DEVICES FOR EX-VIVO MICROSCOPIC EXAMINATION OF SAMPLES AND FOR IN-VIVO MICROSCOPIC EXAMINATION OF THE SKIN

Description

Domaine technique de l'invention

[0001] The present description relates to devices for ex vivo microscopic analysis of samples and in vivo analysis of skin, microscopic analysis systems incorporating such devices and methods for ex vivo microscopic analysis of samples and in vivo analysis of skin using such devices. Etat de la technique

[0002] In the context of a dermatological examination, it is known to carry out a dermoscopic examination, that is to say an observation of the surface of the skin using a magnifying optical instrument, then a local microscopic analysis based on the observations made on the wide-field image obtained by the dermoscopic examination.

[0003] Microscopic analysis includes, for example, microscopic imaging or spectroscopic analysis.

[0004] Among the microscopic imaging techniques, we know in particular, and in a non-limiting manner, confocal microscopy techniques such as for example the technique described in Rajadhyaksha et al [Ref. 1] or K. König et al [Ref 2] for non-linear microscopy. Optical coherence tomographic microscopy techniques (or OCM according to the Anglo-Saxon expression " Optical Coherence Microscopy "), in the time domain ("Time Domain OCM") or in the frequency domain ("Frequency Domain OCM"). Among the OCM techniques, we know techniques combining optical coherence tomography and confocal microscopy (see for example Schmitt et al [Ref. 3]) in order to improve lateral resolution.

[0005] More specifically, patent application WO2015092019 [Ref. 4] describes a technique for visualizing the internal structure of a semi-transparent object arranged at the focus of a microscope objective, for example a biological tissue, in order to obtain vertical sections or " B-Scans » orthogonal to the surface of the object, at a high rate (several sections per second), with a high spatial resolution, i.e. of the order of 1 µm, both axially and laterally, and a satisfactory penetration depth, of the order of a millimeter. This technique is based on optical coherence microscopy but has a linear or one-dimensional confocal filtering configuration (in one direction); for this, the illumination line is optically conjugated, in particular by means of the microscope objective, with a linear detector whose detection surface has a width substantially identical to a width of the image of the line, resulting in a spatial filtering of a region of the object to be observed. Such a technique is thus known under the name of LC-OCT according to the Anglo-Saxon expression (" Line-field Confocal Optical Coherence Tomography ").

[0006] Y. Chen's article et al. [Ref. 5] also proposed a linear scanning optical coherence tomographic microscopy device, but in which a sample is moved in a plane perpendicular to an optical axis of the microscope objective, and in a direction perpendicular to the illumination line, allowing images to be formed en face of the sample (or " C-Scans ").

[0007] Among the techniques for spectroscopic analysis of a sample, and in particular of a biological tissue such as skin, we know for example and in a non-limiting way Raman spectroscopy which makes it possible to form a molecular imprint of biological tissues, as described for example in Schleusener et al. [Ref. 6]. The journal article by E. Drakaki et al. [Ref. 7] presents in general different spectroscopy techniques applied to the microscopic analysis of the skin.

[0008] All of the microscopic analysis techniques described above, whether for imaging or spectroscopy, use a microscope objective having a large nominal numerical aperture, typically greater than or equal to 0.5, for a given field of view, typically between about 0.2 mm and about 1.5 mm.

[0009] In the context in particular of microscopic imaging of the skin, the microscope objective can be movably integrated into a waterproof mount as described in application WO2019138062 [Ref. 8] in the name of the applicant and reproduced on the FIG. 1 The waterproof mount 10 comprises in particular a housing 11, a chamber 12 filled with an immersion medium, an immersed objective 13 axially movable in the mount, and a deformable sealing member 14 which ensures a sealing junction between the immersed objective 13 and the housing 11. In the example of the FIG. 1 , the use of an immersion medium with a refractive index substantially equal to the refractive index of the observed object advantageously makes it possible to reduce optical dispersion deviations, optical path variations and the introduction of aberrations, so that images are obtained with better resolution. The housing 11 comprises a circumferential portion 15 and a light transmission window 16. The housing 11 further comprises an outer portion 17 mechanically fixed to the circumferential portion 15. The circumferential portion 15 and the outer portion 17 of the housing 11 have, for example, a cylindrical shape so as to house the movable immersed objective 13. The light transmission window 16 is coupled to one end of the circumferential portion 15. For example, as illustrated in the FIG. 1 , the end of the circumferential portion 15 has a truncated cone shape, where the base of smaller radius comprises the light transmission window 16. In the frame shown in the FIG. 1 , the deformable sealing member 14 is configured to deform when the submerged lens 13 moves relative to the housing 11.

[0010] Such a mount, perfectly sealed even during axial movement of the microscope objective in the housing, is thus particularly suitable for an in vivo skin analysis probe, intended to be held in the hand by a practitioner to come into contact with the skin.

[0011] In practice, following the patient's examination, if the lesion detected in vivo presents, for example, a suspicion of cancer, the practitioner will perform an excision, that is to say, he will extract the part of the lesion that he has identified as malignant. Once the excision has been carried out, the practitioner must be able to verify that the entire tumor has been extracted. To do this, an ex vivo analysis of the sample taken can be carried out.

[0012] The probe described in [Ref. 8] and incorporating a microscope objective mount as described in the Fig. 1 could allow ex vivo analysis of the sample collected. However, such analysis proves difficult in practice, particularly due to the complexity of moving the sample in a controlled manner relative to the probe.

[0013] Other mounts are known in the context of microscopic analysis of a sample, see for example published patent application EP 3 018 518 [Ref. 9], published patent application WO 2016 / 083003 [Ref. 10] and published patent application DE 10 2009 044 413 [Ref. 11].

