Optical coherence tomography imaging system and associated imaging method

Abstract

The present invention relates to a method for imaging a plurality of samples by means of optical coherence tomography, each sample comprising an air-liquid interface on which a biological material grows.

Description

OPTICAL COHERENCE TOMOGRAPHY IMAGING SYSTEM AND ASSOCIATED IMAGING METHOD FIELD OF INVENTION

[0001] The present invention relates to the field of label-free optical imaging, in particular optical coherence tomography. More specifically, the invention relates to an optical coherence tomography imaging system and an optical coherence tomography imaging method. STATE OF THE ART

[0002] The air-liquid interface is a favorable environment for the development of biological material such as bacterial biofilms. Their structure, composition, and function evolve over time, depending on environmental conditions. A better understanding of the mechanisms of biological material development is essential for controlling or exploiting their properties according to the intended applications.

[0003] However, observing the air-liquid interface using standard optical microscopy presents difficulties due in particular to surface instability, scattering and the low refractive index contrast of the biological material.

[0004] Furthermore, observation by optical microscopy requires cell labeling (e.g., by staining or fluorescence). However, labeling induces phototoxic effects—preventing longitudinal imaging of biological material development—and alters bacterial behavior. Sometimes, it necessitates significant genetic manipulation. Moreover, such labeling is not suitable for the in-depth study of thick biofilms (from 10 µm to a few millimeters).

[0005] Optical coherence tomography (OCT) is an imaging technique that uses a light wave to capture three-dimensional images of a light-scattering material or sample (e.g., biological tissue) with micrometer-level resolution. OCT is based on a low-coherence interferometric technique. OCT does not require cell labeling.

[0006] However, most optical coherence tomography (OCT) imaging setups require the integration of moving components, such as scanners, within the interferometric device to acquire three-dimensional images. Such scanners impose limitations on the size of the optical beams used, and consequently on the size of the objects imaged, as well as on scanning speeds. Furthermore, these components are also expensive and contribute to the high cost of current OCT systems.

[0007] Furthermore, the development of biological material at the air-liquid interface typically takes several days and requires continuous monitoring. For each experiment, multiple biological replicas are necessary to increase the reliability of the findings. The acquisition time for such measurements is therefore very long. Given the fragility of biological material developing at the air-liquid interface and its sensitivity to mechanical disturbances, it is not possible to move the sample to perform a multiplexed measurement of several samples simultaneously. It is therefore necessary to alternate measurements on different replicas and to position successive measurements relative to one another. Moreover, moving the sample could disrupt the imaging.

[0008] The present invention proposes a solution for performing optical coherence tomography imaging on larger objects such as biological material growing at the air-liquid interface, and also in a multiplexed manner on a plurality of samples. ABSTRACT

[0009] A first aspect of the invention relates to a method of optical coherence tomography imaging of a plurality of samples, each sample comprising an air-liquid interface on which biological material develops, the method comprising: provision of an optical coherence tomography imaging system comprising: o an optical coherence tomography imaging device in the frequency domain comprising: • a light emission module, • an interference device, said light emission module being configured to emit an incident beam comprising a plurality of wavelengths, and to illuminate, in operation, said interference device, said interference device comprising an object arm and a reference arm, the reference arm being traversed by a reference beam and the object arm being arranged in a first direction to illuminate, in operation, the air-liquid interface of one of the plurality of three-dimensional samples; said interference device further comprising recombination means configured to recombine the reference beam and a beam reflected by the illuminated air-liquid interface into an interference beam; o an acquisition device configured to acquire an interferometric signal from the interference beam, o a signal processing unit configured to analyze said interferometric signal in order to determine a three-dimensional image of the biological material developing at the illuminated air-liquid interface, o a displacement device comprising a translation stage configured to cooperate with at least a part of the imaging device and configured to move in translation along at least a second direction substantially perpendicular to the first direction, the displacement device being configured to position at least a part of the imaging device in a plurality of acquisition positions; o a linking element configured to ensure cooperation between at least a part of the imaging device and the displacement device, the translation stage including a receptacle configured to receive and retain said linking element, o a sample holder configured to support a plurality of three-dimensional samples in a plane substantially orthogonal to said first direction, placement of the plurality of three-dimensional samples on the sample carrier along an axis parallel to the second direction; for each three-dimensional sample, acquisition and analysis of at least one narrowed interferometric signal using the signal processing unit comprising: to translate, using the translation stage, at least a part of the imaging device to an acquisition position above said three-dimensional sample, o production, by the light-emitting module, of at least one interference beam by illuminating the interference device, o acquisition, by the acquisition device, of at least one interferometric signal from the at least one interference beam produced, o analysis, by the signal processing unit, of at least one interferometric signal so as to obtain a three-dimensional image of the biological material developing at the air-liquid interface of said three-dimensional sample.

[0010] Thus, the method described here allows obtaining a three-dimensional image of a three-dimensional sample by simple translation of the optical coherence partomography imaging device using the displacement device, without internal movement within the imaging device.

[0011] Advantageously, the three-dimensional sample has dimensions greater than 10x10x3 mm 3 These dimensions can be compared to the field of view of a commercial OCT system, which is approximately 5x5x3 mm. 3Thus, the use of a displacement device makes it possible to image samples larger than those achievable with a prior art OCT system. This allows the imaging of larger objects such as biological material growing at the air-liquid interface.

[0012] In addition, the process also allows multiplexed imaging of several samples, which reduces analysis time and avoids transporting and handling samples that may be fragile.

[0013] In one embodiment, the interferometric signal is two-dimensional.

[0014] In one embodiment, the biological material is a bacterial biofilm or a microbial biofilm.

[0015] In one embodiment, the step of acquiring and analyzing at least one interferometric signal is repeated for a plurality of acquisition positions.

[0016] In one embodiment, the step of acquiring and analyzing at least one interferometric signal is repeated for a plurality of times, the imaging process allowing monitoring of the development of the biological material.

[0017] In one embodiment, the method further includes, for each three-dimensional sample, during the step of acquiring and analyzing at least one interferometric signal, a step of translating at least a part of the imaging device along the first direction in order to position at least a part of the imaging device at a predetermined distance from said three-dimensional sample.

[0018] In one embodiment, the method further includes, for each three-dimensional sample, after the step of acquiring and analyzing at least one interferometric signal, a step of translating at least one part of the imaging device along the first direction in order to increase the distance between at least one part of the imaging device and said three-dimensional sample.

