sample plate 108. These fixing means may for example take the form of metal tongues, folded to constrain the sample to be analyzed against the sample plate 108. The sample plate 108 is for example made by means of a transparent or semi-transparent substrate having a transmission rate of at least 30% in the spectral range 400. -700 nm.

An example of a sample plate 108 is shown diagrammatically in FIG. 3. This sample-carrying blade 108 has substantially the shape of a substantially planar blade 1081 having a central groove 1082 forming a receptacle for receiving the sample to be tested. The bottom of the central groove 1082 defines a plane of the sample-carrying blade 108. At its center, the central groove 1082 has a through-hole 1083. This through-hole 1083 makes it possible to fix the sample-holder to an objective, for example an objective of wide angle type as that sold under the name "NA VIT AR NMV-6WA 6 mm F / 1.4 1/2" M30.5 Wide Angle. "Around this central hole 1083, the sample plate 108 has two beads 1084 presenting openings 1085 shaped to receive light emitting diodes (LEDs), such that these light emitting diodes illuminate the sample with grazing incidence light.

These light-emitting diodes form a first light source 110 of the imaging system 102. This first light source 110 makes it possible to illuminate the sample to be analyzed with a grazing incidence light. Grazing incidence means a light having an angle of incidence, measured in a plane perpendicular to the plane of the sample plate 108 - that is to say the plane of Figure 2 - on the sample to be analyzed or the sample holder strip 108 between 0 ° and 30 °, preferably between 0 ° and 15 °, more preferably between 0 ° and 5 °.

Here, as indicated above, this first light source 110 is made in the form of light-emitting diodes, oriented substantially parallel to the plane of the sample holder 108. The LEDs are all arranged on one and the same side of the sample-carrying blade 108. that is to say in the same half-space delimited by the plane of the sample-carrying blade 108.

However, the LEDs are preferably arranged on either side of the sample plate 108, to be able to illuminate the sample with different angles of incidence, measured this time in a plane parallel to the plane of the blade sample holder 108.

The first light source 110 is for example made by 4 LEDs "Nichia NSPW510DS" arranged in parallel which illuminate to the right of the sample in Figure 2, and 4 LEDs "Nichia NSPW510DS" arranged in parallel which illuminate to the left of the sample in Figure 2.

The imaging system 102 further comprises a first light sensor 112 for receiving the light emitted by the sample to be analyzed following the excitation by the first light source 110. Here, this first light sensor 112 is disposed on the opposite side. of the sample plate 108 relative to the first light source to capture the light emitted by the first light source 110 which has passed through the sample to be analyzed. In other words, the first light sensor 112 and the light source 110 are arranged in two distinct half-spaces delimited by the plane of the sample-carrying blade 108.

The first light sensor 112 may be connected to a display screen (not shown) to display the captured image. The operator can thus continuously view the image picked up by this first light sensor 112. In order to be easily connected to a display screen, the first light sensor 112 may notably be of the CMOS type (of the English "Complementary Metal Oxide Semiconductor "). The first light sensor is for example a CMOS sensor "Mightex BCN-C050-U Color 5MP USB2.0 2592 X 1944 pixels" (2.2 pm X 2.2 pm pixel size).

Advantageously, a first optical lens system 114 is interposed between the sample plate 108 and the first light sensor 112, in particular to allow viewing of the sample to be tested with a given magnification, possibly adjustable.

Here, a first optical lens system 114 is selected which allows the first light sensor 112 to capture an extended, in particular complete, image of the sample to be analyzed. In other words, the first optical lens system 114 is chosen so that it has a relatively low magnification, and thus a large field. In particular, the magnification of the first optical lens system 114 can be between 1 and 20, and more particularly be equal to 2. The first optical lens system 114 is for example constituted by a wide-angle lens "NAVITAR NMV-6WA 6 mm F / 1.4 1/2 "M30.5 Wide Angle", placed below the sample to be analyzed, as shown in Figure 2, and at a working distance of 6 mm from this sample to be analyzed.

As illustrated in FIG. 2 and particularly advantageously, the sample plate 108, the first light source 110, the first light sensor 112 and the first optical lens system 114 form a first integral assembly 116, notably integral with translation.

Here, this first assembly 116 is integral in translation in three directions, two directions being parallel to the plane of the sample holder plate 108, and the third being perpendicular to the other two. This is achieved here by fixing the aforementioned elements of the assembly 116 relative to each other, and mounting the assembly 116 on a translation plate 118 in three directions as described above.