[0014] Issued patent US10514532 [Ref. 12] describes a system equipped with a confocal microscopy probe, configured for both ex vivo imaging of tissue samples taken during excision and in vivo imaging of the skin. More specifically, the confocal microscopy probe comprises a mount for receiving a microscope objective and a transmission window arranged at the end of the mount. The confocal microscopy probe is mounted on a horizontal arm coupled to a vertical axis supported by a fixed platform, such that the horizontal arm can rotate about the vertical axis, for example using handles arranged on the probe.This arrangement allows, in a first mode, to position the probe above the platform to allow ex vivo imaging of a sample deposited on the platform, and in a second mode, to rotate the probe around the vertical axis to image a sample outside the platform, for example the skin of a patient.

[0015] However, such a system does not allow the flexibility of a hand-held, manual probe for in vivo skin imaging. Furthermore, it requires adjustment of the optical axis alignment of the microscope objective when switching from skin imaging to imaging an ex vivo sample.

[0016] The present description provides devices for ex vivo microscopic analysis of samples and in vivo analysis of skin, allowing the use of a hand-held microscopic analysis probe for ex vivo analysis of samples, without compromising ergonomics and without requiring alignment of the optical axis of the microscope objective when switching from an in vivo to an ex vivo operating mode. Résumé de l'invention

[0017] In this description, the term "include" means the same as "include", "contain", and is inclusive or open and does not exclude other elements not described or shown. Furthermore, in this description, the term "approximately" or "substantially" is synonymous with (means the same as) a lower and / or upper margin of 10%, for example 5%, of the respective value.

[0018] According to a first aspect, the present description relates to a device configured to operate in a first operating mode comprising an in vivo microscopic analysis of the skin and in a second operating mode comprising an ex vivo microscopic analysis of a sample, the device comprising: a microscope objective comprising an optical axis; a mount comprising a distal portion in which said microscope objective is arranged and a proximal portion secured to said distal portion, said distal portion comprising at one end a first transmission window; a first tip comprising at one end a second transmission window and configured to be removably attached, in the first operating mode, to said distal portion of the mount; a second tip configured to be removably attached, in the second operating mode, to said distal portion of the mount; a sample holder comprising a receiving surface configured to receive a sample in the second operating mode;a support configured to cooperate with the second tip in the second mode of operation so as to receive said mount such that the optical axis of the microscope objective is substantially aligned in a predetermined direction relative to the receiving surface.;

[0019] The device according to the first aspect allows, by means of an original modular arrangement, an in vivo microscopic analysis of the skin and ex vivo of a sample resulting for example from an excision, without compromising on ergonomics in the two operating modes, and without requiring alignment of the optical axis of the microscope objective when switching from an in vivo operating mode to an ex vivo operating mode.

[0020] In practice, in the first operating mode, equipped with the first tip, the device can form a manual probe for in vivo microscopic analysis. In the second operating mode, equipped with the second tip, the device can be housed in the support with automatic alignment of the optical axis in a predetermined direction relative to the receiving surface of the sample holder, thus allowing great ease of use and ensuring repeatability in the way the samples are analyzed.

[0021] The predetermined direction is, for example, a direction perpendicular to the receiving surface, in particular in the case of a substantially planar receiving surface. In the case of a non-planar receiving surface, a predetermined direction may be, for example, a direction perpendicular to a plane tangential to said receiving surface.

[0022] According to one or more exemplary embodiments, the first transmission window comprises a first transparent blade which closes said end of the distal part of the frame. For the purposes of the present description, the term "transparent blade" means any blade capable of transmitting at least part of a spectral band useful for microscopic analysis. A spectral band useful for microscopic analysis comprises at least one detection spectral band and may further comprise an illumination spectral band in the case where an illumination path of the sample comprises said transparent blade. A transparent blade comprises, for example, a glass blade or any other transparent material in the spectral band useful for microscopic analysis.

[0023] A closure of the distal part of the mount makes it possible to protect the microscope objective, particularly when changing from one operating mode to another. A closure also allows the use of an immersion medium for immersion operation of the microscope objective. With a closed transmission window, sealing at the microscope objective can be ensured, for example by means of a sealing device as described in [ref. 12], also when changing from one operating mode to another.

[0024] Thus, according to one or more exemplary embodiments, the device further comprises a first immersion medium in contact with said first transparent slide and said microscope objective. A first immersion medium comprises, for example, a liquid or a gel whose refractive index is between approximately 1.3 and approximately 1.5, that is to say a refractive index generally close to the object of study, skin or sample. The first immersion medium makes it possible in particular, when said microscope objective is configured to move axially in the distal part of the frame for an analysis deep in the skin, to limit the introduction of variation in the optical path and optical aberrations during such a movement. The use of an immersion microscope objective also makes it possible to limit the dispersion difference between the arms of an interferometer, in the case of a microscopic analysis comprising an interferometric system

[0025] In other exemplary embodiments, the medium between said first transparent slide and said microscope objective may simply be air.

[0026] In other exemplary embodiments, said first transmission window may comprise a simple opening formed at said end of the distal portion of the mount. According to one or more exemplary embodiments, said second transmission window comprises a second transparent blade which closes said end of said first tip. The second transparent blade comprises for example a glass slide or any other material transparent in the spectral band useful for microscopic analysis.

[0027] In the first mode of operation comprising in vivo microscopic analysis of the skin, and in the case where the first and second transmission windows respectively comprise first and second transparent blades, the second transparent blade, in combination with the first transparent blade, allows the use of a second immersion medium in contact with said first transparent blade and said second transparent blade.

