Optical device for self-referenced full-field temporal optical coherence tomography microscopic imaging, and associated facility and method

Abstract

The invention relates to an optical device for the full-field optical coherence tomography microscopic imaging of at least one sample, the device comprising a lens for observing the sample when in use, the device comprising a specular interface, the device thus being able, when in use, to allow the production of at least one interference between at least one reference wave obtained by the reflection of light emitted by a light source associated with the device from the specular interface, and at least one object wave obtained by the backscattering of the light emitted by the source from the sample, the specular interface being arranged with respect to the lens in such a way that, when in use, the object wave passes through the specular interface on its path between the sample and the source. The invention also relates to the corresponding facility and method.

Description

[0001] Optical equipment for self-referenced full-field temporal optical coherence tomography microscopic imaging, associated installation and method

[0002] The invention relates to optical equipment for optical coherence tomography microscopic imaging of at least one sample.

[0003] The invention also relates to a corresponding installation and method.

[0004] BACKGROUND OF THE INVENTION

[0005] In the field of optical imaging, optical coherence tomography (better known by the English acronym OCT for "Optical Coherence Tomography") makes it possible to acquire images with great precision and in particular an axial resolution superior to that which can be obtained with a confocal microscope.

[0006] In particular, optical coherence tomography imaging allows:

[0007] - to image many components such as cellular components,

[0008] - to image within a sample in three dimensions,

[0009] - to image with high optical sectioning, independently of the numerical aperture of the associated imaging system,

[0010] - to image non-invasively,

[0011] - to image without marker.

[0012] Optical temporal coherence tomography imaging consists of measuring the interferometric signal between a signal of light backscattered by a sample when illuminated by a source with a reference light signal emitted by this same source and having propagated over the same optical distance.

[0013] Full-field optical coherence tomography imaging makes it possible to parallelize this principle so that the interferometric signal reaches a camera-type sensor (CMOS, CDD, etc.) in order to obtain a two-dimensional image as output.

[0014] Document FR 2 817 030 describes an example of a full-field optical coherence interference microscopic imaging system.

[0015] There are two main variations of full-field optical coherence tomography imaging:

[0016] - static full-field optical coherence tomography imaging (better known by the acronym FFOCT for “static Full-Field Optical Coherence Tomography”), i.e. so-called “static” imaging which is based on different approaches to isolate the interferometric signal between the reference field and the sample field;

[0017] - dynamic full-field temporal optical coherence tomography imaging (better known by the acronym D-FFOCT for “Dynamic FFOCT”) which is based on the temporal analysis of the signal generated by static FFOCT, which makes it possible to quantify the temporal evolution of the signal generated by static FFOCT.

[0018] Thus, by using these two variations, it is just as possible to distinguish morphological structures in a sample (such as collagen fibers, cells and their nuclei, etc.) - by static imaging - as metabolic elements which compose a tissue of one of the said structures of the same sample (such as the organelles of a cell (mitochondria for example) and / or the general metabolic state of one of these structures and in particular the cells and nuclei - by dynamic imaging.

[0019] Full-field optical coherence tomography imaging is particularly advantageous for performing non-invasive three-dimensional structural imaging, enabling multiple applications: the study of organoids, disease modeling, cancer diagnosis, etc.

[0020] Indeed, static FFOCT and D-FFOCT imaging allow us to obtain a lot of information on the functioning of living things thanks to the particularity of its signal, while being non-invasive and non-destructive.

[0021] More specifically, static FFOCT and D-FFOCT imaging provide local amplification - spatially uncorrelated - of the backscattered signal within the sample (usually a three-dimensional volume) and associated local phase information with a transverse resolution similar to that obtainable by confocal microcopy and with an axial resolution superior to that obtainable by confocal microcopy. Thus, static FFOCT and D-FFOCT imaging are sensitive to weak scattering objects and weak longitudinal movements.

[0022] In addition, static FFOCT and D-FFOCT imaging greatly limit the presence of speckles compared to similar imaging techniques with phase resolution (interference scattering microscopy, holographic microscopy, etc.). This allows direct interpretation of the object studied.

[0023] Furthermore, static FFOCT imaging and D-FFOCT imaging are virtually insensitive to spatial aberrations, allowing imaging within three-dimensional structures.

[0024] Static FFOCT imaging and D-FFOCT imaging thus make it possible to detect small organs - such as sub-cellular constituents - within more complex three-dimensional structures, such as tissues, even without labeling. Moreover, in the particular context of D-FFOCT imaging, it is possible, by temporal analysis of the signals obtained, to highlight other contrasts linked for example to cellular activity (such as cellular activity of organelles and for example mitochondria, microvesicles, etc.).

[0025] However, with static FFOCT imaging and D-FFOCT imaging, in the context of imaging in ex-vivo or in vitro conditions, when one wishes to image layers of the samples closest to a face of the sample holder on which the sample rests (conventionally the sample holder being a glass slide or a plastic slide) optical interference artifacts appear. These artifacts, which are also sometimes called "interface artifacts" or "fringe artifacts", greatly hinder the study of the samples or prevent it completely.

[0026] This disadvantage is increased in the case of inverted microscopy (we recall that an inverted microscope is a microscope in which a sample is observed from below). We also specify that a stratum is a two-dimensional section of the sample according to a section plane in the thickness of the sample, i.e. according to a section plane parallel to the face of the sample holder on which the sample rests.

[0027] As a result, static FFOCT and D-FFOCT imaging are not usually used to image these strata close to this surface, especially in the case of studies of very thin samples such as, for example, two-dimensional cell culture samples. However, two-dimensional cell culture remains the overwhelmingly used method in disease modeling and drug development despite the emergence of three-dimensional cell culture. In addition, the study of elements in transition from a thin three-dimensional structure to a thicker three-dimensional structure is key in certain applications, such as the study of intestinal organoids. However, static FFOCT and D-FFOCT imaging seem difficult to use for imaging this latter configuration.

