A module intended to be associated with a microscope and an assembly formed by a microscope and said module

Abstract

The present invention relates to a module associated with a microscope for full-field optical coherence tomography microscopy imaging of at least one sample, the module comprising an interferometric device (4) comprising a non-polarizing beam splitting element (10) for forming two arms when illuminated by a light source, namely a "reference arm" associated with a reflecting surface and an "object arm" associated with the sample, the device further comprising a first adjustment unit (11) arranged upstream of the non-polarizing beam splitting element so that the illuminated arm of the non-polarizing beam splitting element can be modified during operation, and at least one second adjustment unit (18) arranged between the beam splitting element and the reflecting surface so that the reference arm can be modified during operation.

Description

[Technical field]

[0001] The present invention relates to a module intended to be associated with a microscope for full-field optical coherence tomography microscopic imaging of at least one sample.

[0002] The invention also relates to an assembly formed by such a module and a microscope. [Background technology]

[0003] The recent advent of 3D cell culture techniques allows for more accurate mimicking of human physiology compared to traditional 2D cell culture, on which many drug development and other biomedical applications (e.g. tissue transplants) are now based.

[0004] In this field, the advent of recent organoid technology is promising, since it allows to overcome at least some of the problems associated with creating three-dimensional human tissues and, in particular, replicating physiological conditions in vivo. Moreover, this organoid technology allows for the unlimited production of materials that can be described as "human" without any rational constraints, and therefore in the long term, it may lead to fewer or even no studies on animal models. Thus, organoids can be used to model diseases, test the efficacy of new treatments (genetic, pharmaceutical, etc.), transplants, etc. However, despite the maturity of organoid technology, it remains difficult to perform non-destructive controlled imaging with sufficient accuracy to confirm the proper functioning of said organoids. In particular, conventional imaging techniques require fixation of the sample, i.e., the sample must be destroyed or irreversibly altered, which can lead to non-factual analyses and biased decision-making. Furthermore, conventional imaging techniques require growth of the sample (and thus additional cost), resulting in additional variability, and the inability to confirm the viability and quality of the transplanted sample.

[0005] Moreover, the current state of the art does not allow for sufficiently quantitative three-dimensional controlled imaging to ensure a certain level of cellular or subcellular precision. Optical coherence tomography (OCT) imaging allows images to be obtained with greater precision. For this purpose, full-field optical coherence tomography imaging is based on obtaining an image signal from the interference between a light signal backscattered when the sample is illuminated by a light source and a reference light signal emitted by the light source. Patent FR 2 817 030 describes an example of a full-field optical coherence interference microscope imaging system.

[0006] Unfortunately, the use of these systems is not widespread due to their complexity and cost. [Prior art documents] [Patent documents]

[0007] [Patent Document 1] French Patent No. 2817030 Summary of the Invention [Problem to be solved by the invention]

[0008] An object of the present invention is in particular to enable full-field optical coherence tomography microscopic imaging of at least one sample, which is simple to implement. [Means for solving the problem]

[0009] To this end, the present invention provides a module intended to be associated with a microscope for full-field optical coherence tomography microscopic imaging of at least one sample, the module comprising an interferometer equipped with a non-polarizing beam splitting element, the interferometer further comprising a reflecting surface, the interferometer thus comprising, in operation: at least one reference wave obtained by reflecting light emitted by a light source associated with said module on said reflecting surface; At least one interference can be generated between the light emitted by the light source and at least one object wave obtained by backscattering the light onto the sample, Thus, in operation, the non-polarizing beam splitting element, when illuminated by a light source, allows the formation of two arms, a "reference arm" associated with the reflecting surface and an "object arm" associated with the sample; The apparatus further comprises: a first adjustment unit arranged upstream of the non-polarizing beam splitting element so that the illumination arm of the non-polarizing beam splitting element can be modified during operation, and / or A module is provided that comprises at least one second adjustment unit arranged between the beam splitting element and the reflecting surface so that the reference arm can be modified during operation.

[0010] As a result, the present invention can be coupled to existing microscopes to perform full-field optical coherence tomography microscopy imaging. It is sufficient to associate the present invention with a microscope and then use an adjustment unit to ensure interference between the reference wave and the object wave.

[0011] For example, the second unit is adjusted so that when the microscope is coupled to the module, the reference arm is identical to the object arm, i.e., the optical path in the two arms is identical (same as the spatial coherence length of the light source).

[0012] For example, during operation, the first adjustment unit is adjusted to align the action axis of the module (i.e., the axis along which the majority of the light rays propagating on the object arm exit the module and reach the microscope) with the propagation axis of the light source (i.e., the axis along which the majority of the light rays generated by the light source propagate).

[0013] Thus, the use of such optical coherence tomography microscope images is made easier for the user since they are already used in the relevant microscopy tasks.

[0014] Furthermore, once the present invention is coupled to and adjusted with a microscope, anyone using the microscope can benefit from the advantages of the present invention without having to touch it, and as a result, the present invention provides easier access to full-field optical coherence tomography microscopy imaging.

[0015] Advantageously, it is therefore possible to couple the invention to a conventional microscope, ie an existing microscope available on the market, by connecting the invention to the conventional microscope and then adjusting the microscope.

[0016] This again provides easier access to full-field optical coherence tomography microscopy imaging, especially full-field optical coherence tomography microscopy imaging in combination with a conventional microscope (because the user need no longer be concerned with the present invention once it is coupled to a microscope).