[0028] Thus, according to one or more exemplary embodiments, the device further comprises a second immersion medium in contact with said first transparent slide and said second transparent slide. A second immersion medium comprises, for example, a liquid or a gel whose refractive index is between approximately 1.3 and approximately 1.5, that is to say a refractive index generally close to the object of study, skin or sample. A second immersion medium makes it possible to improve the quality of the microscopic analysis in the case of the use of an immersion microscope objective and to limit reflections at the interfaces, such as, for example, the interfaces with the transparent slides.

[0029] According to one or more exemplary embodiments, the second immersion medium is identical to the first immersion medium.

[0030] According to one or more exemplary embodiments, the second immersion medium is different from the first immersion medium and has mechanical, chemical and / or physical characteristics different from the first immersion medium. In particular, the second immersion medium may have a second refractive index substantially identical to the first refractive index of the first immersion medium or have a different refractive index. Using a second immersion medium different from the first immersion medium makes it possible, for example, to choose a second immersion medium which facilitates the transition from one operating mode to the other when the first immersion medium has characteristics making it complicated to handle.

[0031] Alternatively, said second transmission window may comprise a simple opening formed at said end of the first tip.

[0032] According to one or more exemplary embodiments, the sample holder comprises a receptacle with a third transmission window. For example, the third transmission window comprises a third transparent blade that closes the sample holder.

[0033] Such a transparent slide for closing the sample holder allows the sample to be pressed into the sample holder, thus allowing better repeatability of the microscopic analysis. On the other hand, in the second operating mode comprising the ex vivo microscopic analysis of a sample, the third transparent slide, in combination with a first transparent slide for closing the distal part of the mount, allows the use of a third immersion medium in contact with said first transparent slide and said third transparent slide, the third immersion medium not being in contact with the sample.

[0034] According to one or more exemplary embodiments, a thickness of said third blade is substantially identical to a thickness of the second blade; advantageously, said second and third blades also have substantially identical refractive index dispersion curves with wavelength, which makes it possible to limit the introduction of dispersion deviations and / or optical path variations in the case of the use of an interferometric system for microscopic analysis, when switching from one operating mode to another.

[0035] Thus, according to one or more exemplary embodiments, the device further comprises a third immersion medium in contact with said first transparent slide and said third transparent slide which closes the sample holder. A third immersion medium comprises, for example, a liquid or a gel whose refractive index is between approximately 1.3 and approximately 1.5, that is to say a refractive index generally close to that of the sample. A third immersion medium makes it possible to improve the quality of the microscopic analysis in the case of the use of an immersion microscope objective and to limit reflections at the interfaces.

[0036] According to one or more exemplary embodiments, the third immersion medium is identical to the first immersion medium.

[0037] According to one or more exemplary embodiments, the third immersion medium is different from the first immersion medium and has mechanical, chemical and / or physical characteristics different from the first immersion medium. In particular, the third immersion medium may have a third refractive index substantially identical to the first refractive index of the first immersion medium or have a different refractive index. Using a third immersion medium different from the first immersion medium makes it possible, for example, to choose a third immersion medium which facilitates the transition from one operating mode to another when the first immersion medium has characteristics making it complicated to handle.

[0038] According to one or more exemplary embodiments, the third immersion medium is different from the first immersion medium and comprises a third refractive index substantially identical to the refractive index of a transparent overlayer of the sample to be analyzed. According to one or more exemplary embodiments, the third immersion medium comprises a refractive index substantially identical to that of the sample to be analyzed.

[0039] According to one or more exemplary embodiments, the physical characteristics, refractive index and / or dispersions are substantially identical to those of the second immersion medium in order to avoid a modification of the optical path when switching from one operating mode to another.

[0040] Alternatively, said third transmission window may comprise a simple opening in the receptacle formed by the sample holder or the sample holder may comprise only the surface for receiving the sample. In these cases, however, it is always possible to use a third immersion medium which will be in contact with the sample. Advantageously, in this case, a first tip will be chosen in the first operating mode (in vivo microscopic analysis of the skin) without a transparent closing blade, to avoid variations in the optical path when changing from one operating mode to another.

[0041] According to one or more exemplary embodiments, the device further comprises a device configured for illuminating the sample and arranged at one end of said distal portion of the mount. Such a device may comprise light sources, for example light-emitting diodes, or may comprise ends of optical fibers configured to carry light beams. In particular, such an illumination device arranged at the end of the distal portion of the mount may remain in place in both operating modes, in vivo microscopic analyses of the skin and ex vivo of a sample. Such an illumination device arranged at the end of the distal portion of the mount may be part of a sample illumination path for microscopic analysis or may form an illumination of the sample additional to that of an illumination path.

[0042] According to one or more exemplary embodiments, the mount is configured to be handheld in the first operating mode. For example, the proximal portion of the mount comprises a handle. Equipped with a handle, the device makes it possible to form an ergonomic handheld probe for in vivo microscopic analysis of the skin.

[0043] According to one or more exemplary embodiments, the device further comprises means for axially moving the microscope objective in the distal part of the mount, along the optical axis. Such movement is particularly provided in devices configured for microscopy deep in the sample, for example and in a non-limiting manner, optical coherence microscopy with linear confocal filtering, or LC-OCT, with dynamic adjustment of the focus in the sample, in the first operating mode comprising in vivo microscopic analysis of the skin, or in the second operating mode comprising ex vivo microscopic analysis of a sample.

[0044] According to one or more exemplary embodiments, the device further comprises means for axial adjustment of the support in a direction parallel to said optical axis and / or means for lateral adjustment of the support in a plane substantially perpendicular to said optical axis. In the second operating mode, the mount comprising the distal part in which the microscope objective is arranged and the proximal part secured to the distal part is supported by the support also configured to allow the alignment of the optical axis of the microscope objective, thanks to the second end piece fixed to said distal part of the mount with which the support cooperates. Means for axial and / or lateral adjustment of the support also make it possible to adjust the position of the mount relative to the sample holder.