[0028] SUBJECT OF THE INVENTION

[0029] The invention aims in particular to enable full-field optical coherence tomography microscopic imaging which makes it possible to limit the appearance of optical artifacts during the imaging of at least one sample.

[0030] SUMMARY OF THE INVENTION

[0031] For this purpose, a method of full-field optical coherence tomography microscopic imaging of at least one sample is provided, the method comprising the steps of:

[0032] - emit light propagating in an object arm of an installation in which the sample is arranged so that the light backscatters in the sample to produce an object wave,

[0033] - generate at least one interference between the object wave and a reference wave.

[0034] According to the invention, the reference wave is generated by reflection of light on a specular interface arranged on the object arm.

[0035] The inventors were able to observe that such a process made it possible to image the sample while avoiding optical artifacts as much as possible and even completely eliminating the optical artifacts present with the usual static FFOCT and D-FFOCT devices, and in particular fringe artifacts.

[0036] This advantage is further increased when one wishes to image:

[0037] - one of the layers of a thin sample (as indicated above, a layer being a two-dimensional section of the sample along a section plane in the thickness of the sample, i.e. a section plane parallel to a face of the sample holder on which the sample rests); the sample being, for example, a sample of a two-dimensional cell culture, a graphene sample, etc., and more generally a sample having a thickness of less than 15 micrometers, and for example less than 10 micrometers, and for example less than 5 micrometers;

[0038] - one or more interface layers of a sample of greater thickness (such as an organoid for example), i.e. the layers closest to a face of an element carrying the sample (glass slide, plastic slide, incubator, etc.).

[0039] The invention is based on the general principle of FFOCT imaging but improves this principle by being “self-referenced”: in fact the invention works without a specific reference arm as in the FFOCT devices of the prior art since the reference wave is generated from the specular interface which is also cleverly placed on the path of the object wave.

[0040] Furthermore, the particular position of the specular interface allows automatic alignment between a reference field (linked to the reference wave) and an object field (linked to the object wave).

[0041] This automatically produces interference between the reference wave and the object wave.

[0042] This interference is also homogeneous. This interference is also of very good quality since it has no optical aberration.

[0043] The invention can further be implemented by a simple structural installation.

[0044] Furthermore, the invention thus proves to be less sensitive to vibrations than the static and dynamic FFOCT devices of the prior art because the reference waves and the object waves propagate along the same object arm and the specular interface is thus at a less significant distance from the sample (for example the specular interface is at a distance of between 0 and 15 micrometers).

[0045] The invention can thus make it possible to create a uniform mosaic (a mosaic being an assembly of N images acquired from different areas of the same stratum of a given sample), i.e. without overlapping artifacts between two images of the joined mosaic.

[0046] Furthermore, the invention can be implemented in a miniaturized installation according to the intended application. Among other things, the invention uses only a single objective due to its self-referencing.

[0047] The invention can advantageously be implemented in noisy environments such as for example on a vehicle (land, air, sea, etc.), a production line, etc.

[0048] The invention may further be associated:

[0049] - has technologically advanced elements in the field of imaging (whether or not part of the invention) such as, for example, a high numerical aperture objective (better known by the English acronym NA for “numerical aperture”);

[0050] - to technologically less advanced elements in the field of imaging (whether or not part of the invention) such as, for example, a mobile telephone:

[0051] • equipped with a camera to acquire images of the sample after wave interference, and / or

[0052] • equipped with lighting (for example light-emitting diode, commonly referred to by the English acronym LED, and in particular flash LED) to act as the light source, and / or

[0053] • equipped with a processing unit (for example a processor) to process the interference signal(s).

[0054] Optionally, the invention is also configured to perform dynamic full-field optical coherence tomography microscopic imaging.

[0055] For this purpose, the method comprises an additional step for studying the temporal evolution of a resulting signal or of the interference(s) between the reference waves and the object waves.

[0056] The process allows static FFOCT imaging as well as D-FFOCT imaging to be performed.

[0057] The method is configured to perform temporal optical coherence tomography microscopic imaging. Thus, the method makes it possible, for example, to look at morphological elements in a sample (such as collagen fibers, cells and their nuclei) - static imaging - as well as metabolic elements that make up a tissue of the elements of the same sample (such as the organelles of a cell (mitochondria for example)) and / or the general metabolic state of one of these elements and in particular the cells and nuclei - dynamic imaging. The elements can, for example, be cells, structures and in particular subcellular structures such as nuclei, mitochondria, pigments, etc.

[0058] It is further recalled that the invention is less sensitive to vibrations than the static and dynamic FFOCT devices of the prior art.

[0059] This aspect is particularly interesting if the invention is used in a D-FFOCT imaging context.

[0060] Indeed, the D-FFOCT imaging devices of the prior art are difficult to deploy outside of optical laboratories whereas the invention can be installed in demanding and / or noisy environments (a noisy environment being an environment subject to mechanical vibrations).

[0061] For the present application, by "object arm" we mean the part of the installation located between the sample and an optical element making it possible to generate the interfaces such as for example an optical beam splitter element (a light ray can thus propagate in said object arm following a given optical path).

[0062] Optionally, the specular interface is arranged so as to be at a given distance from a plane imaged by the installation, a distance which is greater than or equal to zero and less than or equal to:

[0063] - twice the depth of field of an objective of the installation intended to observe the sample, or

[0064] - twice a temporal coherence length of a light source generating the light if the coherence length is less than the depth of field of the objective.

[0065] By "imaged plane" (which can also be called "image plane"), we mean a plane which is imaged on an acquisition device (whether or not part of the invention), a device adapted to acquire at least one signal resulting from the interference between the reference waves and the object waves. The "imaged plane" therefore corresponds to one of the strata of the sample combined with the acquisition device.

[0066] The "lens depth of field" is defined as 2À / NA 2 (with À the central wavelength of the light emitted by the source) and NA the numerical aperture of the objective).