[0017] Furthermore, the present invention advantageously allows to benefit from the advantages of conventional microscopes, such as special imaging properties (visible light imaging properties, fluorescence imaging properties, etc.), special targets (objectives), etc. In particular, the relevant microscopes can be microscopes for which other optical modules have already been developed, such as differential interference contrast modules (DIC), coherent anti-Stokes Raman scattering modules (CARS), second harmonic generation modules (SHG), third harmonic generation modules (THG), Raman modules, single-photon or two-photon fluorescence modules, etc. The modules of the present invention therefore allow easy coupling with these prior art modules, thereby expanding the possibilities for imaging samples. Furthermore, the relevant microscopes can be microscopes for which accessories have already been developed, so that by connecting the present invention to said microscopes it is possible to take advantage of the accessories of the microscope.

[0018] The present invention can be connected to a standard microscope and therefore advantageously has access to many options already developed for that microscope.

[0019] Furthermore, the invention makes it possible to carry out different types of full-field optical coherence tomography microscopic imaging. In particular, the invention thus makes it possible to make possible both full-field optical coherence tomography (FFOCT) imaging, i.e. so-called "static" imaging, and dynamic full-field optical coherence tomography (D-FFOCT) imaging. The invention thus makes it possible, for example, to observe (statically image) elements present in a sample as well as elements constituting the organization of one of the structures of the sample. These elements may be, for example, cells, structures, in particular subcellular structures such as nuclei, mitochondria, pigments, etc.

[0020] Thus, the present invention can be used in many different applications and / or in different modes of operation (static or dynamic imaging).

[0021] The present invention is therefore particularly modular.

[0022] Additionally, the present invention can reduce the cost of optical coherence tomography microscope imaging since the present invention can be added to existing microscopes.

[0023] For the purposes of the present invention, "reference arm" means the part of the module located between the reflective surface and the non-polarizing beam splitting element (light rays can propagate in said reference arm along a given optical path).

[0024] In the present invention, "object arm" means the part of the assembly formed by the module and the microscope that is located between the sample and the non-polarizing beam splitting element (a light beam can propagate in the object arm along a defined optical path).

[0025] In the present invention, "illumination arm" means the part of the assembly formed by the module and the microscope, which is arranged between the light source and the non-polarizing beam splitting element (a light beam can propagate in the object arm along a predetermined optical path).

[0026] In the present invention, "modification of a propagation characteristic" means, for example, modifying the relative position of the frame of the module with respect to the light source, e.g. modifying the relative position of the frame with respect to the propagation axis of the light source and / or the angle of incidence at which the light rays generated by the light source reach the non-polarizing beam splitting element.

[0027] Optionally, the first adjustment unit comprises at least two reflective surfaces, at least one of which is movable relative to the frame of the module.

[0028] Optionally, the module comprises an objective lens associated with the reference arm, and the second adjustment unit comprises at least one further reflective surface and at least one member for moving said objective lens relative to a frame of the module.

[0029] Optionally, the further reflective surface is also moveable relative to the frame of the module.

[0030] Optionally, the moving member is configured to move the objective lens at least in a plane parallel to a plane in which the first reflecting surface extends.

[0031] Optionally, the light source is part of the module.

[0032] Optionally, the module comprises an acquisition device suitable for acquiring at least one signal resulting from the interference between the reference wave and the target wave.

[0033] Optionally, the interferometric device comprises an optical system arranged between the splitting element and an output of a module intended to be at least optically coupled to the microscope.

[0034] The invention also relates to a microscope and an assembly comprising the above-mentioned module.

[0035] Optionally, the microscope includes a turret, and the module is disposed in one of a plurality of compartments of the turret.

[0036] Optionally, the assembly includes an incubator supported by the stage of the microscope.

[0037] Other characteristics and advantages of the invention will become apparent on reading the following description of particular, non-limiting embodiments of the invention.

[0038] Reference is made to the attached drawings, in which: [Brief description of the drawings]

[0039] [Figure 1] FIG. 1 is a diagram of an assembly formed by a microscope and a module in accordance with a particular embodiment of the invention. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0040] FIG. 1 illustrates a schematic representation of an assembly for full-field optical coherence tomography microscopy imaging of at least one sample, according to a particular embodiment of the invention.

[0041] The assembly 1 therefore comprises at least one microscope 2 and a module 3 connected to the microscope 2 .

[0042] As an example, the module 3 is shaped to be arranged in one of several compartments of a bank of compartments of the microscope 2, said bank forming a turret.

[0043] By shaping the module 3 such that it is a module that can be placed in a compartment of the microscope 2, the module 3 can be coupled (both mechanically and optically) to said microscope 2 in a very simple manner by simply inserting the module 3 into the microscope 2. In particular, the microscope 2 is shaped to have at least one optical output pointing towards at least one bank, such that a module inserted in said bank is effectively optically coupled to the microscope 2.

[0044] The microscope 2 is, for example, an inverted microscope, which is a microscope for observing a sample from below.

[0045] In this case, the microscope is advantageously a conventional microscope, for example the microscope 2 is an IX83 sold by the company Olympus.

[0046] In a manner known per se, the microscope 2 comprises a stage intended to support a sample to be observed, this stage being translatable relative to the frame of the microscope 2 along at least two translation axes.

[0047] As mentioned above, the microscope also comprises a turret for inserting modules for interacting with the microscope 2 .

[0048] The module 3 comprises an interferometric device 4, which in this case comprises a light source 5 and a reflective surface 6, which in operation: A reference wave obtained by reflecting light emitted from a light source 5 on a reflecting surface 6; and Optical interference between the light emitted by the light source 5 and the target wave obtained by backscattering the light onto a sample can be generated.