[0045] Of course, such adjustments can be made alternatively, or additionally, at the sample holder.

[0046] Thus, according to one or more exemplary embodiments, the device further comprises means for lateral adjustment of the sample holder in a plane substantially perpendicular to said optical axis and / or means for axial adjustment of the sample holder in a direction parallel to said optical axis.

[0047] According to one or more exemplary embodiments, the support can be fixed to the mount by other fixing points, for example to a handle arranged on the proximal part of the mount, to allow greater stability of the device in the second mode of operation.

[0048] According to one or more exemplary embodiments, the second end piece comprises an outer surface configured to cooperate with a surface of said support to enable said alignment. The outer surface may, for example, have different shapes to enable this self-alignment. It may, for example, be conical or cylindrical.

[0049] According to one or more exemplary embodiments, the second end piece is an element with rotational symmetry, with a central opening.

[0050] According to one or more exemplary embodiments, the device may further comprise means for fixing the second end piece with the support, for example the second end piece and the support may be magnetized to ensure mechanical cooperation of the two parts allowing alignment.

[0051] According to one or more exemplary embodiments, the device further comprises: an emission source of an illumination path configured to illuminate the sample or the skin; and / or a detector of a detection path comprising said microscope objective, said detector being configured to detect a light beam emitted by the illuminated sample or skin and generate a detection signal; said emission source and / or said detector being arranged in the proximal part of the mount.

[0052] According to one or more exemplary embodiments, the illumination path is configured to illuminate the sample through the microscope objective.

[0053] According to one or more exemplary embodiments, the device only comprises some of the distal elements of the lighting and / or detection paths, the other elements of the lighting and / or detection paths being located outside the device.

[0054] Thus, in order to operate, the device may be connected to external elements, for example and without limitation, one or more of the following elements: lighting source, detector, electrical energy source, controllers, processing unit, for example a computer.

[0055] According to a second aspect, the present description relates to a microscopic analysis system configured for ex vivo microscopic analysis of a sample and in vivo microscopic analysis of skin comprising a device according to the first aspect.

[0056] The microscopic analysis system according to the second aspect comprises: an illumination path configured to illuminate the sample or skin; a detection path comprising said microscope objective, said detection path being configured to detect a light beam emitted by the illuminated sample or skin and generate a detection signal; a processing unit configured to generate microscopic analysis information of the sample or skin from said detection signal.

[0057] All or part of the elements of the lighting and detection channels may be included in the device according to the first aspect.

[0058] According to one or more exemplary embodiments, the illumination path is configured to illuminate the sample through the microscope objective.

[0059] According to one or more exemplary embodiments, the analysis system according to the second aspect is configured for confocal imaging and / or optical coherence tomographic imaging, and said microscopic analysis information comprises at least one image of the sample or the skin.

[0060] For example, the analysis system according to the second aspect is configured for optical coherence tomographic imaging as described in the state of the art and is configured to form images B-Scans , C-Scans (or images en face ) of the sample or in volume. As is known, a cross-sectional image of the sample called " B-Scan ", is an image formed in a plane parallel to the optical axis of the microscope objective; a cross-sectional image of the sample called « C-Scan », or image en face , is an image formed in a plane perpendicular to the optical axis of the microscope objective, and a 3D image of the sample or image en volume results from the acquisition of a plurality of B-Scan images or C-Scan images.

[0061] According to one or more exemplary embodiments, the analysis system according to the second aspect is configured for spectroscopic analysis, and said microscopic analysis information comprises at least one spectrum of said light beam emitted by the sample or the skin at at least one point of the sample or the skin.

[0062] According to a third aspect, the present description relates to a method for ex vivo microscopic analysis of a sample and in vivo microscopic analysis of the skin by means of an analysis system according to the second aspect, comprising: attaching said first tip to the frame and in vivo microscopic analysis of the skin; removing said first tip, attaching said second tip to the frame and ex vivo microscopic analysis of a sample.

[0063] The method according to the third aspect may comprise at least one of the following steps, depending on the characteristics of the analysis system according to the second aspect, the steps being able to be combined with each other: filling the first tip before fixation, if a second immersion medium is used; positioning a third immersion medium between a first transparent blade configured to close the distal portion of the mount and the sample holder if a third immersion medium is used.

[0064] According to one or more exemplary embodiments, the microscopic analysis of the sample or skin comprises confocal and / or optical coherence tomographic imaging of the sample or skin.

[0065] According to one or more exemplary embodiments, the microscopic analysis of the sample or the skin comprises a spectroscopic analysis of the sample or the skin. Brève description des figures

[0066] Other advantages and characteristics of the invention will appear on reading the description, illustrated by the following figures: [ Fig. 1 ], already described, a diagram illustrating a waterproof mount for an in vivo microscopic skin imaging system according to the state of the art; [ Fig. 2A ], a diagram illustrating a first example of a microscopic analysis system according to the present disclosure, when operating in a first mode of operation comprising in vivo microscopic analysis of the skin; [ Fig. 2B ], a diagram illustrating the first example of a microscopic analysis system according to the present disclosure, when operating in a second mode of operation comprising ex vivo microscopic analysis of a sample; [ FIG. 2C ], a diagram illustrating a second example of a microscopic analysis system according to the present description, when operating in the second mode of operation; [ Fig. 3A ], a diagram illustrating a third example of a microscopic analysis system according to the present description, when operating in the first mode of operation; [ Fig. 3B ], a diagram illustrating the third example of a microscopic analysis system according to the present description, when operating in the second mode of operation; [ Fig. 4 ], diagrams illustrating steps of a method for in vivo microscopic analysis of the skin and ex vivo of a sample, using an analysis system according to the present description. Description détaillée de l'invention

[0067] In the following detailed description, numerous specific details are set forth in order to provide a more thorough understanding of the present disclosure. However, it will be apparent to those skilled in the art that the present disclosure may be implemented without these specific details. In other instances, well-known features have not been described in detail to avoid unnecessarily complicating the disclosure.

