Method for dynamic microscopic optical coherence tomography imaging

Abstract

The invention relates to a method for the dynamic microscopic optical coherence tomography imaging of at least one sample in which a difference between an optical path taken by the light rays in a reference branch and an optical path taken by the light rays in an object branch is modified regularly and deliberately during the acquisition of a time series of signals.

Description

[0001] DESCRIPTION

[0002] TITLE OF THE INVENTION: Dynamic optical coherence tomography microscopic imaging method

[0003] The invention relates to a method for dynamic optical coherence tomography microscopic imaging of at least one sample.

[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 which can be 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 without any labeling and in a non-invasive manner,

[0008] - to image in three dimensions a complex sample generally exceeding 100 micrometers in thickness,

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

[0010] Optical coherence tomography imaging in the time domain (including full-field OCT) consists of measuring the interferometric signal, point by point, between a signal of light backscattered by a sample when it is illuminated by a source with a reference light signal emitted by this same source.

[0011] 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 (without point-by-point scanning).

[0012] There are two main variations of full-field optical coherence tomography imaging:

[0013] - 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;

[0014] - dynamic full-field temporal optical coherence tomography imaging (better known by the acronym D-FFOCT for “Dynamic FFOCT”) which quantifies the temporal evolution of the interferometric signal.

[0015] 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 subcellular elements that make up 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.

[0016] D-FFOCT imaging is particularly advantageous for performing non-invasive imaging, enabling multiple applications to be implemented: the study of organoids, disease modeling, cancer diagnosis, etc.

[0017] Indeed, D-FFOCT imaging makes it possible to obtain a great deal of information on the functioning of living things thanks to the particularity of its signal, while being non-invasive and non-destructive.

[0018] More precisely, D-FFOCT imaging makes it possible to obtain a local amplification - spatially decorrelated - of the backscattered signal within the sample (generally a three-dimensional volume) as well as associated local phase information.

[0019] In addition, D-FFOCT imaging allows to limit the presence of speckles compared to similar imaging techniques with phase resolution (interference scattering microscopy, holographic microscopy, etc.) thanks to the use of a spatially incoherent source and the temporal analysis carried out. This allows a direct interpretation of the sample studied.

[0020] However, when scatterers (cells, nuclei, metabolic elements, etc.) are static or not very mobile in the sample, speckles remain a problem (even in the case of D-FFOCT imaging). In addition, optical interference artifacts, sometimes called "interface artifacts" or "fringe artifacts," can also appear.

[0021] These artifacts and speckles therefore greatly hinder the study of the samples or prevent it completely.

[0022] SUBJECT OF THE INVENTION

[0023] The invention aims in particular to enable dynamic optical coherence tomography microscopic imaging which makes it possible to limit the appearance of optical artifacts and / or speckles during dynamic imaging of at least one sample even when diffusers internal to the sample do not move or move little.

[0024] SUMMARY OF THE INVENTION

[0025] To this end, the invention provides a method for dynamic optical coherence tomography microscopic imaging of at least one sample by means of optical equipment comprising an interference device which comprises a beam splitter element and at least one reflection surface, the method comprising the steps of: producing at least one interference between

[0026] • at least one reference wave obtained by reflection of the light emitted by a light source associated with the optical equipment on the reflection surface, and

[0027] • at least one object wave obtained by backscattering the light emitted by said source on the sample, the beam splitter element thus forming two arms, namely a “reference arm” associated with the reflection surface and an “object arm” associated with the sample, acquiring a series of signals of at least two signals succeeding each other in time to form a time series of signals, each signal resulting from the interference between the reference wave and the object wave.

[0028] According to the invention, a difference between the optical path taken by the light rays in the reference arm and the optical path taken by the light rays in the object arm is regularly and deliberately modified during the acquisition of the time series of signals.

[0029] In this way, the invention causes a voluntary variation in the phase shift between the object wave and the reference wave throughout the acquisition of the series of signals.

[0030] This voluntary variation of the phase shift is thus added to any natural oscillations of said phase shift caused by movements of internal diffusers (cells, nuclei, metabolic elements, etc.) in the sample.

[0031] The phase shift between the object wave and the reference wave has a random initial value at the time of launching the acquisition. The invention allows a voluntary modification of this phase shift (this modification can be designated for example by the English formula of "rolling phase"). Thanks to this it is possible to make the measurements carried out on the acquired signals (measurements of light intensity for example) independent of this initial phase shift, initial phase shift which was generally at the origin of the artifacts and / or speckles mentioned above. For example, a regular sampling of the interference function (obtained via the series of signals) makes it possible to homogenize the average response of the interference independently of the initial phase.

[0032] We therefore understand that the phase shift between the object wave and the reference wave sees its value evolve during the acquisition by means of two components: - a natural component due to the movements of the internal diffusers (these movements being defined by a characteristic frequency called natural frequency fo, fo is for example the average frequency of the movements of the internal diffusers),

[0033] - a voluntary component caused by the modification of the difference in optical paths between the reference arm and the object arm (the frequency of the voluntary modification of the phase shift being subsequently called the voluntary frequency f V oi) .

[0034] Subsequently, and for simplification, we will speak of "natural" for the modification of the phase shift due to the movements of the internal diffusers, of "voluntary" for the modification of the phase shift due to the voluntary modification of the differences in optical paths and of "global" for the addition of the voluntary variation and the natural variation of the phase shift.