[0049] The light source 5 is a spatially incoherent light source or a light source with a short spatial coherence length (for example in the range of 0.5 to 120 micrometers, or for example in the range of 0.8 to 100 micrometers, or for example in the range of 1 to 20 micrometers). The light source 5 is for example a block formed by a halogen lamp, a light emitting diode (LED) or a coherent light source (for example a laser) and a structure through which a light beam passes at the output of the coherent light source, making it possible to make the light beam spatially incoherent at the output of the structure (and thus the block). As an example, this structure comprises a multimode cavity, a multimode fiber, a hexagonal bar, etc.

[0050] The reflecting surface 6 is a flat surface, for example a mirror.

[0051] In this case, the assembly 1 also comprises an acquisition device 7, which in this case is part of the module 3. The acquisition device 7 makes it possible to acquire at least one signal resulting from the interference between a reference wave and a target wave.

[0052] For this purpose, the acquisition device 7 comprises an optical sensor, which is preferably a complementary metal-oxide semiconductor (CMOS) optical sensor.

[0053] Preferably, the optical sensor is selected to acquire images at a high rate. This allows to follow a sequence of movements in the sample, if necessary, when the sample contains at least one living cell. For example, the optical sensor can acquire images at frequencies higher than 100 Hz, preferably higher than 200 Hz, preferably higher than 400 Hz.

[0054] Preferably, the optical sensor is selected to acquire images with a high signal-to-noise ratio, which allows the optical sensor to be sensitive to even very small anatomical structures and / or very small movements. For example, the optical sensor may acquire images with a signal-to-noise ratio of greater than 500, preferably greater than 800, preferably greater than 1000.

[0055] As an example, the light sensor is a camera, such as a CMOS camera, such as the Q-2A750 camera or the Q-2HFW camera, both sold by Adimec Corporation.

[0056] In this case, the assembly 1 also comprises a device 8 for processing the signals emitted by the acquisition device 7, for example to generate an image of at least one portion of the sample. However, in this case the signal processing device 8 is external to the module 3. The signal processing device 8 comprises at least one processing unit, such as a processor. By way of example, the signal processing device 8 comprises a computer.

[0057] Preferably, the assembly 1 comprises an incubator 9 for the microscope. By way of example, the incubator 9 is arranged within the microscope 2 such that it is supported by a stage of the microscope 2, and the sample is placed directly within the incubator 9.

[0058] The incubator 9 is preferably portable so that it can be temporarily connected to the microscope 2 .

[0059] The incubator 9 allows samples containing at least one living cell to be studied in a simpler way, in particular by means of the incubator 9 making it possible to keep such samples alive for several days or weeks.

[0060] The incubator 9 is preferably configured to accommodate multi-well plates.

[0061] The incubator 9 is preferably equipped with a temperature control device within the incubator 9 .

[0062] This makes it possible, for example, to prevent heating of the sample, in particular due to the internal illumination of the microscope 2, which could alter or destroy the sample.

[0063] The incubator 9 is preferably equipped with a control for the presence of at least one gas in the incubator, for example the incubator is equipped with a control for the presence of carbon dioxide in the incubator and / or for the presence of nitrogen in the incubator and / or for the presence of oxygen in the incubator.

[0064] If the sample comprises live cells, the incubator 9 is preferably configured to keep the sample at a given temperature, for example a temperature substantially equal to 37°C (e.g. for human, primate or porcine cells, etc.). Preferably, the incubator 9 is configured to allow the sample to be oxygenated (in particular by supplying a mixture of nitrogen and dioxygen and venting carbon dioxide), this oxygenation being ensured by controlling at least the level of carbon dioxide within the incubator. The incubator 9 is preferably configured to allow the humidity level of the sample to be managed so that the sample does not dry out. For example, the incubator 9 is equipped with a sensor to ensure that the humidity level within the incubator 9 is between 70 and 100%.

[0065] In this case, the incubator 9 is advantageously a conventional incubator, for example the H201-K ​​incubator sold by the company Okolab.

[0066] The coupling of the module 3 to a commercially available microscope advantageously makes it possible to benefit from existing equipment capable of cooperating with said microscope, e.g. a portable incubator, without having to develop new equipment. The interferometric device 4 will now be described.

[0067] The interferometer 4 comprises a frame supporting a seat which is immovable relative to the frame.

[0068] The seat supports a non-polarizing beam splitting (NPBS) element 10. By way of example, the splitting element 10 is a non-polarizing splitting cube, a non-polarizing splitting plate, or the like.

[0069] The light source 5 is for illuminating the dividing element 10. However, the light source 5 does not directly illuminate the dividing element 10. Therefore, the light source 5 is offset from the dividing element 10 and from the seat. Therefore, the light source 5 is fixed to a part of the frame.

[0070] In particular, the interference device 4 comprises a first adjustment unit 11 by means of which the light source 5 illuminates the dividing element 10 .

[0071] In the present invention, the first adjustment unit 11 comprises at least one reflecting surface. As an example, the first adjustment unit 11 comprises at least two reflecting surfaces 12, 13 mounted facing each other. The reflecting surfaces 12, 13 are planar. The reflecting surfaces 12, 13 are, for example, mirrors.

[0072] Typically, the first adjustment unit 11 is positioned such that, during operation, the light source 5 directly illuminates one of the reflective surfaces 12, which reflects the light towards the other reflective surface 13, which in turn reflects the light towards the dividing element 10 to illuminate it.