[0067] The “temporal coherence length” L c is defined as the full width at half maximum of the autocorrelation function of the spectrum of the interference(s). For example, for a source with a Gaussian distribution spectrum, it is written as: L c = V2 ln2 A 2 / miAA (with n the refractive index of the medium in which the sample is immersed).

[0068] The method thus proves to be particularly effective for imaging strata distances from zero to two times 2À / NA 2 of the specular interface.

[0069] The invention also relates to optical equipment for full-field optical coherence tomography microscopic imaging of at least one sample, the equipment comprising an objective for in-service observation of the sample, the device comprising a specular interface, the device thus being capable in service of allowing the production of at least one interference between:

[0070] • at least one reference wave obtained by reflection of light emitted by a light source associated with the equipment on the specular interface, and

[0071] • at least one object wave obtained by backscattering of the light emitted by said source on the sample, the specular interface being arranged with respect to the objective so that the object wave crosses said specular interface in use during its journey between the sample and the source.

[0072] Optionally, the specular interface is arranged so as to be at a given distance from a plane imaged by the installation, a distance which is greater than or equal to zero and less than or equal to:

[0073] - at twice the lens depth of field, or

[0074] - twice a temporal coherence length of the light source if the temporal coherence length is less than the depth of field of the objective.

[0075] Optionally, the specular interface of the equipment belongs to a glass slide or a plastic slide. Advantageously, the source is part of the equipment.

[0076] Advantageously, the equipment comprises an acquisition device adapted to acquire at least one signal resulting from interference between the reference waves and the object waves.

[0077] The equipment according to the invention can also be configured to allow dynamic full-field optical coherence tomograph microscopic imaging.

[0078] The equipment according to the invention may further comprise an incubator intended to receive the sample.

[0079] Advantageously, the source of the equipment illuminates a beam splitter element of the installation through an optical block comprising at least one optic and at least one diaphragm.

[0080] Advantageously, the specular interface is part of the sample holder. Even more advantageously, the specular interface is a face of the sample holder facing in use towards the sample.

[0081] The invention also relates to an installation comprising equipment as mentioned above.

[0082] Optionally, the installation comprises a reference arm, the installation thus being capable in service of also allowing the production of at least one interference between: • at least one reference wave obtained by reflection of the light emitted by the light source on a reflection surface of the additional reference arm, and

[0083] • at least one object wave obtained by backscattering of the light emitted by said source on the sample, the installation comprising means for blocking the reference arm.

[0084] These means of locking the reference arm can be temporary.

[0085] At least part of the installation according to the invention can be integrated into a mobile telephone.

[0086] Thus, when we wish to image the sample, we can either resort to creating interference by the specular interface (the blocking means preventing the light paths to and from the reference arm) or resort to creating interference by the reflection surface (the blocking means then being inactive).

[0087] The installation thus makes it possible to benefit advantageously and simply from two different technologies to best image a sample.

[0088] Optionally, the facility is configured to perform dynamic full-field optical coherence tomography microscopic imaging.

[0089] Thus it is possible to implement FFOCT imaging as well as D-FFOCT imaging when using the reference arm, in order to increase the imaging depth.

[0090] Due to its simple structure, the equipment can be advantageously installed relatively simply in existing static or dynamic FFOCT installations.

[0091] For the present application, "reference arm" means the part of the installation located between the reflection surface and an optical element making it possible to generate the interfaces (a light ray can thus propagate in said reference arm following a given optical path).

[0092] For the present application, “lighting arm” means the part of the installation located between the source and the optical element enabling the interfaces to be generated (a light ray can thus propagate in said object arm following a given optical path).

[0093] Other characteristics and advantages of the invention will emerge from reading the following description of particular and non-limiting embodiments of the invention.

[0094] BRIEF DESCRIPTION OF THE DRAWINGS

[0095] Reference will be made to the attached drawings, among which: [Fig. 1] Figure 1 is a schematic view of equipment according to a first embodiment of the invention; [Fig. 2a] Figure 2a is a schematic view of equipment according to a second embodiment of the invention when a reference arm of said equipment is blocked;

[0096] [Fig. 2b] Figure 2b is a schematic view of the equipment shown in Figure 2a when the reference arm is operational.

[0097] DETAILED DESCRIPTION OF THE INVENTION

[0098] With reference to Figure 1, the installation 1 according to a first embodiment of the invention is configured to carry out static and dynamic full-field optical coherence tomography microscopic imaging, in particular at the level of one or more interface layers of a sample 100 (regardless of the thickness of the sample 100). The sample 100 is carried by a sample holder. The sample holder is, for example, a glass slide, a plastic slide, an incubator, etc.

[0099] The installation 1 further comprises optical equipment 2 which is configured to allow observation of the sample 100, here from below the sample 100. The equipment 2 further comprises a frame (not shown here) and a plate (not shown here) intended to carry the sample holder with the sample 100 to be observed, the plate being movable in translation relative to the frame along at least two translation axes.

[0100] The equipment also comprises an interference device which here comprises a source 5 and a specular interface 3, the interference device thus being capable, in service, of producing optical interference between:

[0101] • reference waves obtained by reflection of the light emitted by the source 5 on the specular interface 3, and

[0102] • object waves obtained by backscattering of the light emitted by said source 5 on the sample.

[0103] Source 5 is a source that is said to be spatially incoherent.

[0104] The source 5 is for example a halogen lamp or a light-emitting diode (better known by the acronym LED) or even a block formed of a spatially coherent source (the coherent source being for example a laser) and a structure crossed by the rays at the output of the spatially coherent source, a structure making it possible to make said rays spatially incoherent at the output of the structure (and therefore of the block). The structure is for example provided with a multimode cavity, a multimode fiber, a hexagonal rod, etc.

[0105] Source 5 is here a temporally incoherent source, i.e. a source with a low temporal coherence length (for example in a range of 0.5 to 15 micrometers and preferably 1 to 10 micrometers). We therefore understand that even if source 5 is said to be temporally incoherent, it is still possible to define a coherence length for it (this length will however be low).