[0035] Therefore, even in the case of internal diffusers with little or no movement (usually by little or no movement is meant that a given diffuser has moved by an axial distance less than / 2n during the acquisition time with the central wavelength of a light source illuminating the sample by the optical equipment and n the medium in which the sample evolves), the presence of the variation in the phase shift makes it possible to limit the appearance of optical artifacts and / or speckles in the images obtained by this dynamic optical coherence tomography microscopic imaging method of the invention.

[0036] Advantageously, the inventors were able to observe that the images obtained with this method were of better quality than with the methods of the prior art even in the case of diffusers with high mobility.

[0037] For the present invention, "reference arm" means the part of the equipment located between the reflection surface and the beam splitter element (a light ray can thus propagate in said reference arm following a given optical path called the reference arm optical path).

[0038] For the present invention, "object arm" means the part of the equipment located between the sample and the beam splitter element (a light ray can thus propagate in said object arm along a given optical path called the object arm optical path).

[0039] For the present invention, "lighting arm" means the part of the equipment located between the source and the beam splitter element (a light ray can thus propagate in said lighting arm following a given optical path called the lighting arm optical path).

[0040] Thus, we understand that the value of the phase shift between the reference wave and the object wave is directly linked to the value of the phase shift between the reference arm and the object arm.

[0041] Preferably, the optical path difference is changed during the acquisition of the time series according to a frequency f V oi such that: fo > 2*f vo i, with fo a frequency characteristic of the natural movements of diffusers internal to the sample called natural frequency.

[0042] The inventors were indeed able to observe that to better benefit from the advantages of dynamic optical coherence tomography microscopic imaging, it was preferable that the frequency of the voluntary modification of the phase shift f V oi (voluntary frequency) is less than half the frequency of the involuntary modification of the phase shift fo (natural frequency) due to the movements of the diffusers.

[0043] Thus, it is relatively simple to generate an image from the series of signals because the voluntary variation of the phase shift is known, controlled and slower than that induced by the diffusers within the sample. This makes it possible in particular to distinguish the movements induced by the voluntary variation of the position of the reference arm from those natural ones induced by the sample.

[0044] Preferably, the optical path difference is modified during the acquisition of the time series so that: fo > 10*f voi .

[0045] Optionally at least one element of the optical equipment is moved regularly to voluntarily and regularly modify the difference between the optical path taken by the light rays in the reference arm and the optical path taken by the light rays in the object arm.

[0046] Optionally the difference between the optical path taken by the light rays in the reference arm and the optical path taken by the light rays in the object arm is generated by a modification of the length of the reference arm.

[0047] The reference arm therefore does not remain immobile during the dynamic acquisition, unlike what is done in prior art dynamic optical coherence tomography microscopic imaging. In particular, the reference arm is modified voluntarily and regularly during the dynamic acquisition, so that the overall phase shift (due to the voluntary phase shift) is modified voluntarily and regularly during the dynamic acquisition.

[0048] Optionally, at least one element of the optical equipment is moved regularly to voluntarily and regularly modify the length of the reference arm. Optionally, the series of signals comprises at least twenty signals and preferably at least fifty signals succeeding each other in time to form the time series of signals, each signal resulting from the interference between the reference wave and the object wave. Optionally, the natural frequency (fo) has a value between 0.1 and 50 Hertz and for example between 0.1 and 25 Hertz and for example between 0.1 and 20 Hertz and for example between 0.3 and 20 Hertz and for example between 0.3 and 25 Hertz and for example between 5 and 18 Hertz.

[0049] The natural frequency depends on the nature of the sample being imaged.

[0050] Optionally, the natural frequency (fo) is predetermined.

[0051] Optionally, the natural frequency (fo) is estimated during a step prior to the acquisition of the signal time series.

[0052] Optionally, during a preliminary step:

[0053] - at least one acquisition of a preliminary time series of signals is carried out during dynamic optical coherence tomography microscopic imaging without modification of the length of the reference arm, - an interval of fluctuations frequencies of the internal diffusers in the sample is estimated,

[0054] - the value of the natural frequency is fixed as being the average of the interval or as being the minimum limit of the interval.

[0055] We therefore understand that during this preliminary step we roughly estimate the natural frequency from the images outside the areas of optical artifacts and / or speckles present on the images.

[0056] It is therefore understood that this preliminary step is carried out according to a prior art dynamic optical coherence tomography microscopic imaging method.

[0057] The value of this natural frequency can then be refined once the rest of the method according to the invention has been carried out, and recorded for later use on another sample.

[0058] Optionally, the variation of the difference of the optical path reference arm / object arm has a frequency between 0.1 and 10 Hertz.

[0059] In this way, f V oi is between 0.1 and 10 Hertz.

[0060] Preferably, the voluntary phase shift between the object wave and the reference wave is defined by a periodicity function 2n, the variation of the difference in the reference arm / object arm optical paths being such that the voluntary phase shift covers an interval [0; W] during the acquisition of the time series, W being greater than or equal to n and more preferably so that W is greater than or equal to 2n.

[0061] In this way, the voluntary variation of the phase shift is important.

[0062] The voluntary variation of the phase shift is preferably equal to at least n / N between two successive signals and preferably at least 2n / N between two successive signals, N being the number of signals making up the time series.

[0063] The element is optionally translated between two successive signals by an increment corresponding to at least X / N and preferably to at least 2X / N, being the central wavelength of a source illuminating the beam splitter element.