[0019] The invention also relates to an optical coherence tomography imaging system for at least one three-dimensional sample configured to implement the method described above, the imaging system comprising: - an optical coherence tomography imaging device in the frequency domain, said imaging device comprising: o a light emission module, o an interference device, o said light emission module being configured to emit an incident beam comprising a plurality of wavelengths, and to illuminate, in operation, said interference device, o said interference device comprising an object arm and a reference arm, the reference arm being traversed by a reference beam and the object arm being arranged in a first direction to illuminate, in operation, the air-liquid interface of one of the plurality of three-dimensional samples; o said interference device further comprising recombination means configured to recombine the reference beam and a beam reflected by the illuminated air-liquid interface into an interference beam; - an acquisition device configured to acquire an interferometric signal from the interference beam, - a displacement device configured to cooperate with at least one part of the imaging device, said displacement device comprising a translation stage configured to move in translation along at least one second direction substantially perpendicular to the first direction, the displacement device being configured to position at least one part of the imaging device in a plurality of common positions; - a linking element configured to ensure cooperation between at least one part of the imaging device and the displacement device, the translation stage comprising a receptacle configured to receive and retain said linking element, - a sample holder configured to support a plurality of three-dimensional samples along an axis parallel to the second direction in a plane substantially orthogonal to said first direction, - a signal processing unit configured for: to translate the imaging device to said position using the translation stage, to produce at least one interference beam by illuminating the interference device with the light-emitting module, and to acquire at least one narrowed interferometric signal from at least one interference beam using the acquisition device. to analyze at least one interferometric signal so as to obtain a three-dimensional image of the biological material developing at the air-liquid interface of at least one three-dimensional sample corresponding to said position by the signal processing unit.

[0020] In one embodiment, the movement device is a movement device for a 3D printer.

[0021] In one embodiment, the translation plate is further configured to move in translation along the first direction.

[0022] In one embodiment: said light-emitting module is configured to emit, in operation, a beam of linear cross-section extending along a third direction perpendicular to the first and second directions, such that said interference beam has a linear cross-section, said imaging device further comprises a dispersive element configured to spatially separate the wavelengths of the plurality of wavelengths along the first direction, so as to transform said linear cross-section interference beam into a two-dimensional cross-section beam, said interferometric signal is a two-dimensional signal, said acquisition device is configured to acquire two-dimensional signals parallel to a plane defined by the first direction and the third direction, the two-dimensional signals together forming said three-dimensional image.

[0023] In one embodiment, the light emission module includes a fiber source comprising an output tip, and an optical device comprising a collimator followed by a cylindrical lens, the output tip being positioned in an object focal plane of said collimator.

[0024] In one embodiment, the light-emitting module includes a round-to-linear fiber bundle comprising an inlet tip with a circular arrangement of fibers and an outlet tip with a linear arrangement of fibers, and an optical device comprising a collimator, the outlet tip being positioned in a focal plane of said collimator.

[0025] In one embodiment, the connecting element includes a mounting plate comprising fixing fasteners in positions centered relative to the translation plate.

[0026] In one embodiment: said imaging device further comprises an additional translation means configured to move the incident beam along a third direction perpendicular to the first and second directions, said interferometric signal being a one-dimensional signal, and said acquisition device is a spectrometer.

[0027] In one embodiment, the object arm is mounted in a dedicated mount and in that the linking element comprises a first support portion configured to be fixed on said translation plate and a second support portion configured to retain said mount and to be fixed in the first support portion.

[0028] This disclosure relates, alternatively, to an optical coherence tomography imaging system of at least one three-dimensional sample, comprising: - an optical coherence tomography imaging device in the frequency domain, said imaging device comprising: o a light emission module, o an interference device, said light-emitting module being configured to emit an incident beam comprising a plurality of wavelengths, and to illuminate, in operation, said interference device, said interference device comprising an object arm and a reference arm, the reference arm being traversed by a reference beam and the object arm being arranged in a first direction to illuminate, in operation, said three-dimensional sample; said interference device further comprising recombination means configured to recombine the reference beam and a beam reflected by the illuminated sample into an interference beam; - an acquisition device configured to acquire an interferometric signal from the interference beam, - a signal processing unit configured to analyze said interferometric signal in order to determine a portion of a three-dimensional image of said at least one three-dimensional sample, corresponding to a current position, - a displacement device configured to cooperate with the imaging device, said displacement device comprising a translation stage configured to move in translation along at least one second direction substantially perpendicular to the first direction, - a linking element configured to ensure cooperation between the imaging device and the displacement device, the translation stage comprising a receptacle configured to receive and retain said linking element, - a suitable sample holder configured to support said at least one three-dimensional sample in a plane substantially orthogonal to said first direction, the displacement device being configured to position said imaging device in a plurality of current positions, so as to determine a plurality of portions of a three-dimensional image of said three-dimensional sample forming together a three-dimensional image of said at least one three-dimensional sample.

[0029] Thus, the imaging system according to this variant enables three-dimensional optical coherence tomography imaging without any moving parts in the imaging device, the latter moving as a whole by means of the displacement device. In particular, the imaging system according to the invention does not use any expensive components such as a scanner in its main operation. Furthermore, the imaging system according to the first aspect of the invention allows for imaging large samples or for tracking multiple samples in parallel over time.

[0030] In some embodiments, the movement device is a movement device for a 3D printer.

[0031] Thus, the displacement amplitudes permitted by a 3D printer's displacement device allow for the exploration of much larger imaging observation volumes than more conventional displacement systems such as scanners used in prior art OCT setups. Consequently, the components of the imaging system according to the invention can be appropriately and easily sized to cover such observation volumes. In particular, the components can have dimensions well beyond the order of magnitude of 10 mm imposed by scanner-type components. Typically, components with dimensions on the order of 50.4 mm (2 inches) in diameter are available. Larger custom-made components can also be used.

[0032] As an example, the displacement device is a displacement device for a 3D printer capable of building parts with volumes up to 220x220x250 mm. 3 , so that the displacement device is configured to move between 0.025 mm and 250 mm along the first direction and between 0.01 mm and 220 mm along the second direction and a third direction perpendicular to the first and second dimensions.

[0033] In other examples, the displacement device is a displacement device for a 3D printer capable of building parts with larger volumes, on the order of 500x500x500 mm. 3 , or even beyond.

[0034] Furthermore, the invention overcomes the difficulty and provides a solution that may appear counterintuitive for fixing an interferometric imaging device onto a displacement device of a 3D printer, an interferometric imaging device for which stability and repeatability are critical factors.

[0035] In certain embodiments, the translation stage is further configured to move in translation along the first direction. Thanks to this additional direction of movement, the focusing of the imaging components of the imaging system according to the invention is facilitated, in particular, when there are several samples to be imaged, for example positioned in wells or containers of a given depth, which notably prevents the components of the imaging system from coming into contact with these wells or containers.

[0036] In some embodiments: said light-emitting module is configured to emit, in operation, a beam of linear cross-section extending along a third direction perpendicular to the first and second directions, such that said interference beam has a linear cross-section, said imaging device further includes a dispersive element configured to spatially separate the wavelengths of the plurality of wavelengths along the first direction, so as to transform said linear cross-section interference beam into a two-dimensional cross-section beam, said interferometric signal is a two-dimensional signal, said acquisition device is configured to acquire two-dimensional signals, and / or said portion of said three-dimensional image is a current two-dimensional section parallel to a plane defined by the first direction and the third direction, so that the displacement device is configured to determine a plurality of current two-dimensional images of said three-dimensional sample forming together said three-dimensional image of said three-dimensional sample.