[0073] At least one, and preferably both, reflecting surfaces 12, 13 are mounted movably relative to the frame. The first adjustment unit 11 is shaped so that the two reflecting surfaces 12, 13 can move relative to the frame independently of each other. For example, at least one, and preferably both, reflecting surfaces 12, 13 can rotate relative to the frame. For example, the first adjustment unit 11 comprises a first kinematic mount supporting the reflecting surface 12 (and allowing the reflecting surface 12 to be moved relative to the frame) and a second kinematic mount supporting the reflecting surface 13 (and allowing the reflecting surface 13 to be moved relative to the frame). The movement of the reflecting surfaces 12, 13 can, for example, modify the light path of the light beam generated by the light source 5 to the splitting element 10.

[0074] In particular, the interference device 4 comprises a first optical system 14 arranged between the light source 5 and the first adjustment unit 11 upstream of the first adjustment unit 11 (the concepts of "upstream" and "downstream" are understood in relation to the flow direction of light).

[0075] The first optical system 14 makes it possible in particular to reduce the divergence of the light beam generated by the light source 5, thereby limiting any losses of power in the light output generated by the light source 5.

[0076] As an example, the first optical system 14 is a single lens, a single doublet, or a pair of lenses, or a pair of doublets. If the first optical system 14 is a pair of doublets, the first optical system 14 is composed of, for example, a first achromatic (i.e., an assembly formed by an upstream achromatic converging lens and an infinity-corrected flat lens) and a second achromatic doublet (i.e., an assembly formed by a downstream achromatic converging lens and an infinity-corrected flat lens), the second doublet being disposed downstream of the first doublet. The flat surfaces of the lenses face the light source 5 and the adjustment unit 11, respectively.

[0077] The interference device 4 comprises a second optical system 15 arranged downstream of the first adjustment unit 11 between the first adjustment unit 11 and the splitting element 10. By way of example, the second optical system 15 is a lens, a doublet, etc. For example, the second optical system 15 is an achromatic converging lens, preferably an achromatic doublet, comprising an achromatic converging lens having a flat surface facing the first adjustment unit 11.

[0078] According to a particular embodiment, the interferometric device 4 comprises a first diaphragm 16 arranged between the light source 5 and the first adjustment unit 11, upstream of the first adjustment unit 11. By way of example, the first diaphragm 16 is arranged between the first optical system 14 and the first adjustment unit 11, downstream of the first optical system 14.

[0079] As an example, the first diaphragm 16 is an aperture stop. The first diaphragm 16 is arranged in the focal plane of the second doublet of the first optical system 14, preferably in the image focal plane of the second doublet of the first optical system 14.

[0080] Thus, the first optical system 14 images the light source 5 in the image focal plane of the second doublet, which coincides with the first aperture 16 .

[0081] It should be noted that the light source 5 is conjugated to the first aperture 16 by the first optical system 14 .

[0082] Its aperture therefore makes it possible, for example, to control the degree of spatial incoherence of the light source 5 and / or the amount of light received by the sample and / or the illumination numerical aperture of the assembly 1 by means of the first diaphragm 16. Furthermore, the first diaphragm 16 is arranged in the focal plane of the second optical system 15, preferably in the target focal plane of the second optical system 15.

[0083] Furthermore, the focal length of the second optical system 15 must be equal to or less than the distance between the first optical system 14 and the first aperture 16 .

[0084] For the purposes of this description, the term "diaphragm" refers to any member for controlling the passage of the light output generated by the light source 5, i.e. an iris diaphragm, a hole in a dedicated wall, etc.

[0085] According to a particular embodiment, the interferometer device 4 comprises a second diaphragm 17 arranged downstream of the first adjustment unit 11 between the first adjustment unit 11 and the splitting element 10. By way of example, the second diaphragm 17 is arranged downstream of the second optical system 15 between the second splitting element 10.

[0086] The second aperture 17 is a field aperture.

[0087] The second aperture 17 makes it possible to limit the illumination of the sample to illuminate only that part of the sample that will be imaged by the assembly 1 and / or to reduce incoherent reflections.

[0088] 1, the light source 5, the first optical system 14, the first diaphragm 16, the first adjustment unit 11, the second optical system 15 and the second diaphragm 17 are thus arranged in succession upstream of the splitting element 10. The first optical system 14, the first diaphragm 16, the second optical system 15 and the second diaphragm 17 are fastened to the module 3.

[0089] By way of example, the second optical system 15 and the second aperture 17 are supported by a seat. Typically, the second optical system 15 and the second aperture 17 are arranged in line with the splitting element 10.

[0090] Preferably, the light source 5, the first optical system 14 and the first aperture 16 are supported by the same part of the frame, offset from the seat.

[0091] Thus, the light source 5 illuminates the dividing element 10 and thereby makes it possible to define “illumination arms” of said dividing element 10 .

[0092] Furthermore, the dividing element 10, once illuminated by a light source, has two arms, i.e. a "reference arm" associated with reflecting surface 6; It is known that a "target arm" can be formed that is associated with the sample during operation.

[0093] Thus, the light source 5 is neither on the object arm nor on the reference arm, but on the illumination arm.

[0094] In contrast, the reflecting surface 6 is on the reference arm. The interference device 4 also comprises a second adjustment unit 18, which allows the reflecting surface 6 to interact with the light beam propagating from the splitting element 10 on the reference arm.