[0068] Furthermore, in the figures, the elements are not shown to scale for better visibility.

[0069] THE FIGS. 2A And 2B represent a first example of a microscopic analysis system 200 according to the present description, configured for ex vivo microscopic analysis of a sample and in vivo microscopic analysis of the skin. The FIG. 2A represents the microscopic analysis system when operating in the first mode of operation, namely in vivo microscopic analysis of the skin, and the FIG. 2B represents the microscopic analysis system when operating in the second mode of operation, namely ex vivo microscopic analysis of a sample.

[0070] The microscopic analysis system 200 comprises a device 201 for microscopic analysis according to the present description. The device 201 generally comprises a microscope objective 210 comprising an optical axis Δ, a mount 220 comprising a distal portion 221 in which said microscope objective is arranged and a proximal portion 224 secured to said distal portion. The microscope objective may in certain exemplary embodiments be axially movable in a direction parallel to the optical axis. The proximal portion 224 generally comprises at least a portion of the optical elements of a detection path of the microscopic analysis system and may comprise at least a portion of the optical elements of an illumination path.

[0071] In the example of the FIGS 2A And 2B, at least a portion of the optical elements of the lighting and detection paths arranged within the device are shown diagrammatically by the reference 280. In certain exemplary embodiments, the optical elements 280 may comprise, in a non-limiting manner, one or other of the following elements: a lighting source, an interferometric assembly, a detector, one or more optical systems making it possible to ensure optical conjugations between image planes or pupil planes, one or more deflecting mirrors, one or more scanning systems, etc. The arrangement of such optical elements is known to those skilled in the art and depends on the microscopic analysis that one seeks to carry out.

[0072] In the example of the FIGS 2A And 2B , an illumination channel of the microscopic analysis system 200, configured to illuminate the sample 102 ( FIG. 2B ) or skin 101 ( FIG. 2A ) through the microscope objective 210, comprises for example an illumination source 294, for example a laser source, arranged outside the device 201. Furthermore, a detection path of the microscopic analysis system 200, configured to detect a light beam emitted by the illuminated sample or skin and generate a detection signal, and also comprising the microscope objective 210, comprises for example a detector 292, for example a camera, also arranged outside the device 201. In the example of the FIGS 2A And 2B, the microscopic analysis system 200 also comprises a processing unit 290 configured to generate microscopic analysis information of the sample or of the skin from the detection signal and, optionally, one or more controllers 296 for controlling the optical elements arranged in the proximal part of the device 201. The controller(s) are for example electronic cards configured to control an axial movement of the microscope objective 110 if applicable, or mirrors of a scanning system, etc.

[0073] The controller(s), as well as the source 294, may themselves be controlled by the processing unit 290.

[0074] When the analysis system 200 is configured for confocal imaging and / or optical coherence tomographic imaging, said microscopic analysis information comprises at least one image of the sample or the skin. When the analysis system 200 is configured for spectroscopic analysis, said microscopic analysis information comprises at least one spectrum of said light beam emitted by the sample or the skin at at least one point of the sample or the skin.

[0075] Note that in other embodiments, the lighting source of the lighting path and / or the detector of the detection path can be integrated into the proximal part 224 of the mount 220, with the optical elements 280.

[0076] In other embodiments, the illumination path may not include the microscope objective, with illumination of the sample for microscopic analysis being provided directly at an illumination device 228 arranged in the distal portion of the mount. Such an illumination device may include illumination sources, for example of the light-emitting diode type, or optical fiber ends configured to carry the illumination beam.

[0077] There FIG. 2A represents the microscopic analysis system 200 in an in vivo microscopic analysis operation of the skin, referenced 101 on the FIG. 2A .

[0078] As can be seen on the FIG. 2A , the distal portion 221 of the mount 220 comprises at one end a first transmission window closed by a first transparent blade 222. The device 201 further comprises a first end piece 230 fixed to the distal portion 221 of the mount 220. The first end piece is removably mounted, for example positioned by means of a stop 235 formed in the distal portion 221 of the mount. For example, the first end piece is screwed, and the stop 235 may be formed by a locknut. In exemplary embodiments, the position of the stop may be adjusted on the distal portion of the mount.

[0079] In the example of the FIG. 2A , the first end piece 230 comprises at one end a second transmission window closed by a second transparent blade 231.

[0080] The device 201 also comprises a first immersion medium 241, liquid or gel, in contact with the first transparent slide 222 and the microscope objective 210 and a second immersion medium 242, liquid or gel, in contact with the first transparent slide 222 and the second transparent slide 231.

[0081] The original layout as shown in the FIG. 2A with the first tip 230 fixed on the distal part 221 of the mount allows, in the context of the use of an immersion microscope objective 210, to split the immersion into two parts. In particular, the first immersion medium 241 and second immersion medium 242 may have different characteristics, for example different mechanical, chemical and / or physical characteristics, in particular refractive index and / or dispersion.

[0082] A two-part immersion with different immersion media may for example be useful in a case where the microscope objective is designed to be immersed in an immersion medium 241 whose characteristics are close to those of the skin, but where the first immersion medium 241 also has characteristics (in particular mechanical or chemical) making it complicated or dangerous for a user of the device to handle. A second different immersion medium 242 may then be used, this second immersion medium having to be handled by the user to switch from one operating mode to the other.

[0083] A two-part immersion with different immersion media may also be useful in the case where the first immersion media 241 has physicochemical characteristics making it difficult to separate the distal part 221 of the mount and the first tip 230 in in vivo use mode (first immersion media 241 too sticky, for example).