[0037] Advantageously, the emission of a linear section beam by the light emission module makes it possible to do without an additional scanning direction, and a corresponding moving component such as a scanner, which makes it possible to increase the speed of acquisition of three-dimensional images by the imaging system according to the invention while limiting its cost.

[0038] In one embodiment, the light emission module includes a fiber source comprising an output tip, and an optical device comprising a collimator followed by a cylindrical lens, the output tip being positioned in an object focal plane of said collimator.

[0039] Alternatively, the light-emitting module may include a round-to-linear fiber bundle comprising a circularly arranged fiber inlet tip and a linearly arranged fiber outlet tip, and an optical device comprising a collimator, the outlet tip being positioned in a focal plane of said collimator.

[0040] In one embodiment, the connecting element includes a mounting plate comprising fixing fasteners in positions centered relative to the translation plate.

[0041] Advantageously, the thickness and material of the mounting plate are optimized to meet the mechanical constraints of the translation plate and the displacement device.

[0042] Such an architecture of the linking element makes it possible to stabilize the imaging device on the moving device and to ensure the robustness of the overall imaging system, which is fundamental for optical coherence tomography imaging applications whose principle is based on the phenomenon of interference which is very sensitive to vibrations.

[0043] In some embodiments: said imaging device further includes an additional translation means configured to move the incident beam along a third direction perpendicular to the first and second directions, said interferometric signal is a one-dimensional signal. said acquisition device is a spectrometer, said portion of said three-dimensional image is a row of voxels along the first direction, so that the displacement device and the additional translation means are configured to determine a plurality of rows of current voxels of said three-dimensional sample forming together said three-dimensional image of said three-dimensional sample.

[0044] The object arm can be mounted in a dedicated mount and the linking element includes a first support portion configured to be fixed on said translation plate and a second support portion configured to hold said mount and to be fixed in the first support portion.

[0045] This disclosure also relates, alternatively, to a method for optical coherence tomography imaging of at least one three-dimensional sample, comprising: provision of an optical coherence tomography imaging system for at least one three-dimensional sample as described above; step of placing said at least one three-dimensional sample on the sample support; for a set of N positions along the second direction, for each position, an acquisition and analysis step comprising: translation of the imaging device to said position by means of the translation stage, o production of at least one current interference beam by illuminating the interference device with the light-emitting module, o acquisition of at least one two-dimensional narrowed interferometric signal from at least one current interference beam by the acquisition device, o analysis of at least one two-dimensional interferometric signal so as to obtain at least a portion of a three-dimensional image of at least one three-dimensional sample corresponding to said position by the signal processing unit, so as to obtain a plurality of portions of a three-dimensional image together forming a three-dimensional image of said at least one three-dimensional sample.

[0046] Thus, the method described here allows obtaining a three-dimensional image of a three-dimensional sample by simple translation of the imaging device by optical coherence tomography using the displacement device, without internal movement within the imaging device.

[0047] Advantageously, the three-dimensional sample has dimensions greater than 10x10x3 mm 3 These dimensions can be compared to the field of view of a commercial OCT system, which is approximately 5x5x3 mm. 3 Thus, the use of a displacement device makes it possible to image samples with dimensions larger than those permitted with a prior art OCT system.

[0048] In some embodiments, the method is configured for imaging a plurality of individual samples placed on the sample holder, along an axis parallel to the second direction, each individual sample being centered at a position xi along said axis, and further comprises: translation step of the imaging device to each position xi by control of said translation stage; repetition of the acquisition and analysis step; repetition of the translation and acquisition and analysis steps for each individual sample.

[0049] Advantageously, the imaging method according to the invention allows for the simultaneous acquisition of multiple three-dimensional images of multiple three-dimensional samples by simply moving the entire optically coherent tomography imaging device with the moving device. For example, when the three-dimensional samples are pre-positioned on the sample holder in a calibrated manner, the method allows for the automated and uniform acquisition of three-dimensional images of the samples solely by moving the imaging device, without any movement of the samples themselves. BRIEF DESCRIPTION OF THE FIGURES

[0050] Figure 1 is a schematic representation of an optical coherence tomography imaging system 1 of at least one three-dimensional sample 6 (or several samples 6i,.. 6j, 6L) according to an embodiment of the invention.

[0051] Figure 2 is a schematic representation of an optical coherence tomography imaging device 2 of an optical coherence tomography imaging system 1 of at least one three-dimensional sample 6 according to an embodiment of the invention.

[0052] Figure 3 is a schematic representation of an optical coherence tomography imaging device 2 according to certain embodiments of the invention.

[0053] Figure 4 is a schematic representation, on the left, of a beam intercepted by a dispersive element 25 in the optical coherence tomography imaging device 2 of Figure 3, and on the right, of a beam reflected by the dispersive element 25.

[0054] Figure 5 is a three-dimensional representation of a connecting element 4 according to certain embodiments of the invention.

[0055] Figure 6 is a first example of a light emission module 21 of an optical coherence tomography imaging device 2 according to certain embodiments of the invention.

[0056] Figure 7 is a second example of a light emission module 21 of an optical coherence tomography imaging device 2 according to certain embodiments of the invention.

[0057] Figure 8 is a three-dimensional representation of a first portion of support 42 of a connecting element 4 according to certain embodiments of the invention.

[0058] Figure 9 is a three-dimensional representation of a second support portion of a connecting element 4 according to certain embodiments of the invention.

[0059] Figure 10 is a top view of the second support portion of connecting element 4 in Figure 9.

[0060] Figure 11 is a side view of the second support portion of connecting element 4 of Figure 9 and Figure 10.

[0061] Figure 12 is a flowchart representing steps that can be taken to implement a first embodiment of an optical coherence tomography imaging process of this disclosure.

[0062] Figure 13 is an example of a one-dimensional interferometric signal acquired during the implementation of the optical coherence tomography imaging process according to certain embodiments of the invention to image a first bacterial or microbial biofilm.

[0063] Figure 14 is a two-dimensional section of the first bacterial or microbial biofilm imaged during the implementation of the optical coherence tomography imaging process according to certain embodiments of the invention.

[0064] Figure 15 is a two-dimensional section of a second bacterial or microbial biofilm imaged during the implementation of the optical coherence tomography imaging process according to certain embodiments of the invention.

[0065] Figure 16 is a three-dimensional reconstruction of the second bacterial or microbial biofilm, a two-dimensional section of which is shown in Figure 15, imaged during the implementation of the optical coherence tomography imaging process according to certain embodiments of the invention.

[0066] Figure 17 represents three two-dimensional sections, obtained at three different times, of a first imaged sample during the implementation of the optical coherence tomography imaging process according to certain embodiments of the invention.