[0095] Typically, the second adjustment unit 18 comprises at least one reflecting surface 27. In particular, the second adjustment unit 18 is arranged such that, during operation, light rays from the splitting element 10 are reflected on the reflecting surface 27 and reach the reflecting surface 6.

[0096] The reflecting surface 27 is flat and may be, for example, a mirror.

[0097] Preferably, the reflective surface 27 is mounted so as to be movable relative to the frame of the module 3. By way of example, the reflective surface 27 may rotate relative to the frame. For example, the second adjustment unit 18 may comprise a kinematic mount which supports the reflective surface 27 (and allows the reflective surface 27 to move relative to the frame).

[0098] Thus, by moving the reflecting surface 27, the optical path of the light ray reflected by the reflecting surface 6 to the beam splitting element 10 can be modified.

[0099] Preferably, the reflective surface 6 is itself mounted within the interferometric device 4 such that it is movable relative to the frame, so that the reflective surface 6 can be moved relative to the sample while the sample is being studied. By way of example, the reflective surface 6 is mounted on a base that is translatable relative to the frame. For example, the base can be translated relative to the frame by at least one piezoelectric actuator. For example, the base can be translated parallel to the optical axis of the light source 5, but not coincident with the optical axis.

[0100] In this case, the interference device 4 comprises a third optical system 19 arranged between the splitting element 10 and the reflecting surface 6. By way of example, the third optical system 19 is a lens, a doublet, etc. For example, the third optical system 19 is an achromatic converging lens, preferably an achromatic doublet, comprising an achromatic converging lens having a flat surface facing the splitting element 10.

[0101] The third optical system 19 makes it possible, for example, to simplify the balance between the object arm and the reference arm and / or to properly illuminate the reflecting surface 27 (i.e. over a wide field of view). Furthermore, the assembly 1 comprises a first objective lens 20 and a second objective lens 21. The two objective lenses 20, 21 are identical and each associated with one of the abovementioned arms. Both objective lenses have the same numerical aperture (NA).

[0102] Optionally, the numerical aperture of the objective lens is high. For the purposes of this application, the term "high" means a numerical aperture greater than 0.8, preferably greater than 1.

[0103] It should be noted that the second optical system 15 enables the light source 5 to be imaged onto the focal planes of the two objectives, e.g. the target focal planes of the two objectives 20,21.

[0104] The first objective lens 20 is part of a module 3, in this case part of the interferometer device 4. Thereby, the first objective lens 20 is arranged in the module 3 at the reflecting surface 6. By way of example, the optical axis of the first objective lens 20 is perpendicular to the plane in which the reflecting surface 6 extends.

[0105] More precisely, in this case the first objective lens 20 is arranged such that the reflecting surface 6 is located at one of the focal points of the first objective lens, for example at the second focal point of the first objective lens 20. The first objective lens 20 is therefore on the reference arm.

[0106] Preferably, the second adjustment unit 18 also comprises a member 26 for moving the first objective lens 20 relative to the frame, in particular relative to the reflecting surface 6. By way of example, the first objective lens 20 is supported by a frame which is moved by a moving member. For example, the moving member is configured to be able to move the first objective lens 20 along at least two translation axes. For example, the moving member is configured to be able to move the first objective lens 20 at least in a plane perpendicular to the axes along which the reflecting surface 6 can be moved relative to the frame.

[0107] In the example shown in FIG. 1, the third optical system 19, the second adjusting unit 18, the first objective lens 20 (associated with the movable member 26) and the reflecting surface 6 are therefore located in that order downstream of the splitting element 10, on the side of the reference arm.

[0108] Moreover, the second objective lens 21 is not part of the module 3. In fact, the second objective lens 21 is directly an objective lens (or one of the objective lenses) of the microscope 2.

[0109] The sample is intended to be positioned at one of the multiple focal points of the second objective lens 21, for example the second focal point of the second objective lens 21.

[0110] Thus, when the microscope 2 and the module 3 are coupled, the second objective lens 21 is on the object arm. Therefore, since the second objective lens 21 is the objective lens of the microscope, it is clear that the module 3 alone is not sufficient to form a complete interferometer.

[0111] Since the microscope 2 is an inverted microscope, the second objective lens 21 is arranged to observe the sample from below the sample. As an example, the second objective lens 21 is arranged below the stage, in this case below the incubator 9.

[0112] In a manner known per se, the microscope 2 is provided with a reflecting surface 22 so that light rays passing through the second objective lens 21 along the optical axis of said second objective lens 21 can be reflected up to the optical output of the microscope 2.

[0113] Typically, the reflecting surface 22 is planar. For example, the reflecting surface 22 is a mirror, for example a thick mirror, for example a mirror having a thickness of 3 mm or more, for example a thickness of 4 mm or more. Preferably, the reflecting surface 22 is a plane mirror supported by a prism or a plane mirror supported by a cube.

[0114] In the present case, said reflecting surface 22 is arranged in such a way that light rays propagating along the optical axis of the second objective lens 21 propagate and then, after being reflected at the reflecting surface 22, exit via an optical output of the microscope 2. When the module 3 is connected to the microscope 2, such light rays thus propagate from said output along the object arm to the splitting element 10.