[0064] Instead, the element is translated continuously and not incrementally during the acquisition of the time series.

[0065] Optionally, the element is moved in a translational motion.

[0066] Optionally, the moving element is the reflection surface.

[0067] Optionally, an image is generated from only the series of signals acquired during the movement of the reflecting surface.

[0068] Other characteristics and advantages of the invention will emerge from reading the following description of a particular and non-limiting implementation of the invention373.

[0069] BRIEF DESCRIPTION OF THE DRAWINGS

[0070] Reference will be made to the attached drawings for which:

[0071] [Fig. 1] Figure 1 is a schematic view of optical equipment allowing a particular implementation,

[0072] [Fig. 2] Figure 2 is a graph representing a light intensity as a function of a phase shift between a reference wave and an object wave when using the optical equipment of Figure 1,

[0073] [Fig. 3] Figure 3 is a graph representing the evolution of a variation of a voluntary phase shift between a reference wave and an object wave when using the optical equipment of Figure 1 during a signal acquisition without modification of the reference arm of said optical equipment,

[0074] [Fig. 4] Figure 4 is a graph representing the evolution of a variation of a voluntary phase shift between a reference wave and an object wave when using the optical equipment of Figure 1 during a signal acquisition with modification of the reference arm.

[0075] DETAILED DESCRIPTION OF THE INVENTION

[0076] Figure 1 schematically illustrates optical equipment for full-field optical coherence tomography microscopic imaging of at least one sample allowing a particular implementation of the invention.

[0077] Such optical equipment 1 is for example that described in application WO 2023 / 203283 of the present applicant. It will be described here in less detail than in application WO 2023 / 203283 and reference may be made to said application for further details on the equipment.

[0078] The optical equipment 1 thus comprises at least one microscope 2 and a module 3 connected to the microscope 2. For example, the module 3 is shaped so as to be able to be arranged in one of the racks of the row of racks of the microscope 2, a row forming a turret. In particular, the microscope 2 is shaped so as to have at least one optical output opposite at least one row so that the module inserted in said row is de facto optically coupled to the microscope 2.

[0079] In a manner known per se, the microscope 2 comprises a stage intended to carry a sample to be observed, the stage being movable in translation relative to a frame of the microscope 2 along at least two translation axes.

[0080] The module 3 comprises an interference device 4 which here comprises a source 5 and a reflection surface 6, the interference device 4 thus being capable, in service, of producing optical interference between:

[0081] • reference waves obtained by reflection of the light emitted by the source 5 on the reflection surface 6, and

[0082] • waves obtained by retrodi f fusion of the light emitted by said source 5 on the sample.

[0083] The source 5 is a temporally incoherent source or a source with a low temporal coherence length (for example in a range of 0.5 to 1.2 micrometers or for example in a range of 0.8 to 1 micrometer) and / or spatially incoherent or a source with a low spatial coherence length (for example in a range greater than the value of 100 micrometers and for example in a range greater than 1 millimeter and for example in a range between 1 and 2 millimeters for a light-emitting diode). 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 coherent source (the coherent source being for example a laser) and a structure crossed by the rays at the output of the coherent source, a structure making it possible to make said rays spatially and temporally 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. The reflection surface is diffusing or specular. The reflection surface 6 is flat. The reflection surface 6 is for example a mirror, or for example a silicon surface or a specular surface.

[0084] The optical equipment 1 also comprises an acquisition device 7. The acquisition device 7 allows the acquisition of at least one signal resulting from the interference between the reference waves and the obj waves and .

[0085] 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). The optical sensor thus comprises an active surface.

[0086] Preferably, the optical sensor is chosen to acquire images at a high rate. This makes it possible to follow, if desired, significant movement dynamics within the sample when the latter comprises at least one living cell. For example, the optical sensor is capable of acquiring images at a frequency greater than 50 Hertz and preferably greater than 100 Hertz and for example greater than 200 Hertz and for example greater than 400 Hertz. For example, the optical sensor is capable of acquiring images at a frequency which remains lower than 1000 Hertz. Preferably, the optical sensor is chosen to acquire images 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 images with a signal-to-noise ratio greater than 500 and preferably greater than 800 and preferably greater than 1000.

[0087] The optical equipment 1 also comprises here a device for processing the signal 8 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 8 is external to the module 3. The signal processing device 8 comprises at least one processing unit such as a processor. The signal processing device 8 comprises for example a computer.

[0088] Preferably, the optical equipment 1 comprises a microscope incubator 9.

[0089] The interference device 4 comprises a frame carrying a base which is preferably stationary relative to the frame.

[0090] Said base carries a beam splitter element 10. Said splitter element 10 is here a non-polarizing beam splitter element (better known by the English acronym NPBS for Non-Polarizing Beamsplitters). The splitter element 10 is for example a non-polarizing (alternatively polarizing) splitter cube, a non-polarizing (alternatively non-polarizing) splitter blade, etc.

[0091] In particular, the interference device 4 comprises a first adjustment unit 11 via which the source 5 will illuminate the separating element 10. For example, the first adjustment unit 11 comprises at least two reflection surfaces 12, 13 which are mounted opposite each other. The reflection surfaces 12, 13 are flat. The reflection surfaces 12, 13 are, for example, mirrors.