[0067] Figure 18 represents three two-dimensional sections, obtained at three different times, of a second imaged sample during the implementation of the optical coherence tomography imaging process according to certain embodiments of the invention.

[0068] Figure 19 is a flowchart representing steps that can be performed to implement a second embodiment of an optical coherence tomography imaging process 100 of this disclosure.

[0069] Figure 20 is a flowchart representing steps that can be performed to implement a third embodiment of an optical coherence tomography imaging process 100 of this disclosure.

[0070] Figure 21 is a three-dimensional representation of a sample support 5 for biofilms that can be used in the optical coherence tomography imaging system 1 of at least one three-dimensional sample 6 according to the invention.

[0071] Figure 22 is a top view of sample support 5 for biofilms from Figure 21.

[0072] Figure 23 is a front view of sample support 5 for biofilms from Figure 21 and Figure 22.

[0073] Figure 24 is a side view of sample support 5 for biofilms from Figure 21, Figure 22 and Figure 23.

[0074] Figure 25 is a three-dimensional representation of a mounting bracket configured to receive, position and mechanically hold the sample support 5 of Figure 21, Figure 22, Figure 23 and Figure 24 and to be fixed onto a bed of a three-dimensional printer.

[0075] Figure 26 is a top view of the mounting bracket of Figure 25.

[0076] Figure 27 is a front view of the mounting bracket for Figures 25 and 26.

[0077] Figure 28 is a side view of the mounting bracket for Figures 25, 26 and 27.

[0078] Figure 29 is a two-dimensional section of a third bacterial or microbial biofilm contained in a beaker imaged during the implementation of the optical coherence tomography imaging process 100 according to certain embodiments of the invention. DETAILED DESCRIPTION

[0079] In the detailed description that follows, only certain embodiments are described in detail to ensure clarity of exposition, but these examples are not intended to limit the general scope of the principles arising from this description.

[0080] The various embodiments and aspects described herein can be combined or simplified in numerous ways. In particular, the steps of the different processes can be repeated, reversed, or performed in parallel, unless otherwise specified. Imaging System

[0081] A first aspect of this disclosure relates to an optical coherence tomography imaging system 1 of at least one three-dimensional sample 6, preferably a plurality of samples (6i,.. 6j, 6L), one embodiment of which is illustrated in Figure 1.

[0082] Sample 6, or each sample in the plurality of samples (6i, ..., 6j, 6L), comprises an air-liquid interface on which biological material develops. As this biological material develops, it has a thickness ranging from 10 µm to a few millimeters. The biological material is therefore three-dimensional.

[0083] System 1 will be described below in its use with a sample 6. In the case of a plurality of samples (6i,.. 6j, 6L), a similar use is made for each sample.

[0084] Each sample is preferably contained in a container, for example, a beaker. To limit evaporation of the sample, the container may include a lid configured to allow observation of the sample by optical coherence tomography (OCT). For example, the lid may have a hole. For example, the lid may include at least one observation hole allowing the insertion of at least part of the OCT imaging device described below through the perforated lid. At least one oxygenation hole may also be present in the lid. The observation hole ensures that the biological material receives sufficient oxygen. The container may also include an alignment means, such as a protrusion configured to cooperate with a notch in the lid.This allows, when positioning lids on multiple containers, for the alignment of the viewing holes across all containers. For example, when the container has a spout, the spout can act as a protrusion.

[0085] System 1 includes: - an optical coherence tomography imaging device in the frequency domain 2; - a displacement device 3 configured to cooperate with at least part of the imaging device 2; - a linking element 4 configured to ensure cooperation between at least a part of the imaging device 2 and the displacement device 3; - a sample holder 5 configured to support at least one three-dimensional sample 6.

[0086] The principle of OCT is that of low temporal coherence interferometry. An OCT device comprises an interference pattern illuminated by broad-spectrum, low temporal coherence light. The images of a sample obtained by the OCT device are derived from an interference signal resulting from the interference between a reference beam and a beam reflected and / or backscattered by the sample.

[0087] A frequency-domain optical coherence tomography (OCT) imaging device is an OCT device where the interference spectrum is recorded, either with a spectral detector that analyzes the frequency of the interference signal, or by using a light source whose wavelength varies over time. The principle is to acquire information about the interference values ​​at different wavelengths. The frequency of the signal corresponding to the interference spectrum depends on the optical path difference between the reference beam and the beam reflected and / or scattered by a given slice of the imaged sample.Fourier transform analysis of the frequency variation of the complex interference signal between the reference beam and the beam reflected and / or scattered by the different layers of an imaged sample allows us to reconstruct a signal of the amount of light scattered according to the depth of the imaged sample.

[0088] In more detail, the optical signal resulting from the superposition of a reference beam and a sample beam composed of N individual and sufficiently spaced reflectors is written: with Ri the reflectivity of each of the sample's reflectors, k the wave vector: 2TT k = — with A being the wavelength, and 8 t The path difference between reflector i and reference arm: Si = n ech Zi — the refractive index of the sample and n rey is the refractive index of the medium traversed by the reference beam.

[0089] By measuring the interference spectrum I as a function of Â, we can obtain an expression of the signal I as a function of the wave vector k, which is expressed as the sum of a constant term and N sinusoids of spatial frequencies — .

[0090] We can express I det for a sample composed of continuous structures in depth, and the sum then transforms into an integral: with line the incoherent (non-interferometric) part and S(k) the power spectral density (intensity variations as a function of the wave vector k related to the source and the different optical components of the OCT system), Ri being equal to the reflectivity R ec h(z) of an individual reflector located at a depth z.

[0091] The signal Idet k) can be transformed by taking its complex expression: Idet in which we recognize the definition of the Fourier transform of the function S(k) Ri' evaluated at the depth <5(z).

[0092] Thus, by taking the inverse Fourier transform of the interference spectrum, we obtain:

[0093] This gives us the axial distribution of / R ec h convolved by the Fourier transform of the power spectral density, at a given position (Xo, Yo) of the sample.

[0094] To obtain an axial section and a volume, it is then necessary to scan the illumination beam in the transverse plane of the sample, typically, in the prior art, via the use of an angular scanner placed in the pupil plane of an imaging lens.

[0095] The imaging system 1 proposed by this disclosure allows scanning of the optical beam by translating at least part or all of the imaging device 2 around or over a sample 6, by means of the displacement device 3 and its translation stage 31. In addition, it allows multiplexed translation imaging of at least part or all of the imaging device 2 over or around each of the samples (6i,.. 6j, 6L).

[0096] Scanning refers to the process by which at least part or all of the imaging device 2 is moved to explore different areas of the sample to be imaged. Scanning can therefore be performed in the absence of a scanner, modifying the direction of the optical beam of the imaging device 2 to explore different areas of the sample to be imaged without moving the imaging device 2.