Such a plate can for example be made by the combination of a motorized stage "PI micos P-622.Z PIHera® Precision Z-Stage" to ensure the displacements along the axis perpendicular to the plane of the sample plate 108, fixed on two plates "PI micos

Translation Stage VT-80, 50 mm travel range "to perform translations in a plane parallel to the plane of the sample-holder blade. Such a translation plate 118 makes it possible to move the whole assembly 116 in a non-moving manner, without the movement of one element of the assembly 116 relative to another. This allows in particular the first light sensor 112 to capture an identical image of the sample, regardless of the movements of the sample to be analyzed performed using the translation plate 118.

The imaging system 102 furthermore comprises a light source for analyzing the sample 120. Here, this light source for analyzing the sample takes the form of a laser 120, in particular a "Spectra" laser. -Physics EXLSRONE-532FC-80, 80 mW monomode »longitudinal at 532 nm, adjustable between 0 and 80 mW single mode. This light source for analyzing the sample 120 is here distinct from the first light source 110.

This laser 120 is here associated with a first collimation device 122 for directing the light beam emitted by the laser 120 towards a semi-reflecting plate 124. The semi-reflecting plate 124 makes it possible to direct the laser beam towards the sample to be analyzed. via a second optical lens system 126. Here, the second optical lens system 126 takes the form of an objective, which can optionally be changed, of a microscope 128 including the semi-reflective plate 124. By way of example, the semi-reflecting blade 124 may be a "OPTOPRIM RazorEdge Dichroic beamsplitter LPD01-532RU-25x36xl.l" blade, the first collimator device 122 a "Micro Laser Systems F-H10-VIS-APC" collimator, and the second system optical lenses 126 a lens "Olympus 50x LMPLFLN, NA 0.5 - WD 10.6mm - FN 26.5".

The light emitted by the sample to be analyzed following the excitation by means of the beam emitted by the laser 120 passes through the semi-reflecting plate 124, then is collected by collecting means 130.

These collection means 130 may in particular comprise at least one of a blade dividing the collected beam, in particular the semi-reflecting blade 124, a notch filter, to attenuate or even eliminate the wavelength corresponding to that of the laser 120. , and a collimation lens. The notch filter is for example a filter "OPTOPRIM StopLine single-notch filter NF03-532E-25", the collimation lens is known under the name "Thorlabs AC254-030-ML". The collecting means 130 are optically connected, via a collection fiber, to the spectrometer 104, which can thus measure the intensity of the different wavelengths present in the light emitted by the sample and deduce therefrom emission spectrum.

Moreover, the imaging system 102 as illustrated in FIG. 2 comprises a second light sensor 132 adapted to capture the light emitted by the first light source 110 and / or the laser 120 and reflected by the sample at analyze.

In order to be able to effectively capture these lights, the second light sensor 132 is associated with the semi-reflecting plate 124 which makes it possible to orient a portion of the light beam coming from the sample to be analyzed towards the second light sensor 132. This second sensor 132 of light can also be of the CMOS type and connected to a display screen to allow an operator to view in real time the image captured by the second light sensor 132. The second light sensor 132 is for example also a CMOS sensor "Mightex BCN-C050-U Color 5MP USB2.0 2592 X 1944 pixels" (2.2 pm X 2.2 pm pixel size).

Preferably, the second light sensor 132 is associated with the optical lens system 126 here formed by the objective of the microscope 128, optically disposed between the sample to be analyzed and the second light sensor 132. This second optical lens system 126 allows to apply a fixed or adjustable magnification to the image effectively captured by the second light sensor 132.

This magnification is preferably chosen different from the magnification of the first optical lens system 114, to allow the operator to display two distinct images, in particular having a different magnification, of the sample to be analyzed. The second optical lens system 126 has for example a higher magnification than the first optical lens system 114 and therefore a reduced field but with a better resolution. By way of example, the second optical lens system 126 has a magnification of between 5 and 100, for example equal to 50. Such magnification makes it possible to capture by means of the second light sensor 132 an enlarged, and therefore more resolute, image. , of the sample to be analyzed, in particular an image making it possible to differentiate the cells of the sample to be analyzed. A collimation system 134, in particular a collimating lens, is provided here between the second optical lens system 126 and the second light sensor 132 for focusing the light transmitted by the semi-reflecting plate 124 towards the second light sensor 132. This collimation system 134 may especially take the form of a collimation lens "Thorlabs AC254-050-ML".