[0092] In particular, the interference device 4 comprises a first optic 14 arranged upstream of the first adjustment unit 11 (the notions of “upstream” and “downstream” being understood according to the direction of circulation of the light) between the source 5 and the first adjustment unit 11. The first optic 14 is for example a single lens, a single doublet or a pair of lenses or a pair of doublets.

[0093] The interference device 4 comprises a second optic 15 arranged downstream of the first adjustment unit 11 between the first adjustment unit 11 and the separating element 10. The second optic 15 is for example a lens, a doublet ...

[0094] According to a particular embodiment, the interference device 4 comprises a first diaphragm 16 arranged upstream of the first adjustment unit 11 between the source 5 and the first adjustment unit 11. For example, the first diaphragm 16 is arranged downstream of the first optic 14 between the first optic 14 and the first adjustment unit 11.

[0095] According to a particular embodiment, the interference device 4 comprises a second diaphragm 17 arranged downstream of the first adjustment unit 11 between the first adjustment unit 11 and the separating element 10. For example, the second diaphragm 17 is arranged downstream of the second optic 15 between the second optic 15 and the separating element 10.

[0096] In the example illustrated in the single figure, upstream of the separating element 10 are thus successively the source 5, the first optic 14, the first diaphragm 16, the first adjustment unit 11, the second optic 15 and the second diaphragm 17. The first optic 14, the first diaphragm 16, the second optic 15 and the second diaphragm 17 are fixed in the module 3.

[0097] The source 5 therefore illuminates the separating element 10, which makes it possible to define a “lighting arm” of said separating element 10.

[0098] Furthermore, it is known that the separating element 10 makes it possible to form two arms following its illumination by the source:

[0099] - an arm called the “reference arm” which is associated with the reflection surface 6,

[0100] - an arm called the “object arm” which is associated, in operation, with the sample.

[0101] The interference device 4 also comprises a second adjustment unit 18 via which the reflection surface 6 will be able to interact with rays propagating from the splitter element 10 on the reference arm. Typically the second adjustment unit 18 comprises at least one reflection surface 27. Thanks to the movements of the reflection surface 27, it is thus possible to modify the optical path of the rays reflected by the reflection surface 6 to the beam splitter element 10.

[0102] The interference device 4 here comprises a third optic 19 arranged between the separating element 10 and the reflection surface 6. The third optic 19 is for example a lens, a doublet...

[0103] Furthermore, the optical equipment 1 comprises a first objective 20 and a second objective 21. The two objectives 20, 21 are identical and are associated with one of the arms respectively. The two objectives have a numerical aperture (better known by the English acronym NA for “numerical aperture”) preferably identical. Optionally, the numerical aperture of the objectives is high. By “high” is meant a numerical aperture greater than 0.8 and preferably greater than 1 for the present application.

[0104] The first objective 20 is arranged in the module 3 at the level of the reflection surface 6. The optical axis of the first objective 20 is for example normal to a plane along which the reflection surface 6 extends.

[0105] Preferably, the second adjustment unit 18 also comprises a member 26 for moving the first objective 20 relative to the frame and in particular relative to the reflection surface 6.

[0106] In the example illustrated in the single figure, downstream of the separating element 10, on the reference arm side, there are thus successively the third optic 19, the second adjustment unit 18, the first objective 20 (associated with the displacement member 26) and the reflection surface 6.

[0107] Because the microscope 2 is an inverted microscope, the second objective 21 is arranged so as to observe the sample from below the sample. For example, the second objective 21 is arranged under the stage and in this case under the incubator 9.

[0108] In a manner known per se, the microscope 2 comprises a reflection surface 22. This reflection surface 22 makes it possible to illuminate the sample and also to allow a ray passing through the second objective 21 along the optical axis of said second objective 21 to be reflected up to the optical output of the microscope 2.

[0109] In the present case, said reflection surface 22 is arranged so that a ray propagating along the optical axis of the second objective 21 is then propagated, after reflection on the reflection surface 22, to exit via the optical output of the microscope 2. When the module 3 is connected to the microscope 2, such a ray thus propagates from said output to the separating element 10 along the object arm.

[0110] The interference device 4 further comprises a fourth optic 23 arranged between the separating element 10 and an “object arm” output of the module, i.e. the output of the module coupled at least optically with the optical output of the microscope 2. The fourth optic 23 is, for example, a lens, a doublet, etc.

[0111] Preferably, the fourth optic 23 is associated with at least one member for moving the fourth optic 23 relative to the frame and in particular with respect to the separating element 10 (in particular for moving the fourth optic 23 closer to or further away from the separating element 10).

[0112] Furthermore, the module 3 comprises a fifth optic 24 arranged at the output of the interference device 4, that is to say arranged between the separating element 10 and the acquisition device 7. For example, the fifth optic 24 is a single lens, a single doublet or a pair of lenses or a pair of doublets.

[0113] Preferably, the interference device 4 comprises a third diaphragm 25 arranged upstream of the fifth optic 24, between the fifth optic 24 and the separating element 10.

[0114] It is easily understood that the relative positioning of the second objective 21 on the one hand with respect to the source 5 and the reflection surface 6 on the other hand is essential. When the microscope 2 is coupled to the module 3, the optical axis of the second objective 21 is in fact in a fixed position with respect to the working axis of the module 3. Nevertheless, the two adjustment units 11, 18 make it possible to correctly position the source 5 and the reflection surface 6 with respect to the second objective 21 and this in particular in order to allow the generation of interference between the reference waves and the obj and .