[0097] Thus, the optical coherence tomography imaging system 1 of at least one three-dimensional sample 6 according to this disclosure has several advantages: it allows imaging of large samples; it allows imaging and tracking of a plurality of samples in parallel, for example, several samples in culture wells, and over periods that can extend over several days by automation of image capture which can be carried out continuously; it also allows for real-time sample tracking.

[0098] Furthermore, in its primary configuration, the imaging system 1 described in this disclosure can, thanks to the movement device 3, eliminate the need for expensive moving components such as scanners, thus reducing its cost. This is because part of the scanning is performed by translating the imaging device 2 without requiring a scanner. The imaging system 1 also simplifies and reduces the size of the various optical components. Additionally, the movement device of the imaging system 1 allows the imaging device 2 to be moved in multiple directions, thereby enhancing its versatility.

[0099] The optical coherence tomography imaging system in frequency domain 2, schematically illustrated in Figure 2, comprises: - a light emission module 21 configured to emit an incident beam 71 comprising a plurality of wavelengths; - an interference device 22; - an acquisition device 23; - a 24 signal processing unit.

[0100] The interference device 22 includes a reference arm 81 and an object arm 82.

[0101] In operation, with at least one three-dimensional sample 6 positioned in a current position on the sample support 5 and centered on the object arm 82, the incident beam 71 emitted by the light emission module 21 illuminates the interference device, so that the reference arm 81 is traversed by a reference beam 72 and the object arm 82 is traversed by an object beam 73.

[0102] According to one example, the interference device 22 includes a beam splitter, such as a splitter cube, which, in operation, divides the incident beam 71 emitted by the light-emitting module into two beams, one constituting the reference beam 72 and the other constituting the object beam 73.

[0103] The object arm 82 is arranged so that, during operation, the object beam illuminates at least one three-dimensional sample 6 along a first direction Z. The first Z direction is perpendicular to the sample. The first Z direction is preferably vertical. Following illumination by the object beam 73, the at least one three-dimensional sample 6 reflects the object beam 73 into a reflected beam 74.

[0104] The interference device 22 further includes recombination means configured to recombine into an interference beam 75 the reference beam 72 and the beam reflected 74 by at least one three-dimensional sample 6.

[0105] According to one example, the recombination means consist of the same separator cube that was used to separate the incident beam 71.

[0106] The acquisition device 23 is configured to acquire a narrowed interferom signal from the interference beam 75.

[0107] The signal processing unit 24 is configured to analyze the interferometric signal acquired by the acquisition device 23 when the imaging device 2 is positioned in a current position relative to the at least one sample 6, in order to determine a portion of a three-dimensional image of the at least one three-dimensional sample 6.

[0108] The 24 signal processing unit can be configured to enable continuous and automated acquisition of interferometric signals for a predetermined duration, which can extend over several days. Such automated and continuous acquisition is advantageous for applications such as imaging the development of biological samples and cultures, or the aging of industrial materials.

[0109] The processing unit 24 can be a computer device comprising one or more memories for the storage of digital data and for the storage of program instructions, as well as a data processor, capable of executing program instructions stored in the memory or memories.

[0110] The processing unit 24 can also be implemented as an integrated circuit, comprising electronic components adapted to implement the function(s) described in this disclosure for the processing unit 24. The processing unit 24 can also be implemented by one or more physically separate devices. [YES] In one embodiment, the displacement device 3 comprises a translation stage 31 configured to move in translation along a second direction X and a third direction Y substantially perpendicular to the first direction Z, and perpendicular to each other. Thus, the displacement device 3 makes it possible, in particular, to image all parts of a large sample, or to move the imaging device 2 from one sample to another when the imaging system 1 is used for tracking several samples in parallel.

[0112] Advantageously, the translation plate 31 includes a receptacle configured to receive and retain the linking element 4.

[0113] According to one embodiment, the translation stage 31 is configured to move in translation further along the first Z direction. This allows the imaging device 2 to be positioned at a predetermined distance from the sample 6.

[0114] As an example, the translation platform 31 can be motorized and include a motor controlling its movement. When the processing unit 24 is a computer device, the motor can be controlled via this computer device, possibly coupled to a controller. Alternatively, the motor can be controlled via a computer device other than the processing unit 24.

[0115] Advantageously, the translational movements of the translation plate 31 have a greater amplitude than that permitted by standard moving components integrated into OCT assemblies. For example, the translational movements of the translation plate 31 can be performed over a volume of 220x220x250 mm 3 at a speed of 250 mm / s.

[0116] The amplitude, acceleration and / or speed of movement of the translation stage 31 can be controlled in order to avoid vibration of the sample 6.

[0117] According to one embodiment, at least a part of the imaging device 2 is fixed and held by the connecting element 4, so that when the translation stage 31 moves, at least a part of the imaging device 2 moves in a fixed manner with the connecting element 4.

[0118] The displacement device 3 is thus configured to move and position at least a part of the imaging device 2 in a plurality of current positions relative to at least one sample 6, so as to determine a plurality of portions of a three-dimensional image of at least one three-dimensional sample 6, together forming a three-dimensional image of at least one three-dimensional sample 6. The displacement device 3 is also configured to move and position the imaging device 2 in order to image several samples positioned on the sample holder 5.

[0119] The sample holder 5 can be configured to align several samples along the second X direction. The sample holder 5 allows for reproducible sample positioning. An example of the sample holder 5 is shown in Figure 21. The sample holder 5 is fixed in the XY plane. Figures 22, 23, and 24 show, respectively, a top view, a front view, and a side view of the sample holder 5 in the embodiment of Figure 21. A circular receptacle 52, visible in Figures 21, 22, 23, and 24, is configured to receive each sample container. The sample holder 5 can be configured to be fixed in a mounting bracket, a three-dimensional view of which is shown in Figure 25. Figures 26, 27, and 28 show, respectively, top, front, and side views of the mounting bracket.The mounting support may include at least one groove 51 into which the sample support 5 is fitted. The mounting support may be attached to a bed of a three-dimensional printer comprising the displacement device 3 as described below.

[0120] The sample support 5 can be mechanically decoupled from the displacement device 3 in order to limit sample vibrations during the displacement of the imaging device 2 by the displacement device 3.

[0121] In addition, a mat configured to dampen vibrations can be placed under the sample support 5 to limit sample vibrations during the movement of the imaging device 2 by the movement device 3.

[0122] As an example, when the processing unit 24 is a computing device, this computing device can also be configured, as will be seen later, to control the movement of the movement device 3 in accordance with at least one of the imaging methods 100 described later in this disclosure.

[0123] According to one embodiment, the displacement device 3 is a displacement device of a three-dimensional printer (3D printer).