[0106] The installation also includes an acquisition device 7. In this case, the acquisition device 7 is part of the equipment. The acquisition device 7 allows the acquisition of at least one signal resulting from the interference between the reference waves and the object waves.

[0107] For this purpose, the acquisition device 7 comprises an optical sensor. The optical sensor is preferably a complementary metal-oxide-semiconductor optical sensor (better known by the English term CMOS for Complementarity metal-oxide-semiconductor).

[0108] Preferably, the optical sensor is chosen to acquire images at a high rate. This makes it possible to follow, if desired, dynamic movements within the sample when it contains at least one living cell. For example, the optical sensor is capable of acquiring images at a frequency greater than 100 Hertz and preferably greater than 200 Hertz and preferably greater than 400 Hertz.

[0109] Preferably, the optical sensor is chosen to acquire raw images (i.e. at the input of the acquisition device) according to a high signal-to-noise ratio. This allows the optical sensor to be sensitive even to very small living structures and / or to even very small movements. For example, the optical sensor is capable of acquiring raw images with a signal-to-noise ratio greater than 500 and preferably greater than 800 and preferably greater than 1000.

[0110] For example, the optical sensor is a camera, and for example, a high well capacity camera (better known by the English term “full well capacity”) and for example, a CMOS camera and for example, a Q-2A750 camera or a Q-2HFW camera, both marketed by the company Adimec.

[0111] The assembly 1 also comprises here a signal processing device (not shown here) emitted by the acquisition device 7 for example to generate an image of at least part of the sample. In the present case, the signal processing device is external to the equipment 2. The signal processing device comprises at least one processing unit such as a processor. The signal processing device comprises for example a computer.

[0112] Preferably, the installation 1 comprises a microscope incubator (not shown here) then forming the sample holder for the sample 100. The incubator 1 is for example arranged in the equipment 2 so as to be carried by the stage, the sample 100 being placed directly in the incubator.

[0113] The incubator is preferably portable so that it can be temporarily brought back to equipment 2.

[0114] The incubator facilitates the study of samples containing at least one living cell. Indeed, the incubator allows such samples to be kept alive for several days or even weeks.

[0115] Preferably, the incubator is shaped to accommodate multi-well plates.

[0116] Preferably, the incubator is equipped with temperature control within the incubator.

[0117] This makes it possible, for example, to avoid heating the sample 100, which could alter or destroy the sample 100. Preferably, the incubator is 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 the presence of nitrogen in the incubator and / or the presence of oxygen in the incubator.

[0118] In the case where the sample comprises living cells, the incubator is preferably configured to maintain the sample 100 at a given temperature and for example at a temperature substantially equal to 37 degrees Celsius (for example for human, primate, pig cells, etc.). Preferably, the incubator is configured to allow oxygenation of the sample (in particular by supplying a nitrogen and oxygen mixture and removing carbon dioxide), this oxygenation being ensured by at least controlling the carbon dioxide level in the incubator. Preferably, the incubator is configured to allow the humidity level of the sample to be managed so that the sample does not dehydrate. For example, the incubator is equipped with a sensor to ensure that the humidity level in the incubator 9 is between 70 and 100%.

[0119] The incubator here is advantageously a conventional incubator. For example, the incubator is an H201-K ​​incubator marketed by the company Okolab.

[0120] The interference device will now be detailed.

[0121] The interference device comprises a base which is fixed to the frame.

[0122] Said base carries a beam splitter element 10 which is here a non-polarizing beam splitter element 10 (better known by the English acronym NPBS for Non-Polarizing Beamsplitters). The splitter element 10 is for example a non-polarizing splitter cube, a non-polarizing splitter plate, etc.

[0123] The source 5 is intended to illuminate the separating element 10 via an optical unit 4.

[0124] In the present case, the optical block 4 like the source 5 are arranged in the extension of the separating element 10. The optical block 4 like the source 5 are here carried by the base and are therefore fixed relative to the separating element 10 and therefore relative to the frame.

[0125] The optical block 4 comprises, inside a housing of the optical block which is the part of the optical block 4 fixed to the base, successively between the source 5 and the separating element 10:

[0126] - a first diaphragm 12 arranged downstream of the source 5,

[0127] - a first optic 13 (such as for example a single lens, a single doublet or a pair of lenses or a pair of doublets) arranged downstream of the first diaphragm 12,

[0128] - a second diaphragm 14 arranged downstream of the first optic 13,

[0129] - a second optic 15 (such as for example a single lens, a single doublet or a pair of lenses or a pair of doublets) arranged downstream of the second diaphragm 14 and upstream of the separating element 10.

[0130] The four aforementioned elements are fixed in the casing and are therefore fixed relative to the separating element 10 and therefore relative to the frame.

[0131] The first optic 13 makes it possible, for example, to reduce the divergence of the rays generated by the source 5. This limits a loss of power of the light radiation generated by the source 5.

[0132] The first diaphragm 12 is for example an aperture diaphragm. The first diaphragm 12 is arranged in a focal plane of the first optic 13 and preferably in the image focal plane of the first optic 13. Thus the first optic 13 images the source 5 at the image focal plane of the first optic 13 which coincides with the first diaphragm 12. It is thus retained that the source 5 is conjugated to the first diaphragm 12 by means of the first optic 13. The first diaphragm 12 therefore makes it possible, by its aperture, for example to control the degree of spatial incoherence of the source 5 and / or the quantity of light received by the sample 100 and / or the numerical aperture of illumination of the installation 1 ... Furthermore, the first diaphragm 12 is also arranged in a focal plane of the second optic 15 and preferably in the object focal plane of the second optic 15.Furthermore, the focal length of the second optic 15 must be less than or equal to the distance separating the first optic 13 from the first diaphragm 12.

[0133] The second diaphragm 14 is for example a field diaphragm. The second diaphragm 14 makes it possible to restrict the illumination of the sample to illuminate only the portion of the sample which will be imaged by the installation 1 and / or to reduce incoherent reflections. The second diaphragm 14 is arranged in a focal plane of the second optic 15 and preferably in the object focal plane of the second optic 15.