The second set 136 including in particular the laser 120, the collimation device 122, the semi-reflecting plate 124, the second optical lens system 128, the collecting means 130, the second light sensor 132 and the collimation system 134 is integral, in particular integral in translation in a direction lying in a plane parallel to the plane of the sample-carrying blade 108, preferably in two directions included in this plane, more preferably in three directions, two of which lie in a plane parallel to the plane plane of the sample plate 108 and one is perpendicular to these two directions. This second set 136 may be displaced, at least in one direction, preferably in two, preferably in three directions, with respect to the assembly 116. It is thus possible to move the beam emitted by the laser 120 relative to the sample to be analyzed and / or improve the sharpness of the image captured by the second light sensor 132 and move the position of the collection fiber, to improve the quality of the Raman signal.

Thus, in the spectroscopy device 100 of FIG. 2, the first light sensor 112 captures a substantially constant signal from the sample to be analyzed, irrespective of the movements of the second set 136 relative to the first set 116, while the second light sensor 132, it, captures a signal that varies according to these movements.

The spectroscopy device 100 thus makes it possible to simultaneously provide the user with a wide-field view (for example a field of the order of 1 × 1 cm 2) of the sample to be analyzed in order to define the zones of interest (which have dimensions of the order of 100 × 100 μm 2), and a view at high magnification (for example a field of the order 100 × 100 μm 2) for placing the excitation laser beam very precisely on the areas of interest.

The spectroscopy device 100 is also compact, easy to use, robust and automatable, thus making it possible to implement it as an analysis tool in hospitals, analysis laboratories, and private medical practices as a complementary tool. and rapid assistance with diagnosis and / or determination of a treatment.

The spectroscopy device 100 makes it possible to implement the spectroscopy method comprising the following steps: (i) exciting a sample to be analyzed by means of a beam emitted by the analysis light source, (ii) collecting the light emitted by the sample to be analyzed following excitation by the beam emitted by the light source, and (iii) to analyze the light collected using the spectroscope.

In the case of a Raman spectroscopy method, the collected light is filtered to suppress the wavelength of the analysis light source. However, other spectroscopy methods can also be implemented in such a device. For example, the light emitted by the sample to be analyzed may be a fluorescent light. In another example, the analysis light source emits an infrared wavelength.

The spectroscopy method may further comprise two steps prior to step (i) above, comprising: a) determining an area of interest of the illuminated sample to be analyzed using the light source 110 d illumination of the sample, in particular from the image displayed by the display device of the image captured by the first light sensor 112, and b) turn off the illumination light source 110 of the sample.

Thus, an operator can visualize a large field image of the sample, which is invariable, permanently illuminated by the illumination light source 110 of the sample and determine, with the aid of the corresponding displayed image, the zones of interest of the sample to be analyzed. The displacement of the sample to be analyzed (in fact of the sample holder) with respect to the analysis light source 120 is also achieved by keeping lit the illumination light source 110 of the sample, the wide field image displayed. remaining identical. It is thus easier for the operator to identify the zones of interest and to correctly dispose the sample by supplying the analysis light source 120. This analysis light source 120 can also be lit during the search for zones of interest of the sample, in order to ensure correct positioning of the sample. Then, once this correct positioning of the sample to be tested with a zone of interest excited by the analysis light source 120, the operator can turn off the light source 110 for illuminating the sample and carry out an analysis. spectroscopic using the light source of analysis.

Once the analysis is complete, the light source 110 can be turned on again to allow a new analysis cycle.

The spectroscopy device 100 was implemented to carry out the simultaneous collection of the wide field image and small field images (respectively with a 20x and 50x magnification objective) of FIGS. 4 and 5.

In FIG. 6, the laser is in operation, the spectrometric analysis being in progress. These figures illustrate Raman spectroscopic analyzes of prostate biopsies. As can be seen in these figures, the wide-field image on the left in these figures is the same in all three cases, while the image at high magnification is different. This last point is explained by a displacement of the sample to be analyzed with respect to the objective of the microscope used, this displacement being able notably to make it possible to align precisely the laser beam on the cells or tissues to be analyzed.

Figure 7 illustrates an example of visualization of a tissue sample in a mouse implanted with HNSCC carcinoma ("Head and Neck Squamous Cell Carcinoma").

Figure 8 shows the Raman spectrum of this visualization.

It should be noted that the objective here is the characterization of biopsy slides by Raman spectroscopy. However, thanks to the first and / or second light sensors, it is conceivable to couple the information of the spectrum with a parallel morphological characterization, from the analysis of the wide field image, or rather of the image. small field according to the resolution reached.

The present invention is however not limited to the single example described above with regard to Figure 2 and many variants can be made.