[0124] Advantageously, in this embodiment, when the movement device 3 comprises the translation plate 31 equipped with its receptacle, the receptacle is initially designed to receive a print head, including a nozzle, of the 3D printer associated with the movement device 3. Thus, the connecting element 4 replaces the nozzle of the 3D printer associated with the movement device 3. This configuration is advantageous for the application of this disclosure, for which the weight on the movement device 3 must be limited. In other words, when the movement device 3 is a movement device for a 3D printer, the imaging device 2 advantageously replaces the print head (and therefore the nozzle) of the 3D printer.The weight of the imaging device 2 on the movement device 3 does not add to, but rather replaces, the weight of the 3D printer nozzle. Furthermore, the imaging system 1 has a smaller footprint and is less expensive than a traditional OCT system with a moving scanner component.

[0125] Furthermore, replacing the print head of a three-dimensional printer with an imaging device 2 facilitates the integration of the imaging device 2 onto the linking element 4 due to the limited available space in the effective volume of a complete three-dimensional printer.

[0126] Advantageously, in this embodiment, the displacement device 3 is controlled by one or more motors ensuring displacements of the translation plate 31 parallel to the first direction Z, the second direction X and the third direction Y.

[0127] For example, when the processing unit 24 is a computer device, the motor(s) can be controlled via this computer device, possibly coupled to a controller. Alternatively, the motor(s) can be controlled via a computer device other than the processing unit 24.

[0128] Advantageously also, in this embodiment, the sample support 5 can be integrated into a bed of the three-dimensional printer associated with the displacement device 3.

[0129] According to some embodiments, schematically illustrated in Figure 3, the light emission module 21 is configured to emit, in operation, a beam with a linear cross-section extending along the third direction Y, so that the interference beam has a linear cross-section along the third direction Y, as illustrated in Figure 4.

[0130] In some embodiments where the light-emitting module 21 is configured to emit a linear-section beam, the imaging device 2 further includes a dispersive element 25 configured to spatially separate the wavelengths of the plurality of wavelengths along the second X direction, as illustrated in Figure 4, so as to transform the linear-section interference beam into a two-dimensional-section beam.

[0131] In one example, the dispersive element 25 is a diffraction grating. In another example, the dispersive element is a prism.

[0132] In some embodiments where the light emission module 21 is configured to emit a beam of linear cross-section, at least two configurations are possible.

[0133] In a first configuration, the narrowed interferometry signal is one-dimensional, and the acquisition device is configured to acquire one-dimensional signals. For example, the acquisition device is a line camera. In this case, the light source 21 is a source whose wavelength varies over time (in English: "swept laser source").

[0134] In a second configuration, the interferometric signal is a two-dimensional signal. The acquisition device is then configured to acquire two-dimensional signals. For example, the acquisition device is an image sensor, such as a two-dimensional CMOS or CCD camera. In this case, the source 21 is a broadband spectral source. In OCT setup terminology, the acquisition device acquires a B-scan. When the imaging device 2 is positioned in a current position, the three-dimensional image portion determined by the signal processing unit is a current two-dimensional section parallel to a plane defined by the first Z direction and the third Y direction.

[0135] According to an example shown in Figure 3, the object arm 82 further includes an imaging lens 91, such as a microscope objective, so as to obtain a high-resolution transverse image of the sample 6. The three-dimensional sample 6 is then placed in the focal plane of the imaging lens 91 to obtain a sharp image of the three-dimensional sample 6. The measured OCT signal is then sharp according to the depth of field of the imaging lens 91. Thus, preferably, an imaging lens with a limited numerical aperture is chosen, in order to increase the depth of field and therefore to widen the range of depth values ​​where the measured OCT signal is sharp.

[0136] A motor can advantageously be integrated into the sample holder 5 or into the imaging device 2 in order to dynamically adjust the distance between the focal plane and the sample.

[0137] According to the example in Figure 3, the reference arm 81 includes an imaging lens 92, such as a microscope objective, configured to image a high-resolution reference mirror 93, and in such a way as to make the interference device 22 as symmetrical as possible, for example in terms of dispersion and optical path, so as to avoid digital correction steps.

[0138] Alternatively to figure 3, the reference arm 81 may not include an imaging lens and consist solely of a reference mirror 93.

[0139] Advantageously, as shown in Figure 3, an additional lens is positioned downstream of the recombination means configured to produce the interference beam 75. Such an additional lens allows an image of the sample 6 and the line to be formed at the level of a diaphragm, which serves to filter out all light not originating from the focal plane of the sample (stray reflections, multiple scattering, etc.) and thus increase image contrast. For example, the additional lens has a focal length of 200 mm.

[0140] Advantageously, the dispersive element 25 can be placed in the pupil plane of the imaging device 2. In this position, the configuration of the imaging system 1 is optimal.

[0141] As illustrated in Figure 3, a first lens with focal length fi is positioned upstream of the dispersive element 25, and a second lens with focal length fz is positioned downstream of the dispersive element 25. The diaphragm described above is positioned in the focal plane of the first lens, i.e., at a distance fi upstream of the first lens. The second lens converts the information from the output of the dispersive element 25, which disperses wavelengths at different angles, into position information for the acquisition device. This second lens thus allows the image of different wavelengths to be captured on the different pixels of the sensor. The first lens, upstream of the dispersive element 25, conjugates the sample 6 with the detection plane of the acquisition device 23. In this example, the distance fi is 50 mm and the distance fz is 100 mm.

[0142] The dispersive element 25 is positioned at a distance fi from the first lens, and at a distance fz from the second lens. The acquisition device 23 is positioned at a distance f2 downstream of the second lens.

[0143] In one embodiment, the connecting element 4 comprises a breadboard, illustrated in Figure 5, and includes mounting brackets centered relative to the translation stage. The structure and weight of the breadboard are optimized to be compatible with the maximum load of the translation stage 31 of the displacement device 3 and to limit mechanical vibrations. The mounting brackets are positioned to facilitate the positioning of components, such as optical components, of the imaging device 2 onto the breadboard.

[0144] In the embodiment where the connecting element 4 includes a mounting plate, screw threads can be made in the mounting plate so as to be able to fix it on the fixing plate 31 of the displacement device 3.

[0145] The following components of the imaging device 2 are mounted and fixed on the mounting plate: the interference device 22 and / or the acquisition device 23.

[0146] Advantageously, in some cases, if the weight of the light source 21 is limited, the light source 21 can also be fixed to the mounting plate.

[0147] In other cases, the light source 21 is not fixed to the mounting plate. In this case, the light source 21 can be connected with an optical fiber. The interference device 22 then further includes an illumination arm configured to illuminate the reference arm 81 and the object arm 82. The optical fiber is then connected to the illumination arm.

[0148] In one embodiment, the signal processing unit 24 is not mounted on the mounting plate.

[0149] In another embodiment, the signal processing unit 24 is mounted on the mounting plate. The signal processing unit 24 is, for example, a mini-computer weighing a few grams, such as a Raspberry Pi.