[0134] For the present application, the concepts of “upstream” and “downstream” are understood according to the direction of circulation of the light.

[0135] For the purposes of this description, the term "diaphragm" means any organ enabling the passage of light radiation generated by the source 5 to be controlled: iris diaphragm, hole in a dedicated wall, etc.

[0136] In the example illustrated, upstream of the separating element 10 are thus successively the source 5, the first diaphragm 12, the first optic 13, the second optic 15 and the second diaphragm 14.

[0137] As already indicated, the first optic 13, the first diaphragm 12, the second optic 15 and the second diaphragm 14 are fixed in the equipment.

[0138] The source 5 therefore illuminates the separating element 10 (via the optical unit) which makes it possible to define a “lighting arm” of said separating element 10.

[0139] Furthermore, the separating element 10 allows, following its illumination by the source, to form an arm called the “object arm” which is associated, in operation, with the sample. The source 5 is not on the object arm but is on the illumination arm.

[0140] Furthermore, installation 1 includes a first objective 21 associated with the object arm.

[0141] Preferably, the numerical aperture of the first objective 21 is high. By “high” is meant a numerical aperture greater than 0.8 and preferably greater than 1 and preferably greater than 1.4 for the present application.

[0142] It is noted that the second optic 15 makes it possible to image the source 5 on the focal plane of the objective and for example on the object focal plane of the objective.

[0143] The first objective 21 is for example part of equipment 2.

[0144] The sample 100 (or at least the stratum of the sample 100 that we are seeking to image) is intended to be positioned at one of the foci of the first objective 21 and for example at the image focus of the first objective 21.

[0145] Accordingly, the first objective 21 is on the ob- j arm and .

[0146] Preferably, the second diaphragm 14 is arranged in a focal plane of the second optic 15, and for example in the object focal plane of the second optic 15. Thus the second diaphragm 14 is conjugated to the sample by means of the first objective 21 (the sample being at a focus of the first objective 21 and for example at the image focus of the first objective 21). Furthermore, the source 5 is conjugated to a focus of the first objective and for example at the object focus (pupil plane) of the first objective 21 (by means of the first optic 13.

[0147] Because the equipment here is “inverted observation”, the first objective 21 is arranged so as to observe the sample 100 from below the sample 100. For example, the first objective 21 is arranged under the stage and in this case under the incubator.

[0148] The equipment 2 comprises a reflection surface 22 so that a ray passing through the first objective 21 along the optical axis of said first objective 21 can be reflected up to the separating element 10. This makes it possible to have the first objective vertical.

[0149] Typically, the reflection surface 22 is flat. For example, the reflection surface 22 is a mirror, for example a thick mirror, for example a mirror having a thickness greater than or equal to 3 millimeters, for example a thickness greater than or equal to 4 millimeters. Preferably, said reflection surface 22 is a plane mirror carried by a prism or a plane mirror carried by a cube.

[0150] In the present case, said reflection surface 22 is arranged so that a ray propagating along the optical axis of the first objective 21 is then propagated, after reflection on the reflection surface 22, to then propagate to the separating element 10 along the ob- j et arm.

[0151] The interference device also comprises the specular interface 3 previously described arranged between the first objective 21 and here the sample 100.

[0152] The specular interface 3 is for example part of a glass slide. The specular interface 3 is for example part of the sample holder carrying the sample 100.

[0153] Preferably, the specular interface 3 is movable relative to the first objective 21 and is therefore movable in the frame.

[0154] For this purpose, the specular interface 3 is associated with at least one member for moving the specular interface 3 relative to the frame and in particular with respect to the first lens 21 (in particular to move the specular interface 3 closer to or further from the lens 21). For example, the specular interface 3 is carried by a base which is moved via the moving member. For example, the moving member is configured to move the specular interface according to at least one translation. For example, the moving member is configured to move the specular interface 3 according to at least one translation along the optical axis of the first lens 21.

[0155] Preferably, the plate and the specular interface 3 are fixed relative to each other. Here, the plate is therefore linked in translation to the specular interface 3: the plate is for this purpose fixed to the base on which the specular interface is also fixed. Consequently, when the specular interface is brought closer to, or moved further away from, the first objective 21, the plate (and therefore the sample) is identically brought closer to or further away from the first objective 21.

[0156] Furthermore, the specular interface 3 is arranged in the equipment 2 so that the distance between the specular interface and the plane imaged by the installation is here (limits included) between zero and three times or more the depth of field of the first objective 21 and preferably is here (limits included) between zero and two times the depth of field of the first objective 21 and preferably is here (limits included) between zero and one times the depth of field of the first objective 21.

[0157] For this purpose, the plate + specular interface assembly 3 can advantageously be moved if necessary in relation to the first objective 21 to fulfill this function.

[0158] In a particular case, the imaged plane is the image focal plane of the first objective 21. The imaged plane may however be different from said image focal plane depending on the relative position of the acquisition device 7 in the installation 1.

[0159] The stratum of the sample 100 that we wish to image is located here in the image focal plane of the first objective 21, we therefore ensure that the distance between said stratum and the specular interface 3 is preferably less than or equal to twice the depth of field. Preferably, we therefore ensure that the distance between said stratum and the specular interface 3 is less than one time the depth of field of the first objective 21.

[0160] This allows the specular interface 3 to serve as a reference field for the reference waves.

[0161] In the case where the source 5 emits light rays according to a spectral distribution obeying a Gaussian law, we can define Ào as the central wavelength of said distribution and AA as the spectral width of said definition.

[0162] In this case, the first objective 21 and the specular interface 3 are arranged so that the distance AZ between the image focal plane of the first objective 21 and the face of the specular interface 3 facing the sample 100 is preferably less than twice the depth of field of the first objective 21, i.e. less than twice

[0163] 2Ào / NA 2 (with NA the aperture field of the first lens 21).