[0115] In particular, the reflection surface 27 is configured to (by virtue of its relative displacement with respect to the frame) align the splitter element 10 / third optic 19 / reflection surface 18 / first lens 20 group with the splitter element / fourth optic 23 / reflection surface 22 / second lens group.

[0116] Module 3 is thus easily inserted into microscope 2 and then the characteristics of module 3 are adjusted via the two adjustment units 11, 18 to optically align module 3 and microscope 2.

[0117] In another aspect, the reflection surface 6 is itself mounted movably in the interference device 4 relative to the frame so as to be able to be moved relative to the sample during the study thereof. For example, the reflection surface 6 is movable in translation and for example movable in translation relative to the frame. For example, the reflection surface is mounted on a plate which is movable in translation relative to the frame. For example, said plate is movable by means of at least one piezoelectric actuator.

[0118] Preferably, the reflection surface 6 is movable in translation according to a translation movement:

[0119] - parallel but not coincident with the optical axis of the source 5, and / or

[0120] - parallel to the optical axis of the first objective 20, and / or

[0121] - parallel to at least part of the optical path of the reference arm and in particular parallel to at least the start of the optical path of the reference arm (the optical path starting at the level of the reflection surface 6).

[0122] It is therefore understood that the reflection surface 6 is movable so that it can be moved closer to or further away from the second adjustment unit 18.

[0123] It is therefore understood that the reflection surface 6 is movable so as to be able to modify the length of the reference arm.

[0124] The optical equipment 1 thus described is an improvement of a Linnik interferometer in a Koehler illumination configuration.

[0125] From the optical equipment 1 described, it is advantageously possible to perform static optical coherence tomography imaging as well as dynamic optical coherence tomography imaging.

[0126] Furthermore, in the case of dynamic optical coherence tomography imaging, it is also possible, from the optical equipment 1 described, to carry out dynamic optical coherence tomography imaging with a fixed reference surface 16 as well as dynamic optical coherence tomography imaging with a moving reference surface 16.

[0127] Furthermore, in the case of dynamic full-field optical coherence tomography imaging, it is also possible, from the optical equipment 1 described, to carry out dynamic full-field optical coherence tomography imaging with a fixed reference surface 16 or "D-FFOCT with fixed reflection surface" as well as dynamic full-field optical coherence tomography imaging with a moving reference surface 6 or "D-FFOCT with moving reflection surface".

[0128] Optical equipment 1 is thus particularly modular.

[0129] D-FFOCT with fixed reflection surface

[0130] A sample is placed in microscope 2.

[0131] A temporal succession of N interferometric signals (each signal N± arising from the interference between an object wave and a reference wave at an instant t±) is acquired by the acquisition device 7 with a reference arm and an object arm which remain fixed throughout the acquisition of said temporal succession of the N signals. The N interferometric signals are those of a slice of the sample. The number N of interferometric signals is for example greater than 20 and for example greater than 50 and for example greater than 300 and for example greater than 500 and is for example 512. Optionally, the number N of interferometric signals is greater than 800 and for example greater than 1000 and is for example 1024.

[0132] The duration of the acquisition of the temporal succession of the N signals is defined by a duration AT which has a predetermined value (defined by the acquisition frequency of the optical sensor).

[0133] For example, 512 or 1024 images are acquired during a time interval of 512 / (100 Hz) or (1024 / 100Hz), 100 Hertz being the acquisition frequency of the optical sensor.

[0134] It is thus understood that the variation in the optical path between the reference arm and the object arm is not voluntarily modified during the entire acquisition of the temporal succession of the N signals. In particular, there is no relative voluntary variation in the position of the sample with respect to the reflection surface 6 during the entire acquisition of the temporal succession of the N signals. In particular, the reflection surface 6 remains fixed with respect to the frame during the entire acquisition of the temporal succession of the N signals.

[0135] From the sole temporal succession of the N signals transmitted by the acquisition device 7 to the processing device 8, an image is then generated by the processing device 8.

[0136] The image is thus defined by pixels, each pixel P comprising a position (x,y) in the image. It is understood that a two-dimensional reference frame can be associated with the image so that a pixel P(x,y) of the image generated by the processing device 8 corresponds to a corresponding portion of the active surface of the optical sensor. Each pixel P(x,y) thus has a light intensity I(x,y) (also called dynamic light intensity) calculated from the different light intensities I±(x,y,ti) of the N interferometric signals, acquired at time t± for the corresponding portion on the active surface of the acquisition device.

[0137] With the D-FFOCT with fixed reflection surface, the optical path variation between the reference arm and the object arm is not voluntarily modified. Figure 3 thus shows that the voluntary variation value is zero throughout the acquisition (in Figure 3 only a part of the N acquired signals is represented for visibility reasons).

[0138] It should nevertheless be noted that there is nevertheless an overall phase shift between the object wave and the reference wave which will have a random initial value at the start of the acquisition, a value which will naturally change, if there is mobility, during the acquisition of the temporal succession of the N signals due to the movements of the diffusers internal to the sample which themselves introduce oscillations around this initial value. As illustrated in Figure 2, the light intensity I (x,y) of a pixel P(x,y) is a function of the phase shift cp between the reference waves and the object waves. More precisely, said function is a periodic sinusoidal function.Therefore, it is understood that depending on the initial value of the phase shift cp, for the same oscillation Az around said initial phase shift value (oscillation induced by the movements of the diffusers internal to the sample) over a given time interval At, the variation in light intensity AI during this time interval At may be more or less significant.