[0150] As an example, the mounting plate can be manufactured by 3D printing using PLA (polylactic acid) material and drilled with holes positioned at the intended locations of the various optical components of the interference device 22, and its weight is between 200 and 300 grams. For example, the dimensions of the mounting plate are 150x150x6 mm. 3 For added stability and to prevent weakening the mounting plate, the various holes in the mounting plate can also be created using a 3D printer. Metal threaded inserts are then inserted into the holes in the mounting plate. For example, the inserts are standard sizes M4 or M6. Advantageously, the total weight of the mounting plate and the components of the imaging device 2 mounted and secured to the mounting plate does not exceed 3 kilograms.

[0151] In certain embodiments illustrated in Figure 6, the light-emitting module 21 comprises a fiber source 21a, for example, a spatial single-mode source, including an output tip, and an optical device comprising a collimator 26 followed by a cylindrical lens 27 or a Powell lens arranged to form a beam with a linear cross-section along the third direction Y. For example, the cylindrical lens 27 has a focal length of 50 mm. The output tip is aligned with an axis of the collimator 26. Advantageously, to optimize the image quality determined with the imaging system 1, the output tip is positioned in the object focal plane of the collimator 26.Advantageously, to further optimize the image quality determined with the imaging system 1, when the object arm 82 includes an imaging lens 91, the cylindrical lens 27 is positioned so that its focal plane coincides with the rear focal plane of the imaging lens 91, or alternatively with the pupil plane of the imaging lens 91. In this case, the beam has a linear cross-section shape along the second direction X, but a linear cross-section shape along the third direction Y in the plane of the sample 6.

[0152] According to one example of these embodiments, the fiber source 21a is a spectrally broad light source of the LED, SLED, or supercontinuum laser type. The spectral width of the source determines the axial resolution of the imaging system 1 (due to the convolution of the signal with the inverse Fourier transform of the spectrum—if the spectrum is broad, its Fourier transform is narrow, and vice versa). For example, a light source centered around 560 nm and with a spectral width of 104 nm (e.g., a green LED) will provide an axial resolution on the order of 1 micrometer, while an SLED source centered around 840 nm with a spectral width of 70 nm will have an axial resolution on the order of 5 micrometers.

[0153] According to another example of these embodiments illustrated in Figure 7, the light-emitting module 21 comprises a round-to-linear fiber bundle, including an input tip with a circular arrangement of fibers 21b and an output tip with a linear arrangement of fibers 21c, and an optical device including a collimator 28. The output tip 21c is aligned on an axis of the collimator 28. Advantageously, to optimize the image quality determined by the imaging system 1, the output tip 21c is positioned in the object focal plane of the collimator 28.

[0154] In some embodiments, the resolution of the three-dimensional images obtained is on the order of 2.5x2.5x1 pm 3In the case where the optical components of the imaging device 2 include 4X 0.1 NA microscope objective-type imaging lenses, the size of the samples that can be imaged by a single, uninterrupted scan performed by moving the imaging device 2 via the movement device 3 is on the order of 1.5 x 1.5 x 1 mm. 3 .

[0155] According to other embodiments, the optical coherence tomography imaging device 2 further includes an additional translation means configured to move the object beam in translation along the third direction Y. Thus, the additional translation means allows an additional degree of freedom to be added in the scanning of at least one sample 6 by the optical coherence tomography imaging device 2.

[0156] According to some of these embodiments, the additional translation means is further configured to move the object beam in translation along the second X direction.

[0157] According to one example, the additional translation means includes a scanner configured to move in rotation coupled to a lens and acting in rotation on the object beam 73 when the latter illuminates the three-dimensional sample 6. The lens is configured to transform the rotational movement of the scanner into translational movement of the object beam.

[0158] Advantageously, the additional translation means allows for smaller amplitude displacements at higher speeds than the displacements of the translation plate of the displacement device. Thus, two types of displacement are possible in these embodiments: large amplitude displacements at relatively slow speeds with the displacement device 3, and smaller amplitude displacements at higher speeds with the additional translation means. This allows for the assembly of precise volumes. More specifically, it is possible to record a first volume of, for example, 5x5x3 mm. 3 then move with a 5 mm translation to record the adjacent field and move to a 10x5x3 mm field 3, and so on, tiling the space. This configuration is advantageous and useful if a fine resolution is expected, because the displacement over small amplitudes can be accurate to within 100 nm, and complemented by a displacement to image other fields by translation of the displacement device 3, when this is a displacement device of a three-dimensional printer, less precise, on the order of 10 m.

[0159] In some embodiments where the imaging device 2 further comprises an additional translation means, the object arm 82 includes an imaging lens. For example, the imaging lens has a focal length of 30 mm.

[0160] In some embodiments where the imaging device 2 further includes an additional translation means, the interferometric signal is a one-dimensional signal.

[0161] According to some of these embodiments, the acquisition device 23 is a spectrometer. For example, a grating spectrometer may be used. In OCT setup terminology, for a current position of the imaging device 2, the spectrometer acquires an A-scan signal. When the imaging device 2 is positioned at a current position relative to at least one three-dimensional sample 6, the three-dimensional image portion determined by the signal processing unit is a row of voxels along the first Z direction.

[0162] In some embodiments, the emission module 21 includes an SLED type light source, for example centered at a wavelength Xo equal to 840 nm and width Az equal to 85 nm.

[0163] In certain embodiments where the imaging device 2 further comprises an additional translation means, the object arm 82 is mounted in a dedicated mount. At least a portion of the imaging device 2 held and secured by the connecting element 4 consists of the dedicated mount with the object arm 82. The connecting element 4 comprises a first support portion 42, a three-dimensional view of which is shown in Figure 8, configured to be fixed to the translation stage 31, and a second support portion, a three-dimensional view of which is shown in Figure 9, configured to hold the dedicated mount and to be fixed in the first support portion 42. Figures 10 and 11 show, respectively, a top view and a side view of the second support portion of Figure 9.According to the example of figures 9, 10 and 11, the second support portion may include an annular mount 44 configured to hold the dedicated mount containing the object arm 82, attached to a base 45 configured to be fixed in a receptacle 43 of the first support portion 42. According to one example, the weight of the first support portion 42 and the second support portion is on the order of 50 grams.

[0164] The structure of the first support portion and the second support portion allows us to meet the mechanical stability and weight criteria of the imaging device 2 on the displacement device 3.

[0165] Advantageously, in certain embodiments where the imaging device 2 further comprises an additional translation means, the interference device is fiber-connected; in other words, the object arm and the reference arm are equipped with optical fibers. Thus, the dedicated mount can include, in addition to the fiber-connected object arm, the additional translation means (consisting, for example, of a scanner coupled to a lens) so as to form a portable imaging probe. The portable imaging probe is fixed in the secondary support.