[0164] We note that in the case where the depth of field would be greater than the coherence length L c of the central wavelength of the light source 5, then the distance AZ would preferably be taken less than twice said coherence length L c . As a reminder L c is worth:

[0165] AZ is for example between 0 and 5 micrometers. This value interval is not limiting and will depend mainly on the characteristics of the first objective 21. In reality AZ is linked to the maximum wavelength emitted by the light source 5 for which it is still possible to form interferences.

[0166] We therefore understand with this configuration, which we find on the object arm, between the separating element 10 and the sample 100 successively the reflection surface 22, the first objective 21 and the specular interface 3. Thus, the specular interface 3 is found on the object arm. We therefore understand that the specular interface 3 must be sufficiently transparent to allow the light rays to pass through it so that they can reach the sample 100 and leave via the source 5.

[0167] Furthermore, we note that the specular interface 3 is the element of the equipment 2 allowing the reference waves to be obtained. However, the specular interface 3 is cleverly arranged on the object arm.

[0168] From then on, the equipment 2 thus described does away with a dedicated reference arm thanks to the use of the cleverly placed specular interface 3.

[0169] Furthermore, the equipment 2 here comprises a third optic 23 arranged at the output of the interference device, that is to say arranged between the separating element 10 and the acquisition device 7. The third optic 23 may be part of the interference device or may not be part of said interference device.

[0170] For example, the third optic 23 is a single lens, a single doublet or a pair of lenses or a pair of doublets.

[0171] The acquisition device 7 is arranged in the focal plane of the third optic 23 and for example in the object focal plane of the third optic 23.

[0172] In the present case, the optical axis of the acquisition device 7 is perpendicular to the illumination arm. The installation 1 thus described is an improvement of a Linnik interferometer in a Koehler illumination configuration.

[0173] From the assembly 1 described, it is possible to perform static full-field optical coherence tomography imaging as well as dynamic full-field optical coherence tomography imaging. In the case of dynamic full-field optical coherence tomography imaging, a sample is placed in the equipment 2 and a temporal succession of N two-dimensional interferometric signals of a stratum of the sample 100 is acquired by the acquisition device 7. Furthermore, the installation 1 thus described makes it possible in particular (although not exclusively) to be able to image the stratum or strata of the sample 100 closest to the specular interface 3.

[0174] Additionally, full-field optical coherence tomography imaging can be performed with or without the incubator.

[0175] Consequently, the installation 1 thus described allows the implementation of numerous imaging possibilities.

[0176] With the described setup 1, it is possible, for example, to acquire images of cells, but also to visualize cellular activity and distinguish the metabolic state of a cell. The cells can, for example, be two-dimensional cultures such as two-dimensional monolayer cultures, three-dimensional cultures such as organoids or other three-dimensional multi-layer cultures...

[0177] It is indeed possible to study cells in an invasive but non-destructive manner.

[0178] One of the strengths of the installation 1 described is therefore its ability to image without disturbing the natural environment. The installation 1 thus described can be used for example for the study of organoids, two-dimensional monolayer cultures, three-dimensional multi-layer cultures, fibroblasts, retinas and corneas, the study of retina and cornea explants of mice, pigs, macaques, etc., quality control of large-scale organoid production, assistance in the microfluidic field, disease modeling, to carry out efficacy tests of new treatments (genetic, pharmaceutical, etc.), for transplantation, etc.

[0179] Furthermore, the installation 1 described makes it possible to generate both a static signal allowing the three-dimensional structure of a tissue to be visualized and a dynamic signal allowing the cells of a tissue to be identified and their metabolism to be measured, for example.

[0180] Other applications of the installation 1 described are conceivable, such as for example all microscopy studies with high spatial resolution (for example a resolution between 100 and 400 nanometers) and / or temporal resolution (for example a resolution of the order of a millisecond, such as for example 2 milliseconds), in particular those excluding the destruction of the sample and / or integrating the absence of endogenous markers.

[0181] In addition, it is possible to generate real-time tracking of a sample with, for example, the generation of an image presenting a contrast reflecting movement mechanisms of the order of X milliseconds, X being between 100 and 200 milliseconds, X being, for example, 160 milliseconds.

[0182] Furthermore, installation 1 is of reduced dimensions due to its absence of a reference arm (and in particular due to the absence of a second objective arranged on the reference arm). Installation 1 can thus be miniaturized.

[0183] Furthermore, the installation 1 described proves to be simple in its structure and is therefore easy to be implemented even with means other than those mentioned above.

[0184] According to a first option, the acquisition device 7 can be a simple mobile phone equipped with a camera, such as a smart phone or "smartphone". The acquisition device 7 is then not part of the installation 1. The installation 1 could, for example, include a location (optionally arranged on the equipment frame) and even an inexperienced user will only need to place their phone in the location provided for this purpose to be able to produce high-quality imaging simply and quickly.

[0185] It is noted that the third optic 23 can optionally also be part of the mobile phone.

[0186] Installation 1 is of reduced dimensions due to its absence of a reference arm. Installation 1 can thus be miniaturized. Optionally, light source 5 can also be part of the mobile phone.

[0187] Optionally, the processing device can also be part of the mobile phone.

[0188] According to a second option (possibly combinable with the first option mentioned above), the installation 1 and / or the equipment 2 is manufactured from an existing microscope. For example, the stage, the frame, the reflection surface 3 and the first objective 21 are those of an existing microscope such as for example an inverted microscope. For example the inverted microscope is a turret microscope. However, said microscope is modified so that:

[0189] - the specular interface 3 (as well as optionally its associated displacement member) is arranged between the first objective 21 and the plate,

[0190] - so that the interference device is added to the microscope as well as possibly the source 5 and the acquisition device 7,

[0191] - that we adjust the specular interface distance 3 / first objective 21 as indicated above.

[0192] This advantageously allows the construction costs of installation 1 to be reduced.

[0193] Furthermore, this makes it easier for a user who is already familiar with working with microscopes to use Installation 1.