[0115] The interference device 4 also comprises a fourth optical system 23, which is arranged between the splitting element 10 and the module's "object arm" output, i.e. the module output optically coupled to at least the optical output of the microscope 2. By way of example, the fourth optical system 23 is a lens, a doublet, etc. For example, the fourth optical system 23 is an achromatic doublet with an achromatic converging lens, preferably with a flat surface facing the splitting element 10. Preferably, the fourth optical system 23 is associated with at least one member for moving the fourth optical system 23 relative to the frame, in particular relative to the splitting element 10 (in particular for moving the fourth optical system 23 towards or away from the splitting element 10). By way of example, the fourth optical system 23 is supported by a frame which is moved by a moving member. For example, the moving member is configured to move the fourth optical system 23 in at least one translation. For example, the moving member is configured to move the fourth optical system 23 with at least one translation along the action axis of the module (i.e., the axis along which the majority of the light rays propagating on the object arm exit the module and reach the microscope).

[0116] Preferably, the fourth optical system 23 is arranged in the module such that it is located as close as possible to the reflecting surface 22. In this manner, the fourth optical system 23 is preferably arranged such that the distance between the second objective lens 21 and the fourth optical system 23 is equal to the focal length of said fourth optical system 23.

[0117] This prevents the light source from being unevenly illuminated, and furthermore, it limits any loss of power in the light output generated by the light source 5 at the sample.

[0118] The third optical system 19 is positioned so that the focal plane of the third optical system 19 coincides with the focal plane of the first objective lens 20, for example, so that the image focal plane of the third optical system 19 coincides with the target focal plane of the first objective lens 20.

[0119] The fourth optical system 23 is positioned so that the focal plane of the fourth optical system 23 coincides with the focal plane of the second objective lens 21, for example, so that the image focal plane of the fourth optical system 23 coincides with the target focal plane of the second objective lens 21.

[0120] Preferably, the second aperture 17 is arranged in the focal plane of the fourth optical system 23, e.g. by means of the splitting element 10 being in the target focal plane of the fourth optical system 23. The second aperture 17 is thus conjugated to the sample by means of the second objective lens 21 (the sample is at the focal point of the second objective lens 21, e.g. the second focal point of the second objective lens).

[0121] Preferably, the second diaphragm 17 is arranged in the focal plane of the third optical system 19, for example by means of a splitting element 10 which is in a target focal plane of the third optical system 19.

[0122] To make the object and reference arms symmetrical, the third optical system 19 and the fourth optical system 23 must be positioned identically with respect to the splitting element 10 .

[0123] Furthermore, the module 3 comprises a fifth optical system 24 arranged at the output of the interferometric device 4, i.e. between the splitting element 10 and the acquisition device 7. The fifth optical system 24 may or may not be part of the interferometric device 4.

[0124] As an example, the fifth optical system 24 is a single lens, a single doublet, or a pair of lenses, or a pair of doublets. When the fifth optical system 24 is a pair of doublets, the fifth optical system 24 is composed of, for example, a first achromatic doublet (i.e., an assembly formed by an upstream achromatic converging lens and an infinity corrected flat lens) and a second achromatic doublet (i.e., an assembly formed by a downstream achromatic converging lens and an infinity corrected flat lens), with the second doublet being located downstream of the first doublet. The flat surfaces of the lenses face the acquisition device 7 and the splitting element 10, respectively.

[0125] The fifth optical system 24 is arranged so as to be conjugate in the same plane at the output of the interference device 4 with a plane located at the focal points of the two objective lenses 20, 21 (e.g. a plane located at the second focal points of the two objective lenses 20, 21). Preferably, the optical sensor of the acquisition device 7 is arranged in this plane. The optical sensor is thus arranged in the focal plane of the fifth optical system 24, e.g. in the image focal plane of the fifth optical system 24.

[0126] Preferably, the focal plane of the fourth optical system 23 is conjugate with the focal plane of the fifth optical system 24 , for example the focal plane of the fourth optical system 23 is conjugate with the target focal plane of the fifth optical system 24 .

[0127] Preferably, the interference device 4 comprises a third diaphragm 25 arranged upstream of the fifth optical system 24 and between the fifth optical system 24 and the splitting element 10. The third diaphragm 25 is a field diaphragm.

[0128] This third aperture makes it possible to adjust the optical position of the light sensor, for example to reduce incoherent reflections.

[0129] In this manner, the second diaphragm 17 is disposed so as to be conjugate with the third optical system 19, the fourth optical system 23 and the fifth optical system 24 via the dividing element 10.

[0130] Furthermore, the third diaphragm 25 is arranged by the splitting element 10 in a focal plane of the third optical system 19, for example in an image focal plane of the third optical system 19. This makes the third diaphragm 25 conjugate with the reflecting surface 6 by the first objective lens 20 (the reflecting surface 6 is at one of the focal points of the first objective lens 20, for example at the second focal point of the first objective lens 20).

[0131] Furthermore, the third diaphragm 25 is arranged in the focal plane of the fourth optical system 23 by the splitting element 10. For example, the third diaphragm 25 is arranged in the image focal plane of the fourth optical system 23 by the splitting element 10.

[0132] Furthermore, the third aperture 25 and the light sensor are conjugate (through the fifth optical system 24).

[0133] It should be noted that with the above arrangement, the light source 5 is "imaged" (i.e., a magnified image of it is made at the first aperture 16). This magnification is produced by the pair consisting of the second optical system 15 and the third optical system 23.

[0134] This makes it possible to reduce the divergence of the light beam generated by the light source 5 and / or to reduce the illumination numerical aperture generated by the light source 5.

[0135] However, it is necessary that the magnified image of the light source 5 produced by the pair consisting of the second optical system 15 and the third optical system 23 is not larger than the size of the user's pupil at the target focal planes of the first objective lens 20 and the second objective lens 21.