[0139] Consequently, if the movements of the internal diffusers are weak or non-existent (implying that the value of Az is also weak or non-existent) and the initial value of the phase shift is located at a peak or a trough of the sinusoidal function, the variation of the light intensity AI will be weak and the calculation of the dynamic light intensity I (x,y) of a pixel P(x,y) may thus suffer (we then speak of an inhomogeneous response from the sample). This could lead in particular to the appearance of optical artifacts and / or speckles on the image due to this inhomogeneous response.

[0140] To avoid such occurrences, it is also possible to use optical equipment 1 to perform D-FFOCT imaging with a moving reflection surface.

[0141] D-FFOCT with moving reflection surface

[0142] A sample is placed in microscope 2.

[0143] A temporal succession of N interferometric signals (each signal N± arising from the interference between an object wave and a reference wave at a time t±) is acquired by the acquisition device 7 with a reference arm which is modified throughout the acquisition of said temporal succession of the N signals. The N interferometric signals are those of a slice of the sample. The number N of interferometric signals is greater than that of static optical coherence tomography imaging. The number N of interferometric signals is for example greater than 20 and for example greater than 50 and for example greater than 300 and for example greater than 500 and is for example 512. Optionally, the number N of interferometric signals is greater than 800 and for example greater than 1000 and is for example 1024.

[0144] The duration of the acquisition of the temporal succession of the N signals is defined by a duration AT which has a predetermined value (defined by the acquisition frequency of the optical sensor).

[0145] For example, 512 or 1024 images are acquired during a time interval of 512 / (100 Hertz) or (1024 / 100Hz), 100 Hertz being the acquisition frequency of the optical sensor.

[0146] In fact, the number N of interferometric signals is identical to that of the D-FFOCT protocol with fixed reflection surface disseminated above. In particular, the acquisition parameters by the acquisition device 7 are identical whether we are in the case of D-FFOCT with fixed reflection surface or D-FFOCT with mobile reflection surface (acquisition frequency and number of images acquired in particular).

[0147] On the other hand, the difference in optical paths between the reference arm and the object arm is modified during the entire acquisition of the temporal succession of the N signals in the present case of the D-FFOCT with a mobile reflection surface, thus causing a voluntary modification of the phase shift between the reference waves and the object waves. In particular, there is a relative variation in the position of the sample with respect to the reflection surface 6 during the entire acquisition of the temporal succession of the N signals. In particular, the reflection surface 6 is moved with respect to the frame during the entire acquisition of the temporal succession of the N signals.

[0148] We therefore understand that this movement is known, desired and controlled.

[0149] As will be seen below, the reflection surface 6 is moved more slowly than the temporal fluctuations induced by the internal scatterers inside the sample.

[0150] Preferably, the processing device 8 is configured to synchronize the acquisition device 7 (and in particular its optical sensor) with the actuator allowing the movement of the reflection surface 6. For example, in addition to a computer, the processing device 8 may also comprise an acquisition card connected to the computer to ensure this synchronization.

[0151] In particular, the processing device 8 is configured to synchronize the acquisition device 7 (and in particular its optical sensor) with the actuator allowing the movement of the reflection surface 6 so that the reflection surface 6 is moved between two successive signals.

[0152] The reflection surface 6 is thus moved continuously throughout the acquisition of the temporal succession of the N signals by successive increments. This means that for each signal N± of the N signals, the reflection surface 6 is in a different relative position with respect to the frame (and / or the interference device and / or the sample) between two successive signals of the N signals. The reflection surface 6 is thus moved regularly throughout the acquisition of the temporal succession of the N signals by successive increments all having the same value. The reflection surface 6 is thus moved homogeneously and regularly throughout the acquisition of the temporal succession of the N signals.

[0153] With the D-FFOCT with a moving reflection surface, there is therefore a voluntary variation in the difference in optical paths between the reference arm and the ob j arm.

[0154] As a result, the phase shift between the object waves and the reference waves is voluntarily modified during the acquisition of the temporal succession of the N signals. With the D-FFOCT with a moving reflection surface, there is therefore a voluntary variation of said phase shift which is added to the natural variation induced by the scatterers within the sample. Figure 4 thus makes it possible to visualize that the voluntary variation is modified throughout the acquisition (in Figure 4 only a part of the N signals acquired is represented for visibility reasons).

[0155] Indeed, during the acquisition of the temporal succession of the N signals, movements of the diffusers internal to the sample themselves introduce oscillations of the phase shift between the object wave and the reference wave, oscillations which are added to the voluntary variation of said phase shift.

[0156] The displacement of the reflection surface 6 is thus defined so that a frequency of the voluntary modification of the phase shift (or voluntary frequency fvoi) during the acquisition of the time series of the N signals is lower than a frequency of an additional natural modification of phase shift (or natural frequency fo) induced by these movements of the diffusers.

[0157] Typically fo is between 5 and 18 Hertz. We therefore move the reflection surface 6 so that f V oi is less than 5 Hertz and preferably less than 1 Hertz and preferably less than 0.5 Hertz and preferably less than 0.1 Hertz. This therefore amounts to moving the reflection surface 6 during the acquisition of the time series of the N signals at a frequency less than 0.1 Hertz.