[0084] When a second immersion liquid is used, the first tip can be tightly fixed to the distal part of the mount, for example by means of a seal provided on one and / or the other of the two parts.

[0085] Note that the transmission window of the first tip can also consist of a simple opening. It will nevertheless be possible to use a microscope objective 210 configured to operate in immersion thanks to the tank formed by the distal part 221 of the mount closed by the blade 222. In this case, the immersion medium of the skin will be air. As illustrated in the FIG. 2A , the distal part 221 of the mount can also integrate elements useful for the operation of the system and which need to be close to the skin or the sample to be analyzed, such as for example a lighting device 228. The lighting device 228 can remain in place when changing the ex vivo microscopic analysis operating mode ( FIG. 2B ).

[0086] There FIG. 2B represents the microscopic analysis system 200 when it operates in a second operation, namely the ex vivo microscopic analysis of a sample, referenced 102 on the FIG. 2B .

[0087] As can be seen on the FIG. 2B , the device 201 comprises, for this mode of operation, a second tip 250 fixed to said distal part 221 of the frame 220 in place of the first tip 230 ( FIG. 2A ). In this example, the second end piece is a part with rotational symmetry which includes a central opening. The second end piece 250 is for example positioned by means of the stop 235. For example, the second end piece is screwed and the stop 235 can be formed by a lock nut.

[0088] The device 201 further comprises a sample holder 260 comprising a receiving surface 263 configured to receive the sample 102 and a support 270. The support 270 cooperates with the second end piece 250 so as to receive the mount 220 such that the optical axis of the microscope objective is substantially aligned with a predetermined direction relative to the receiving surface, for example a direction substantially perpendicular to the receiving surface. More specifically in this example, the alignment of the optical axis is obtained by making an outer surface of the second end piece 250 cooperate with a surface of said support 270. In this example, the shapes of the surfaces which cooperate are conical.

[0089] In exemplary embodiments (not shown in the figures), the device may comprise a system for fine adjustment of the alignment of the optical axis of the microscope objective, to refine the adjustment after the self-alignment obtained thanks to the cooperation of the second end piece 250 and the support 270.

[0090] For example, as illustrated in the FIG. 2B , the device 201 further comprises (optional) means 275 for axial adjustment of the support 270 in a direction parallel to the optical axis Δ and / or means for lateral adjustment of the support in a plane substantially perpendicular to the optical axis. The device may also comprise (optional) means 265 for lateral adjustment of the sample holder in a plane substantially perpendicular to said optical axis and / or means for axial adjustment of the sample holder in a direction parallel to said optical axis. These adjustment means aim, in operation, to adjust the relative position of the microscope objective 210 and the sample. In particular, the axial adjustment makes it possible to ensure that the part of the sample for which microscopic analysis is desired is positioned at the required working distance from the microscope objective.

[0091] In the example of the FIG. 2B , the adjustment means 265 and 275 are integral with the same platform 20 which may or may not be part of the device 201.

[0092] The adjustment means 265 and / or 275 can themselves be controlled by the processing unit 290.

[0093] Generally speaking, the support 270 can be configured to support the mount 220 alone by means of the second end piece 250. However, to reinforce the stability of the device, in operation, it is possible to provide other points of attachment of the support 270 with the mount 220. Thus, for example, once the mount 220 is positioned on the support 270, it will be possible to attach the support 270 to the handle 225 of the device 201, as shown diagrammatically with dotted lines on the FIG. 2B .

[0094] As illustrated in the FIG. 2B , the sample holder 260 comprises a receptacle with a third transmission window closed by a third transparent blade 262. The third transparent blade is for example arranged in a cover 261 of the sample holder. For example, a thickness of the third transparent blade 262 is substantially identical to a thickness of the second transparent blade 231. With substantially identical blade thicknesses, the effects of dispersion deviations and optical path variations are limited, when switching from one operating mode to another, in particular in the case where the microscopic analysis uses an interferometric system.

[0095] The third transparent slide 262 allows the sample to be placed in the sample holder, thus allowing better repeatability of the microscopic analysis. On the other hand, as illustrated in the FIG. 2B , the third transparent blade 262, in combination with the use of the first transparent blade 222 to close the distal portion 221 of the mount, allows the use of a third immersion medium 243 in contact with the first transparent blade 222 and the third transparent blade 262, without the third immersion medium 243 being in contact with the sample 102, which can be troublesome.

[0096] The third immersion medium 243 comprises, for example, a liquid or a gel whose refractive index is between approximately 1.3 and approximately 1.5, that is to say a refractive index generally close to that of the sample. The third immersion medium 243 makes it possible to improve the quality of the microscopic analysis in the case of the use of an immersion microscope objective and to limit reflections at the interfaces.

[0097] The third immersion medium may be identical to the first immersion medium 241 or have mechanical, chemical and / or physical characteristics different from the first immersion medium. In particular, the third immersion medium may have a third refractive index substantially identical to the first refractive index of the first immersion medium or have a different refractive index. In the ex vivo microscopic analysis operating mode as shown in the FIG. 2B , a two-part immersion with different immersion media 241, 243 may be useful in the case where the first immersion media 241 has physicochemical characteristics making it difficult to separate the distal part 221 from the mount and the sample holder 260, for example because the first immersion media 241 is too sticky.

[0098] A two-part immersion with different immersion media 241, 243 is also useful if the first immersion media 241 has characteristics that make it difficult to interface the distal part of the mount with the sample holder 260, for example due to insufficient surface tension to place a drop that will maintain a sufficient working distance.