[0136] This makes it possible to preserve the incoherent (or insufficiently coherent) nature of the light source 5 and / or to limit any losses in power in the light output generated by the light source 5 .

[0137] Preferably, the focal length of the first optical system 14 is selected depending on the diverging nature of the light source 5, the distance between the light source 5 and each of the objective lenses 20, 21, and the size of the user's pupil at the target focal plane of the objective lenses 20, 21.

[0138] Furthermore, the first aperture 16 is conjugate with the focal plane of the objective lenses 20, 21, e.g. the target focal plane of the objective lenses 20, 21, by means of the pair consisting of the second optical system 15 and the third optical system 19 and the fourth optical system 23.

[0139] Furthermore, the second aperture 17 can be imaged on the sample by a fourth optical system 23. Preferably, the second aperture 17 is located at the focal length of the second optical system 15.

[0140] The arrangement thus described is a practical application of the principle of Keller illumination.

[0141] It is clear that the relative position of the second objective lens 21 with respect to the light source 5 and the reflecting surface 6 is crucial. When the microscope 2 is coupled to the module 3, the optical axis of the second objective lens 21 is in a practically fixed position with respect to the working axis of the module 3. Nevertheless, the two adjustment units 11, 18 allow the light source 5 and the reflecting surface 6 to be accurately positioned with respect to the second objective lens 21, in particular so that interference between the reference wave and the object wave can be generated.

[0142] In particular, the reflecting surface 27 is configured (by relative movement with respect to the frame) to align the group consisting of the splitting element 10, the third optical system 19, the reflecting surface 18 and the first objective lens 20 with the group consisting of the splitting element, the fourth optical system 23, the reflecting surface 22 and the second objective lens.

[0143] The module 3 is thus inserted in a simple manner into the microscope 2, after which the properties of the module 3 are adjusted using the two adjustment units 11, 18 in order to optically align the module 3 with the microscope 2. As an example, the adjustment unit 18 (which in particular comprises a moving member 26) is used to make the reference arm identical to the object arm (which is more difficult to act on given that on the object arm the distance between the second objective lens 21 and the optical output of the microscope 2 is fixed), and the adjustment unit 11 is used to correct the illumination arm, if necessary, in order to align the working axis of the module 3 with the propagation axis of the light source 5.

[0144] The adjustment units 11, 18 allow the module 3 to be connected to a variety of microscopes available on the market.

[0145] The assembly 1 thus described is an improvement of the Linnik interferometer in a Keller illumination configuration.

[0146] In the assembly 1 thus described, the splitting element 10 is much further away from the two objective lenses 20, 21 than in prior art systems. This advantageously allows the module 3 to be coupled to a conventional microscope. It also allows the use of objective lenses (both the first objective lens 20 and the second objective lens 21) with a larger numerical aperture.

[0147] Furthermore, the presence of at least one optical system downstream of the splitting element 10, on the object arm side, allows to improve the quality of the image derived from the signal generated in the assembly 1. In particular, in this configuration, incoherent reflections are reduced by the third aperture 25 and then spatially confined to the center of the acquisition surface of the optical sensor (when said acquisition surface is centered with respect to an axis perpendicular to the splitting surface of the splitting element). It is therefore easier for the processing device 8 to separate said incoherent reflections from the coherent reflections and a better quality image can be obtained.

[0148] Starting from the above described assembly 1, it is possible to perform both static and dynamic full-field optical coherence tomography imaging.

[0149] In the case of dynamic full-field optical coherence tomography imaging, the sample is placed on the microscope 2 and a time sequence of N two-dimensional interference signals from a portion of the sample is acquired by the acquisition device 7 with a fixed path difference between the object arm and the reference arm. This fixed path difference is maintained, for example, by holding both the reflecting surface 6 and the sample in a constant position. To this end, the processing device 8 is in this case configured to synchronize the acquisition device 7 (in particular its optical sensor) and an actuator for moving the reflecting surface 6. As an example, in addition to a computer, the processing device 8 may also comprise an acquisition card connected to a computer to ensure said synchronization.

[0150] Furthermore, full-field optical coherence tomography imaging can be performed either with or without an incubator.

[0151] The above assembly therefore makes it possible to implement a large number of imaging possibilities.

[0152] The assembly 1 described above makes it possible to obtain, for example, images of cells, but also to visualize cell activity and identify the metabolic state of the cells. The cells may be in two-dimensional cultures, such as two-dimensional monolayer cultures, three-dimensional cultures, such as organoids, or other three-dimensional multi-layer cultures.

[0153] Specifically, it is possible to study cells invasively, but non-destructively. Thus, one of the several strengths of the described assembly 1 is the ability to create images without disturbing the natural environment. The thus described assembly 1 can be used, for example, to study organoids, two-dimensional monolayer cultures, three-dimensional multilayer cultures, retinas and corneas, retinal and corneal explants from mice, pigs, macaques, etc., to perform quality control on large-scale organoid production, for microfluidics, to support the fields of optogenetics and disease modeling (e.g., by light stimulation for photophysiology), for grafts, etc., to test the efficacy of new treatments (genetic, pharmaceutical, etc.).

[0154] Furthermore, the described assembly 1 makes it possible to generate both static signals for visualizing the structure of the tissue in three dimensions and dynamic signals for, for example, identifying the cells of the tissue and measuring their metabolism.

[0155] Other applications of the described assembly 1 can be envisaged, for example, all microscopy studies with high spatial resolution (e.g., resolution between 100 nanometers and 400 nanometers) and / or temporal resolution (e.g., resolution in the millisecond range, e.g., 2 milliseconds), in particular microscopy studies in which the sample is not destroyed and / or in which the lack of endogenous markers is incorporated.