[0158] It is indeed important that the voluntary variation of the phase shift is very slow so as to be able to algorithmically separate in the N signals the movements due to the diffusers from those due to the displacements of the reflection surface 6.

[0159] Preferably, we choose f V oi such that: fo > 2*fvol.

[0160] Preferably, the voluntary phase shift between the object wave and the reference wave is defined by a periodicity function 2n.

[0161] Preferably, the reflection surface 6 is thus moved during the acquisition of the time series of N signals so that the voluntary phase shift covers an interval [0; W] during the acquisition of the time series, W being greater than or equal to n and preferably greater than or equal to 2 n.

[0162] The voluntary variation of the phase shift thus undergoes a modulation of at least n during the acquisition time of the AT signal and preferably of at least 2n.

[0163] The periodicity function 2n of the voluntary variation of the phase shift obeys, for example, a cosine function.

[0164] If we look again at Figure 2, we understand that even if the movements of the internal diffusers are weak or non-existent over a given time interval At, we will nevertheless move significantly during this time interval At on the function represented in Figure 2 thanks to the voluntary variation of the phase shift: in this way the variation of light intensity AI during At will be able to be much more homogeneous than in the case of D-FFOCT imaging with a fixed reflection surface.

[0165] This will allow to better calculate the light intensity I (x,y) of each pixel P(x,y) and thus to obtain a better quality image in order to avoid the appearance of optical artifacts and / or speckles on the image. Indeed, the variations in light intensity AI during At of the same diffuser will be much more homogeneous from one pixel to another limiting the appearance of optical artifacts and / or speckles on the image such as for example one or more fringes.

[0166] In this case, the reflection surface 6 is translated. This translation is carried out incrementally between two successive images. This will allow the voluntary variation function of the phase shift to be sampled.

[0167] By moving the reflection surface 6, it is thus ensured that at least half a period of the voluntary phase shift variation function and preferably at least a whole period of said function can be scanned.

[0168] Thus, the voluntary variation of the phase shift is preferably equal to at least n / N between two successive signals (in the case of half-period scanning) and preferably at least 2n / N between two successive signals (in the case of full-period scanning).

[0169] For the reflection surface 6, this results in a translation between two successive signals of the series of N signals of an increment corresponding to at least X / N (in the case of scanning the half-period) and preferably to at least 2X / N (in the case of scanning the full period), being the central wavelength of the source 5. The number of displacement increments of the reflection surface 6 therefore depends on the number N of signals.

[0170] From the sole temporal succession of the N signals transmitted by the acquisition device 7 to the processing device 8, an image is then generated by the processing device 8. It is thus understood that the generated image depends only on this series of N signals, without other additional signals.

[0171] It is therefore understood that the generation of the image is based both on the temporal quantification of the variations in light intensity induced by the sample (as for the D-FFOCT with fixed reflection surface) and on the temporal quantification of the variations in light intensity induced by the variation in the phase between the reference wave and the object wave caused by the movements of the reflection surface 6 (unlike the D-FFOCT with fixed reflection surface). It is therefore understood that the acquisition module 8 must take into account this variation in the phase between the reference wave and the object wave caused by the movements of the reflection surface 6 in order to be able to generate the image. It should be noted that said variation is known and controlled, which facilitates its identification to generate the image. The image is thus defined by pixels, each pixel P comprising a position (x,y) in the image.It is understood that a two-dimensional reference frame can be associated with the image so that a pixel P(x,y) of the image generated by the processing device 8 corresponds to a corresponding portion of the active surface of the optical sensor. Each pixel P(x,y) thus has a light intensity I(x,y) calculated from the different light intensities I±(x,y,ti) of the N interferometric signals, acquired at time t± for the corresponding portion on the active surface of the acquisition device.

[0172] The light intensity of a pixel P(x,y) is for example obtained by a processing similar to that carried out during D-FFOCT imaging with a fixed reflection surface by ignoring the highest frequency or the X highest frequencies of the N signals acquired (X being for example between 0 and 10).

[0173] Alternatively, a Fourier transform is performed on the time series of the light intensity of a pixel P(x,y) given either on the set of values ​​I±(x,y,ti) of the N signals. A low-pass filter is then applied. They are then removed from the transform to determine the light intensity of a pixel P(x,y) from the transform from which the highest frequency(ies) have been removed. Indeed, it is preferable to remove the highest frequency(ies) because they generally correspond to experimental noise and not to real natural phase shifts.

[0174] In all cases, and unlike D-FFOCT imaging with a fixed reflection surface, processing must also be carried out to remove the lowest frequency or frequencies which are characteristic of the voluntary phase shift. For example, a high-pass filter is used whose value G is determined to be greater than x times fvoi, x being for example equal to or greater than 2. The optical equipment 1 thus described proves to be particularly modular since it makes it possible to carry out different types of static and dynamic optical coherence tomography imaging and in particular dynamic optical coherence tomography imaging with a static or mobile reflection surface.

[0175] Advantageously, the optical equipment 1 thus described makes it possible to carry out any microscopy study excluding the destruction of the sample and / or the absence of markers.

[0176] Advantageously, the optical equipment 1 thus described makes it possible to image a sample without disturbing its natural environment.

[0177] Advantageously, the optical equipment 1 thus described makes it possible to identify, for example, the scatterers of a sample (such as a tissue) and to measure their metabolism.

[0178] Of course, the invention is not limited to the embodiment described but encompasses any variant falling within the scope of the invention as defined by the claims.