For example, one can imagine performing a Raman analysis with only the first light sensor, excluding the second light sensor. This solution can make the spectroscopy device even more compact and simple to implement. Instead of the second light sensor, a second analysis system can be provided. This second analysis system is thus coupled with the Raman spectrometer. This second analysis system may especially be of the phase-imaging type, with fluorescence imaging, in particular autofluorescence imaging, infrared spectroscopy, LIBS (Laser Induced Breakdown Spectroscopy), UV spectroscopy.

In addition, the spectrometer used here is suitable for performing Raman spectroscopy. It may, however, be replaced by any device for performing an analysis by infrared spectroscopy or fluorescence spectroscopy, in particular.

Claims (14)

A spectroscopy device (100) comprising: - a first set (116) comprising: • a preferably planar sample holder (108) for receiving the sample to be analyzed by spectroscopy, the sample holder (108) being at least partially translucent, • a light source (110) for illuminating the sample, capable of illuminating the sample, • a first light sensor (112) for receiving the light emitted by a light source (110) of illuminating the sample and passing through the sample and the sample holder (108), the first light sensor (112) being preferably associated with an image display device sensed by the first light sensor (112). a second set (136) comprising: an analysis light source of the sample (120), and / or a second light sensor (132) adapted to capture the light transmitted through or reflected by the sample to be analyzed, Means (130) for collecting the light scattered by the sample to be analyzed, excited by the light emitted by the analysis light source (120), associated with a spectrometer (104) for analyzing the light diffused by the sample to be analyzed, device in which the elements of the first set (116) are mounted integral in translation in at least one direction, preferably in two directions lying in a plane parallel to the sample holder (108), preferably in three directions two of which lie in a plane parallel to the sample holder (108) and the third is perpendicular to the other two, in which the elements of the second set (136) are mounted integral in translation, and in which the second set (136) is independent in relative translation of the first set (116) in at least one direction, preferably at least two directions in a plane parallel to the port e-sample (108), preferably in three directions, two of which are in a plane parallel to the sample holder (108) and the third is perpendicular to the other two. 2. Spectroscopy device according to claim 1, wherein the light source (110) for illuminating the sample is able to illuminate the sample with a grazing incidence light, in particular with an angle of incidence between 0 ° and 30 °. Spectroscopy device according to claim 1 or 2, wherein the light source (110) for illuminating the sample is integrated in the sample holder. 4. Spectroscopy device according to one of the preceding claims, the light source (110) for illuminating the sample comprising one or more diodes located on the same side of the plane of the sample holder (108), preferably fixed on the sample holder (108), preferably on either side of a zone adapted to receive the sample to be analyzed. The spectroscopy device according to one of the preceding claims, the first set (116) further comprising a first optical lens system (114) disposed between the sample holder (108) and the first light sensor (112). Spectroscopy device according to claim 5, wherein the first lens system (114) has a magnification of between 1 and 20, in particular 10. A spectroscopy device according to any one of the preceding claims, wherein the second light sensor (132) is associated with a second optical lens system (126) disposed between the sample holder (108) and the second light sensor (132). light (132), the second optical lens system (126) preferably having a different magnification, especially greater than the magnification of the first optical lens system (114). 8. Spectroscopy device according to claim 7, wherein the second lens system (126) has a magnification between 20 and 100, in particular equal to 50. Spectroscopy device according to one of the preceding claims, in which the sample analysis light source is a laser (120), preferably associated with a collimator (122) of the beam emitted by the laser (120). disposed between the laser (120) and the sample holder (108). The spectroscopy device according to claims 7 and 9, wherein the second optical lens system (126) is disposed between the laser (120), preferably between the collimator, and the sample holder (108). A spectroscopy device according to any one of the preceding claims, wherein the collection means (130) comprises at least one of a beam splitter from the sample to be analyzed, a notch filter and a lens of collimation. A spectroscopy device according to any one of the preceding claims, wherein at least one, preferably both light sensors (112; 132) are CMOS sensors, preferably further connected to display screens. 13. Spectroscopy method, in particular Raman spectroscopy method, implemented by means of a spectroscopy device according to any one of the preceding claims, comprising the steps of: (i) exciting a sample to be analyzed by means of a beam emitted by the analysis light source (120), (ii) collecting the light scattered by the sample to be analyzed following excitation by the beam emitted by the analysis light source (120), and (iii) ) analyze the light collected using the spectroscope (104). The method of spectroscopy according to claim 13, comprising two steps prior to step (i) and comprising: a) determining an area of interest of the illuminated sample to be analyzed using the light source (110); ) illuminating the sample, in particular from the image displayed by the display device of the image picked up by the first light sensor (112), and b) turning off the light source (110) of sample lighting.