[0156] Furthermore, it is possible to create real-time tracking of the sample by generating images with contrast that reflects moving mechanisms around X ms, for example, where X is 100-200 ms and X is, for example, 160 ms.

[0157] Naturally, the invention is not limited to the described embodiments, but also covers any variant embodiment falling within the scope of the invention as defined by the claims.

[0158] In particular, the incubator may or may not be part of the assembly, and / or the acquisition device may or may not be part of the assembly, and / or the processing device may or may not be part of the assembly, and / or the light source may or may not be part of the assembly.

[0159] Additionally, the capture device may or may not be part of the module, and / or the processing device may or may not be part of the module, and / or the light source may or may not be part of the module.

[0160] The module may comprise one or more glass plates to limit dispersion phenomena.

[0161] If the microscope used does not have a flat reflecting surface between its objective and its optical output, it may of course have such a flat reflecting surface, which, in the context of the present invention, can be arranged, for example, in the module between the fourth optical system and the working output of the module, or in another module which is itself inserted in one of the banks of the microscope.

[0162] In the present case, the module is coupled to the microscope simply by inserting the module into the microscope, however, the module and microscope may additionally or alternatively be coupled together through the interaction of male and female connectors that may interact to connect the module to the microscope. For example, the module may comprise a male connector and the microscope may comprise a female connector. For example, the module may comprise a threaded rod that may be screwed into a corresponding threaded hole in the microscope.

[0163] The microscope of the assembly may be a turretless microscope.

[0164] The microscope of the assembly does not have to be an inverted microscope. In this way, the sample can be illuminated from above (in which case the second objective lens also receives a signal from above) as well as from below (in which case the second objective lens also receives a signal from below).

[0165] The third optical system does not have to be present, and can be included in the microscope rather than in the module.

[0166] The light sensor may be different from that specified, for example the light sensor may be a charge-coupled device (CCD) sensor. The light sensor may operate in another range, such as the visible range and / or infrared. The light sensor may therefore be a near-infrared image sensor (or a short-wave infrared sensor, SWIR). As an example, the light sensor may be an InGaAs (indium gallium arsenide) sensor or an InGaAs SWIR sensor. The module and / or assembly may be shaped so that the light sensor (and / or the acquisition device) is interchangeable, for example to be able to function in the visible range and then to be able to function in a range other than the visible range, for example infrared.

[0167] The assembly may not include an incubator (portable or not). The assembly may include a heating chamber arranged to enable at least the microscope to, for example, keep the sample at a predetermined temperature.

[0168] The optical systems in this case all have lenses with at least one flat surface, but the lenses can be shaped differently. Furthermore, the lenses can have orientations different from those specified. Preferably, the lenses are arranged in the module such that the light beam with the largest aperture angle (between the incoming or outgoing light beam) is associated with the flattest surface of the lens.

[0169] Here, a high numerical aperture objective is used, although objectives with a lower numerical aperture can also be used.

Claims

1. A module intended to be associated with a microscope for full-field optical coherence tomography microscopic imaging of at least one sample, said module comprising an interferometric device (4) equipped with a non-polarizing beam splitting element (10), said interferometric device further comprising at least one first reflective surface (6), said interferometric device thus performing, in operation: at least one reference wave obtained by reflecting light emitted by a light source associated with said module on said at least one first reflecting surface; At least one interference can be generated between the light emitted by the light source and at least one object wave obtained by backscattering the light onto the sample, Thus, when illuminated by the light source in operation, the non-polarizing beam splitting element allows the formation of two arms, a "reference arm" associated with the reflecting surface and an "object arm" associated with the sample; The interference device further comprises: a first adjustment unit (11) arranged upstream of the non-polarizing beam splitting element so that the illumination arm of the non-polarizing beam splitting element can be modified during operation, and / or The module comprises at least one second adjustment unit (18) arranged between the non-polarizing beam splitting element and the reflecting surface so as to be able to modify the reference arm during operation.

2. 2. The module according to claim 1, wherein the first adjustment unit (11) comprises at least two reflective surfaces (12, 13), at least one of the at least two reflective surfaces being movable relative to a frame of the module.

3. 2. The module of claim 1, wherein the module comprises an objective lens (20) associated with the reference arm, and the second adjustment unit (18) comprises at least one further reflective surface (18) and at least one moving member (26) for moving the objective lens relative to a frame of the module.

4. 4. The module of claim 3, wherein the at least one further reflective surface (18) is also movable relative to a frame of the module.

5. 4. The module according to claim 3, wherein the moving member (26) is configured to move the objective lens at least in a plane parallel to a plane in which the first reflecting surface (6) extends.

6. The module of claim 1, wherein the light source (5) is part of the module.

7. 2. The module according to claim 1, wherein the module comprises an acquisition device (7) suitable for acquiring at least one signal resulting from the interference between the reference wave and the target wave.

8. 2. The module of claim 1, wherein the interference device (10) comprises an optical system (23) arranged between the non-polarizing beam splitting element (10) and a module output intended to be at least optically coupled to the microscope.

9. An assembly comprising a microscope (2) and a module according to any one of the preceding claims.

10. 10. The assembly of claim 9, wherein the microscope (2) comprises a turret, and the module is disposed in one of a plurality of compartments of the turret.

11. 10. An assembly as claimed in claim 9, comprising an incubator (9) supported by the microscope stage.

Images