[0179] In particular, although here the equipment described makes it possible to implement a dynamic full-field optical coherence tomography microscopic imaging method of at least one sample, the equipment described may make it possible to implement another type of imaging method and for example another dynamic temporal optical coherence tomography microscopic imaging method or a dynamic optical coherence tomography microscopic imaging method in the Fourier domain, in the spectral domain, or with source scanning of at least one sample.

[0180] The optical equipment making it possible to implement the invention may be different from what has been indicated. It will thus be possible to implement the invention in optical equipment such as that described in the second embodiment of the application PCT / FR2022 / 000084 of the present applicant and in general in any optical equipment making it possible to carry out dynamic optical coherence tomography microscopic imaging, in particular with the possibility of moving the reflection surface of the reference arm in the optical equipment. The optical equipment may thus not be based on an optical microscope as indicated above.

[0181] 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 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 module and / or optical equipment 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.

[0182] Although high numerical aperture objectives are used here, lower numerical aperture objectives can of course be used.

[0183] Although here the beam splitter element is non-polarizing, the beam splitter element may be polarizing.

[0184] Time signals can be two-dimensional or three-dimensional.

[0185] Although here the reflection surface is moved to modify the reference arm length and therefore the phase shift between the reference arm and the object arm, one or more other elements of the optical equipment may be used to modify the reference arm length (in addition to or as an alternative to the reflection surface). For example, it may be the adjustment unit linked to the reference arm which may be used (by moving one or more elements of said unit) to modify the reference arm. For example, the entire frame may be moved to modify the reference arm.

[0186] The movement of at least one of the elements of the optical equipment may be linear and / or rotary.

[0187] With the method described, and whatever the optical equipment allowing it to be implemented, it will also be possible to rely on the series of interference signals received during dynamic imaging with a moving reflection surface to carry out static imaging by simple processing of said series (without taking new signal measurements since the value of the phase shift voluntarily induced between each signal is known).

[0188] Although here the difference between the optical path taken by the light rays in the reference arm and the optical path taken by the light rays in the object arm is generated by a modification of the length of the reference arm, it is possible alternatively or additionally to generate the difference in optical paths by modifying the length of the object arm and / or by modifying the length of the object arm and the reference arm differently from each other and / or by modifying the index of at least the reference arm or the object arm...

[0189] Although here the reflection surface is moved discontinuously in increments, the reflection surface can be moved continuously.

[0190] Although here the reflection surface is moved by successive increments all having the same value, the reflection surface can be moved by increments with at least two successive increments of different values. Preferably, care will then be taken to ensure that the voluntary phase shift stops (for the acquisition of a signal) at as many different values ​​as in the case of successive increments of the same value.

Claims

CLAIMS 1. Method for dynamic optical coherence tomography microscopic imaging of at least one sample by means of optical equipment comprising an interference device which comprises a beam splitter element and at least one reflection surface, the method comprising the steps of: producing at least one interference between • at least one reference wave obtained by reflection of the light emitted by a light source associated with the optical equipment on the reflection surface, and • at least one object wave obtained by backscattering the light emitted by said source on the sample, the beam splitter element thus forming two arms, namely a “reference arm” associated with the reflection surface and an “object arm” associated with the sample, acquiring a series of signals of at least two signals following one another in time to form a time series of signals, each signal resulting from the interference between the reference wave and the object wave, characterized in that a difference between the optical path taken by the light rays in the reference arm and the optical path taken by the light rays in the object arm is modified regularly and voluntarily during the acquisition of the time series of signals.

2. The method of claim 1, wherein the optical path difference is modified during the acquisition of the time series according to a frequency fvoi such that: fo > 2*f vo i, with fo a frequency characteristic of the natural movements of diffusers internal to the sample called natural frequency.

3. Method according to one of the preceding claims, in which at least one element of the optical equipment is moved regularly to voluntarily and regularly modify the difference between the optical path taken by the light rays in the reference arm and the optical path taken by the light rays in the object arm.

4. Method according to claim 3, in which the element is moved in a translational movement.

5. A method according to claim 3 or claim 4, wherein the movable element is the reflecting surface (6).

6. Method according to one of the preceding claims, in which the difference between the optical path taken by the light rays in the reference arm and the optical path taken by the light rays in the object arm is generated by a modification of the length of the reference arm.

7. Method according to one of the preceding claims, in which an image is generated from the single series of signals acquired during the movement of the reflection surface (6).

8. Method according to one of the preceding claims, in which the variation of the difference of the optical path reference arm / object arm has a frequency between 0.1 and 10 Hertz.

9. Method according to one of the preceding claims, in which the voluntary phase shift between the object wave and the reference wave is defined by a periodicity function 2n, the variation of the difference of the reference arm / object arm optical paths being such that the voluntary phase shift covers an interval [0; W] during the acquisition of the time series, W being greater than or equal to n.

10. The method of claim 9, wherein W is greater than or equal to 2 n.

11. Method according to one of the preceding claims, in which the voluntary variation of the phase shift is equal to at least n / N between two successive signals.

12. Method according to one of the preceding claims, in which the variation of the phase shift passes through at least all of its values ​​of a period 2n during the duration of the acquisition of the time series.

13. Method according to one of the preceding claims, in which the series of signals contains N signals, the voluntary variation of the phase shift being equal to at least n / N between two successive signals.

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