[0099] Furthermore, it may happen that certain samples for which microscopic analysis is sought are conditioned with an overlayer of a transparent medium, which may be different from the first immersion medium 241 and of variable thickness. In this case, a third immersion medium may be used whose physical characteristics, for example refractive index and / or dispersion, are substantially identical to the medium of the overlayer, and the thickness of the third immersion medium may be adapted to the thickness of the overlayer to limit the variation of the optical path when switching from one operating mode to another. This is particularly important when the microscopic analysis system comprises an interferometer with a fixed reference arm for example.

[0100] In the example of the FIG. 2B , the alignment of the optical axis is obtained by making a conical outer surface of the second end piece 250 cooperate with a conical surface of said support 270.

[0101] There FIG. 2C shows another exemplary embodiment in which a cylindrical outer surface of the second end piece 250 cooperates with a cylindrical surface of the support 270. Of course, other embodiments may be envisaged by those skilled in the art to obtain self-alignment of the optical axis thanks to the cooperation of the second end piece 250 and the support 270, and in particular to make an outer surface of the second end piece 250 cooperate with a surface of the support 270 so as to allow the alignment of the optical axis of the microscope objective 210 in a predetermined direction relative to the receiving surface 263. Thus, for example, one surface may be equipped with a rolling system (for example balls) configured to fit into grooves of the other surface, or any other means known to those skilled in the art.

[0102] Furthermore, the device may further comprise means for fixing the second end piece with the support, means not shown in the FIGS 2B , 2C , for example the second end piece and the support can be magnetized to ensure mechanical cooperation of the two parts allowing alignment.

[0103] THE FIGS. 3A And 3B represent a second example of a microscopic analysis system 200 according to the present disclosure, configured for ex vivo microscopic analysis of a sample and in vivo microscopic analysis of skin. The FIG. 3A represents the microscopic analysis system in a first operating mode of in vivo microscopic analysis of the skin and the FIG. 3B represents the microscopic analysis system in a second mode of operation for ex vivo microscopic analysis of a sample.

[0104] The elements of the FIGS 3A And 3B similar to those of FIGS 2A And 2B are referenced identically and are not described again.

[0105] As can be seen on the FIG. 3A , in this example of realization and unlike the FIG. 2A , the distal portion 221 of the mount 220 comprises a transmission window formed by a single opening 322. In the same way, the first tip 230 comprises a transmission window formed by a single opening 331. Thus in this example, the microscope objective and the skin are immersed in the air. The distal portion 221 makes it possible in this example to protect the microscope objective 210, in particular when changing from the first operating mode of in vivo microscopic analysis to the second operating mode of ex vivo microscopic analysis, a change during which an operator removes the first tip to position the second tip (250, FIG. 3B ). Moreover,

[0106] The distal portion 221 may incorporate a lighting device, as previously described by means of the FIGS 2A - 2C .

[0107] Moreover, as can be seen on the FIG. 3B , the sample holder 260 may also not be closed by a transparent blade.

[0108] There FIG. 4 represents diagrams illustrating steps of a method for in vivo microscopic analysis of the skin and ex vivo of a sample, using an analysis system according to the present description. On the FIG. 4 , only the distal part of the device is shown. Furthermore, the device shown is the one illustrated on the FIGS. 2A , 2B but of course, the process illustrated by means of the FIG. 4 may apply to other examples of realization. As illustrated in the FIG. 4 , the microscopic analysis method comprises fixing 41 the first tip 230 on the mount 220 and an in vivo microscopic analysis of the skin, then removing 42 the first tip 230 and fixing (steps 43, 44) the second tip 250 on the mount 220. The device with the second tip 250 can then be mounted on the support (270, FIG. 2B ) in order to carry out ex vivo microscopic analysis of a sample. The device can then be used again for in vivo microscopic analysis of the skin by removing the second tip and replacing it with a first tip. Note that in practice, the first tip may be a consumable item that will be changed for each new in vivo use.

[0109] There FIG. 4 illustrates how, by means of the device according to the present description, self-alignment of the optical axis of the microscope objective can be achieved when switching from the first operating mode of in vivo microscopic analysis to the second operating mode of ex vivo microscopic analysis.

[0110] There FIG. 4 also shows an advantage of the presence of the first tip 230 fixed on the distal part 221 of the mount. Indeed, thanks to the presence of the distal part, when removing the first tip to fix the second tip 250, the microscope objective 210 is protected.

[0111] Furthermore, in the case of a 210 immersion microscope objective, as illustrated in the FIG. 4 , the seal is ensured thanks to the distal part 221 of the frame closed by the transparent window 222 and which remains in place when the first tip 230 is removed to put in place the second tip 250.

[0112] For in vivo microscopic analysis of the skin, the first tip 230 is used as a "plug" which simply needs to be filled with the second immersion medium 242, liquid or gel. The first tip can be tightly attached to the distal portion of the mount.

[0113] For ex vivo microscopic analysis of a sample, a third immersion liquid (not shown in the FIG. 4 ) can be arranged between the sample holder and the transparent slide 222.

[0114] For example, for samples conditioned with an overlayer of a transparent medium different from the first immersion medium 24, it will be possible to choose a third immersion medium whose physical characteristics, for example refractive index and / or dispersion, are substantially identical to the medium of the overlayer, and to adapt the thickness of the third immersion medium to the thickness of the overlayer to limit the variation of the optical path when changing from one operating mode to another.