[0194] Furthermore, this makes it possible to take advantage of the benefits of a conventional microscope such as, for example, the particular imaging characteristics (imaging under visible light, fluorescence imaging, etc.), particular objectives, etc. In particular, the microscope may be a microscope for which optical modules [differential interference contrast module, better known by the English acronym DIG module (for Differential Interference Contrast), coherent anti-Stokes Raman scattering module, better known by the acronym CARS module (for Coherent AntiStokes Raman Scattering), second harmonic generation module, better known by the English acronym SHG module (for Second Harmonic Generation), third harmonic generation module, better known by the English acronym THG module (for Third Harmonic Generation), Raman module, one- or two-photon fluorescence module, etc.] have already been developed.

[0195] With this second option, the installation 1 can thus easily be coupled to these modules of the prior art, which makes it possible to enrich the imaging possibilities of the sample 100. In addition, the associated microscope can be a microscope for which accessories have already been developed so that, starting from such a microscope, the installation makes it possible to benefit from the accessories of the microscope. For example, the aforementioned incubator can be such an accessory of the prior art.

[0196] A second embodiment will now be described with reference to Figures 2a and 2b.

[0197] Whereas in the first embodiment, the installation 1 was devoid of an additional physical reference arm (the specular interface 3 being carried by the object arm), in the second embodiment the installation 1 also comprises an additional physical reference arm. The entire installation which was described according to the first embodiment is therefore also present here.

[0198] With the additional reference arm, the interference device is thus also capable, in service, of producing optical interference between: • reference waves obtained by reflection of the light emitted by the source 5 on a reflection surface 6 of the additional object arm, and

[0199] • object waves obtained by backscattering of the light emitted by said source 5 on the sample.

[0200] The reflection surface 6 is flat. The reflection surface 6 is for example a mirror.

[0201] Furthermore, the installation includes a second objective 24. The two objectives 21, 24 are identical and are associated with one of the arms respectively. The two objectives 21, 24 have an identical numerical aperture (better known by the English acronym NA for "numerical aperture").

[0202] Optionally, the numerical aperture of the second objective 24 is high. By “high” is meant a numerical aperture greater than 0.8 and preferably greater than 1 for the present application.

[0203] It is noted that the second optic 15 makes it possible to image the source 5 on a focal plane of the two objectives and for example on the object focal plane of the two objectives 21, 24.

[0204] The second lens 24 is part of the interference device. Thus, the second lens 24 is arranged at the level of the reflection surface 6. The optical axis of the second lens 24 is, for example, normal to a plane along which the reflection surface 6 extends.

[0205] More precisely here, the second lens 24 is arranged so that the reflection surface 6 is located in one of the foci of the second lens 24 and for example at the image focus of the second lens 24.

[0206] The second objective 24 is therefore on the reference arm.

[0207] In the example illustrated, downstream of the separating element 10, on the reference arm side, there are thus successively the second objective 24 and the reflection surface 6.

[0208] In the present case, the acquisition device 7, the third optic 23, the interface element 10, the second objective 24 and the reflection surface 6 are in the extension of one another.

[0209] Preferably, the reflection surface 6 is movable in a translational movement along the optical axis of the second objective 24.

[0210] For this purpose, the reflection surface 6 is mounted movably in the interference device relative to the frame so that it can be moved relative to the second objective 24. For example, the reflection surface 6 is mounted on a base which is movable in translation relative to the frame. For example, the base is movable in translation relative to the frame by means of at least one piezoelectric actuator.

[0211] Furthermore, the reference arm is associated with blocking means (not visible here) for said reference arm in order to temporarily prevent light rays from moving along the reference arm. For example, a cover or a shutter or a mirror can be temporarily arranged at the level of the separating element 10 or the separating element 10 can be oriented differently or the reference arm is mounted on a base which can be moved relative to the separating element 10 ... Thus, according to a first possibility of use of the installation illustrated in FIG. 2a, it is possible to image the sample 100 as in the first embodiment without using the reference arm. The blocking means are then activated to prevent light rays from moving along the reference arm.

[0212] In this configuration the distance AZ is defined between the imaged plane and the specular interface 3. This distance is preferably less than twice the depth of field of the first objective 21 (or equal to twice the coherence length of the central wavelength of the source 5 if the depth of field of the first objective is greater than said coherence length).

[0213] Preferably this possibility is primarily used to image the interface layer(s) of the sample 100 or to image samples 100 in two dimensions. According to a second possibility of using the installation illustrated in FIG. 2b, it is possible to image the sample 100 in a more traditional manner using the reference arm. The blocking means are then deactivated to allow the light rays to move along the reference arm.

[0214] For example, specular interface 3 remains in place in installation 1 and this characteristic is taken into account in the adjustment of the reference arm.

[0215] In this configuration the distance AZ is defined between the imaged plane and the reflection surface 6. For example the imaged plane corresponds to the image focal plane of the second objective 24 (but the imaged plane may correspond to another plane depending on the position of the acquisition device 7 in the installation).

[0216] This distance AZ is preferably less than at least twice the depth of field of the second objective 24 (or twice the coherence length of the source if the coherence length of the source is less than the depth of field of the second objective).

[0217] Preferably this possibility is primarily used to image sample 100 in the strata furthest from the first objective.

[0218] Advantageously, it is thus possible to easily switch from a first possibility of use to a second, which further increases the imaging possibilities of the sample 100. It is noted in particular that for the first possibility of use as for the second, it is thus possible to carry out FFOCT imaging as well as D-FFOCT imaging, whether one is in the first possibility of use or in the second possibility of use.

[0219] It is noted that the first option and the second option mentioned above for the first embodiment are also applicable to the present second embodiment.

[0220] Of course, the invention is not limited to the embodiments described but encompasses any variant falling within the scope of the invention as defined by the claims.

[0221] In particular, the incubator may or may not be part of the installation and / or the acquisition device may or may not be part of the installation and / or the processing device may or may not be part of the installation and / or the source may or may not be part of the installation and / or the mobile phone may or may not be part of the installation.

[0222] Furthermore, the acquisition device may or may not be part of the equipment and / or the processing device may or may not be part of the equipment and / or the source may or may not be part of the equipment.

[0223] The equipment may not be inverted. The illumination of the sample may thus be carried out from above (the collection of the signal by the first objective then being carried out preferentially from above as well) as from below (the collection of the signal by the first objective then being carried out preferentially from below as well).

[0224] The optical sensor may be different from what has been indicated, for example, the optical sensor may be a charge-coupled device (CCD). The optical sensor may be capable of working in the visible and / or in another domain, such as in the infrared. Thus, the optical sensor may be a near-infrared image sensor (SWIR for Short-Wave-Infrared). The optical sensor may, for example, be an InGaAs sensor (for indium-gallium arsenide) or an InGaAs SWIR sensor. The equipment and / or installation may be designed so that the optical sensor (and / or the acquisition device) is interchangeable, for example, to be able to work in the visible and then to be able to work in a domain other than the visible, for example, in the infrared.The installation may not include an incubator (portable or not). The installation may include a heating enclosure in which at least the microscope is arranged to be able, for example, to maintain the sample at a given temperature.

[0225] Although high numerical aperture objectives are used here, lower numerical aperture objectives can of course be used.

[0226] The microscope can be without a turret.

[0227] The equipment may be devoid of a reflection surface arranged on the object arm as described.

[0228] The first objective could be in the alignment of the source.

[0229] The specular interface may include a particular surface treatment allowing its reflection coefficient to be modified.

[0230] The specular interface may be temporarily removable from the equipment or may be fixed without the possibility of disassembling the equipment.

[0231] Although here it is the specular interface that moves relative to the first lens, it could be the first lens that moves relative to said specular interface. Alternatively, the first lens and the specular interface could be at a fixed distance in the frame relative to each other.

[0232] Although here the specular interface belongs to a glass slide, the specular interface could belong to a slide made of another material and for example a plastic slide. The specular interface could belong to an element made of glass, plastic or any other material allowing light rays to pass through it, the material preferably being flat and / or smooth. The specular interface could be part of a slide or be part of another element shaped differently and for example a plate or any other shape allowing light rays to pass through said specular interface.

[0233] The specular interface may or may not be integrated into the sample holder, particularly when the latter is a slide (glass, plastic, etc.). Although here the specular interface is separate from the stage, the specular interface may be integrated into the stage.

[0234] Although here the specular interface is the face of the slide facing the sample, the specular interface could be the face of the slide facing the first objective or any other layer inside the slide. We could also, for the same sample, consider alternatively the specular interface formed by the face facing the first objective and the specular interface formed by the face facing the sample, which would make it possible to image at least two different layers of the same sample (an interface layer and a distance layer from the said interface layer by a distance equal to the distance separating the two interfaces).

[0235] Although here the beam splitter element is non-polarizing, the beam splitter element may be polarizing and associated with an additional device ensuring non-polarization (such as delay plates such as quarter-wave plates). The beam splitter element may thus be any splitter element such as a non-polarizing beam splitter (NPBS), a polarizing beam splitter, a “Polka dot” type beam splitter, one or more membranes, etc.

[0236] The installation will be able to do without the reflection surface 22.

[0237] Although here the specular interface remains in place in the second configuration, alternatively the specular interface can be temporarily removed from the installation as long as the second configuration is used.

Claims

CLAIMS 1. A method of full-field optical coherence tomography microscopic imaging of at least one sample, the method comprising the steps of: - emit light propagating in an object arm of an installation in which the sample is arranged so that the light backscatters in the sample to produce an object wave, - generating at least one interference between the object wave and a reference wave, the method being characterized in that the reference wave is generated by reflection of the light on a specular interface arranged on the object arm.

2. Method according to claim 1, in which the specular interface is arranged so as to be at a given distance from a plane imaged by the installation, distance which is greater than or equal to zero and which is less than or equal to: - has twice the depth of field of an objective of the installation intended to observe the sample, or - at twice a coherence length of a light source generating the light if the coherence length is less than the depth of field of the objective.

3. Optical equipment for full-field optical coherence tomography microscopic imaging of at least one sample, the equipment comprising an objective for in-service observation of the sample, the device comprising a specular interface, the device thus being capable in service of allowing the production of at least one interference between: - at least one reference wave obtained by reflection of light emitted by a light source associated with the equipment on the specular interface, and - at least one object wave obtained by backscattering of the light emitted by said source onto the sample, the specular interface being arranged with respect to the objective so that the object wave crosses said specular interface in use during its journey between the sample and the source.

4. Equipment according to claim 3, in which the specular interface (3) belongs to a glass slide or a plastic slide.

5. Equipment according to one of claims 3 to 4, in which the source (5) is part of the equipment.

6. Equipment according to one of claims 3 to 5, comprising an acquisition device (7) adapted to acquire at least one signal resulting from interference between the reference waves and the object waves.

7. Equipment according to one of claims 3 to 6, wherein the equipment is also configured to allow dynamic full-field optical coherence tomography microscopic imaging.

8. Equipment according to one of claims 3 to 7, comprising an incubator intended to receive the sample.

9. Equipment according to one of claims 3 to 8, in which the source (5) illuminates a beam splitter element of the installation through an optical block comprising at least one optic and at least one diaphragm.

10. Equipment according to one of claims 3 to 9, in which the specular interface is part of the sample holder.

11. Equipment according to claim 10, in which the specular interface is a face of the sample holder facing in use towards the sample.

12. Installation comprising equipment according to one of the claims 3 to 11 as well as a reference arm, the installation thus being capable in service of also allowing the production of at least one interference between: - at least one reference wave obtained by reflection of the light emitted by the light source (5) on a reflection surface (6) of the additional reference arm, and - at least one object wave obtained by backscattering of the light emitted by said source on the sample.

13. Installation according to claim 12, comprising means for temporarily blocking the reference arm.

14. Installation according to one of claims 8 to 13, in which at least part of the installation is integrated into a mobile telephone.

Images