[0115] Although described through a certain number of exemplary embodiments, the devices and methods according to the present description include various variants, modifications and improvements which will be obvious to those skilled in the art, it being understood that these various variants, modifications and improvements are part of the scope of the invention as defined by the following claims. Références

[0116] Ref. 1: M. Rajadhyaksha et al, “In vivo confocal scanning laser microscopy of human skin II: Advances in instrumentation and comparison with histology”, J Invest Dermatol, 1999. Ref 2: K. König et al, “High-resolution multiphoton tomography of human skin with subcellular spatial resolution and picosecond time resolution,” J. Biomed. Opt. 8, 432-439 (2003). Ref. 3: Schmitt et al., "Subsurface Imaging of Living Skin with Optical Coherence Microscopy", Dermatology 1995;191:93-98. Ref. 4: Published patent application WO2015092019. Ref 5: Y. Chen et al. "High-resolution line-scanning optical coherence microscopy", Optics Letters, Vol. 32, No. 14, 1971 - 1973 (2007). Ref 6: J. Schleusener et al., "Raman spectroscopy for the discrimination of cancerous and normal skin", Photon Lasers Med (2015). Ref 7: E. Drakaki et al. "Spectroscopy methods for the photodiagnosis of nonmelanoma skincancer", Journal of Biomedical Optics 18(6), 061221 (June 2013). Ref 8: Published patent application WO2019138062.Ref. 9: EP 3 018 518 Ref. 10: WO 2016 / 083003 Ref. 11: DE 10 2009 044 413 Ref. 12: Patent US10514532.

Claims

1. A device (201, 301) which is configured to operate in a first operating mode comprising an in vivo microscopic analysis of the skin and in a second operating mode comprising ex vivo microscopic analysis of a sample, the device comprising: - a microscope lens (210) comprising an optical axis (Δ); - a mounting (220) comprising a distal portion (221), in which the microscope lens is arranged, and a proximal portion (224) which is fixedly joined to the distal portion, the distal portion (221) comprising at one end a first transmission window; - a first end piece (230) which comprises at one end a second transmission window and which is configured to be fixed in a removable manner, in the first operating mode, to the distal portion (221) of the mounting (220); - a second end piece (250) which is configured to be fixed in a removable manner, in the second operating mode, to the distal portion (221) of the mounting (220); - a sample carrier (260) comprising a receiving surface (263) which is configured to receive a sample in the second operating mode; - a support (270) which is configured to cooperate with the second end piece (250) in the second operating mode so as to receive the mounting (220) so that the optical axis of the microscope lens is substantially aligned in a predetermined direction with respect to the receiving surface.

2. The device as claimed in claim 1, wherein the first transmission window comprises a first transparent plate (222) which closes the end of the distal portion (221) of the mounting.

3. The device as claimed in claim 2, further comprising a first immersion medium (241) in contact with the first transparent plate and the microscope lens (210).

4. The device as claimed in one of the preceding claims, wherein the second transmission window comprises a second transparent plate (231) which closes the end of the first end piece (230).

5. The device as claimed in claim 4, wherein - the first transmission window comprises a first transparent plate (222) which closes the end of the distal portion (221) of the mounting; and - the device further comprises a second immersion medium (242) in contact with the first transparent plate (222) and the second transparent plate (231).

6. The device as claimed in one of the preceding claims, wherein the sample carrier comprises a third transparent plate (262) which closes the sample carrier.

7. The device as claimed in claim 6, wherein - the first transmission window comprises a first transparent plate (222) which closes the distal portion (221) of the mounting; and - the device further comprises a third immersion medium (243) in contact with the first transparent plate (222) and the third transparent plate (262).

8. The device as claimed in one of the preceding claims, further comprising a device (228) which is configured for illuminating the sample and which is arranged at one end of the distal portion (221) of the mounting.

9. The device as claimed in one of the preceding claims, wherein the mounting (220) is configured to be handheld in the first operating mode.

10. The device as claimed in one of the preceding claims, further comprising axial displacement means for the microscope lens (210) in the distal portion (221) of the mounting, along the optical axis (Δ).

11. The device as claimed in one of the preceding claims, further comprising axial adjustment means (275) of the support (270) in a direction parallel with the optical axis (Δ) and / or lateral adjustment means of the support in a plane substantially perpendicular to the optical axis and / or lateral adjustment means (265) of the sample carrier in a plane substantially perpendicular to the optical axis and / or axial adjustment means of the sample carrier in a direction parallel with the optical axis.

12. The device as claimed in one of the preceding claims, wherein the second end piece (250) comprises an external surface which is configured to cooperate with a surface of the support (270) to allow said alignment.

13. A microscopic analysis system (200) which is configured to operate in a first operating mode comprising in vivo microscopic analysis of the skin and in a second operating mode comprising ex vivo microscopic analysis of a sample, the system comprising: - a device as claimed in one of claims 1 to 12; - an illumination path which is configured to illuminate the sample or the skin; - a detection path comprising the microscope lens, the detection path being configured to detect a light beam which is emitted by the sample or the skin which is illuminated and to generate a detection signal; - a processing unit (290) which is configured to generate a microscopic analysis information item for the sample or the skin from the detection signal; and wherein - all or some of the elements of the lighting and detection paths are included in the device.

14. The analysis system as claimed in claim 13, which is configured for confocal imaging and / or optical coherence tomography imaging, wherein the microscopic analysis information item comprises at least one image of the sample or the skin and / or configured for spectroscopic analysis, wherein the microscopic analysis information item comprises at least one spectrum of the light beam which is emitted by the sample or the skin at least at one location of the sample or the skin.

15. A method for ex vivo microscopic analysis of a sample and in vivo microscopic analysis of the skin by means of an analysis system as claimed in claim 13, comprising: - removing the second end piece (250), fixing the first end piece (230) to the mounting (220) and doing an in vivo microscopic analysis of the skin; - removing the first end piece (230), fixing the second end piece (250) to the mounting (220) and doing an ex vivo microscopic analysis of a sample.

16. The method for analysis of a sample as claimed in claim 15, wherein the microscopic analysis of the sample or the skin comprises confocal imaging and / or optical coherence tomography imaging of the sample or the skin and / or comprises spectroscopic analysis of the sample or the skin.