[0166] In certain embodiments where the imaging device 2 further comprises an additional translation means, the resolution of the three-dimensional images obtained is on the order of 15x15x5 pm 3, for a system with imaging lenses of 30 mm focal length and a diameter of 12.5 mm. The size of the samples that can be imaged, by a single, uninterrupted scan performed by moving the imaging device 2 via the displacement device 3, coupled with moving the object beam 73 with the additional translation means, is on the order of 5x5x3 mm 3 Advantageously, this order of magnitude of the volume of samples that can be imaged with the imaging system 1 according to this disclosure can be tailored, in particular by choosing the characteristics of the imaging lens present in the object arm 82, in particular, its focal length.

[0167] At least part of the imaging system 1 can be placed in an incubator comprising a hermetically sealed enclosure configured to control the humidity and / or temperature inside the incubator. For example, the sample holder 5 and the object arm 82 cooperating with the movement device 3 are placed inside the incubator, with the remaining elements of the imaging system 1 being placed outside the incubator.

[0168] For example, the incubator is configured to maintain a humidity level above 50%, preferably above 60%, and even more preferably above 70%. This advantageously limits evaporation of the sample.

[0169] For example, the incubator is configured to maintain a temperature between 25°C and 33°C, preferably between 27°C and 29°C, and even more preferably between 27.8°C and 28.2°C. This advantageously allows for rapid growth of the biological material. Imaging process

[0170] A second aspect of the present invention relates to a method of optical coherence tomography imaging 100 of at least one three-dimensional sample 6, preferably, of a plurality of three-dimensional samples (6i,.. 6j, 6L).

[0171] In some embodiments, process 100 allows obtaining a three-dimensional image of a three-dimensional sample 6.

[0172] In other embodiments, the process 100 allows for the simultaneous acquisition of a plurality of three-dimensional images of a plurality of three-dimensional samples 6j, for j ranging from 1 to L. L is the number of three-dimensional samples. For example, L is an integer between 1 and 4 if only one translation direction, the second X direction, of the translation stage 31 is used, or between 1 and 16 if two translation directions, the second X direction and the third Y direction, are used. In another example, where images of cultures are contained in a 96-well plate array, L can range from 1 to 96.

[0173] The main steps of a first embodiment of the process 100 when it enables obtaining a three-dimensional image of a three-dimensional sample 6 according to the present description are described with reference to Figure 12. In this first embodiment, the process 100 can be implemented with an imaging system 1 as described previously.

[0174] During an initial placement step El 10, the imaging process is initialized and a three-dimensional sample 6 is placed on the sample support 5. For example, when a single sample 6 is imaged, the sample 6 is placed opposite the object arm 82.

[0175] Preferably, the imaging device 2 is translated along the first Z direction in order to position itself at a predetermined distance from the sample 6.

[0176] According to a first variant, the imaging process 100 is implemented by the imaging system 1 in which the light emission module 21 is configured to emit an incident beam 71 of linear section.

[0177] In this first variant, during an acquisition and analysis step E120, the imaging device 2 is successively translated into a set of current positions xi, also called acquisition position xi, for i between 1 and N, where N is an integer between 1 and 512, or 1 and 1024, or 1 and 1536, or 1 and 4x512, i.e., 2048, along the second direction X by controlling the translation stage 31, so as to form N successive interference beams in the interference device and to acquire, by the acquisition device 23, a temporal succession of N interferometric signals, each corresponding to a current interference beam formed. In other words, the translation of the imaging device 2 allows the sample 6 to be scanned along the second direction X.The analysis by the signal processing unit 24 of the N interferometric signals makes it possible to obtain a plurality of portions of a three-dimensional image of the three-dimensional sample E, together forming a three-dimensional image of the three-dimensional sample E.

[0178] In this first variant, for each position xi, for i between 1 and N, occupied by the imaging device 2 along the second direction X, the interferometric signal is a two-dimensional signal, as explained above. The analysis of an interferometric signal corresponding to a current or acquisition position of the imaging device 2 includes a one-dimensional Fourier transform along the first dimension Z of the interferometric signal, which allows for the generation of a slice in a ZY plane.

[0179] The plurality of slices in the ZY planes corresponding to each position of the imaging device 2 along the second X direction allows to reconstruct a volume forming a three-dimensional image of the three-dimensional sample 6.

[0180] According to a second variant, the method 100 is implemented by an imaging system 1 in which the imaging device 2 includes an additional translation means configured to move the incident beam 71 along the second direction X and along the third direction Y.

[0181] In this second variant, during an acquisition and analysis step E120, the imaging device 2 is translated successively, via the additional translation means, into a set of current positions or acquisition positions (xi, y m), for i between 1 and N, where N is an integer, for example, between 1 and 128, and m between 1 and M, where M is an integer, for example, between 1 and 512. Through this translation, a set of NxM are formed such that the acquisition device acquires NxM one-dimensional interferometric signals. In other words, the translation of the imaging device 2 allows the sample 6 to be scanned along the second direction X and the third direction Y. The signal processing unit thus reconstructs, for each position xi, for i between 1 and N, M rows of voxels along the third direction Y.

[0182] According to an example, for each position (xi, y m ), swept by the incident beam 71, a plurality of wavelengths, for example 1024 wavelengths, are recorded for a plurality of pixels, for example 512 pixels, of data along the third Y direction.

[0183] Figure 13 shows a raw interferometric signal acquired by the acquisition device 23, and corresponding to a position (xi, y m ) in a first bacterial or microbial biofilm.

[0184] Figure 14 shows a two-dimensional cross-section of this first bacterial or microbial biofilm obtained by translation along the third Y direction, for a given position along the X axis. A column of the two-dimensional cross-section in Figure 14 is obtained from a similar raw interferometric signal such as that shown in Figure 13.

[0185] Figure 15 shows a two-dimensional section of a second bacterial or microbial biofilm obtained by translation along the third Y direction, for a given position along the X axis.

[0186] Figure 16 shows a three-dimensional volume reconstruction of the second bacterial or microbial biofilm of Figure 15, obtained by implementing the imaging process 100 according to this second variant.

[0187] Thus, the set of NxM rows of voxels allows us to reconstruct a volume forming a three-dimensional image of the three-dimensional sample 6.

[0188] Advantageously, thanks to parameter settings for the acquisition device 23 and the signal processing unit 24, the imaging process 100 allows for monitoring the evolution of samples over time periods. The acquisition device 23 and the signal processing unit 24 can indeed be configured to continuously acquire interferometric signals from a sample 6 over a predetermined time period. In other words, the acquisition and analysis step E120 is repeated for a plurality of times.

[0189] Figure 17 shows three two-dimensional slices (b-scans) obtained with the imaging system 1 according to the invention, at three different times: the first two-dimensional slice was acquired 15 hours and 30 minutes after the start of recording, the first and second two-dimensional slices were acquired 2 hours and 30 minutes apart, and the second and third two-dimensional slices were acquired approximately 13 hours apart. The temporal evolution of the first sample can be observed from the three two-dimensional slices. In the experiment producing the three two-dimensional slices in Figure 17, a two-dimensional slice is acquired every 5 minutes.

[0190] Figure 18 shows three two-dimensional slices (b-scans) obtained with the imaging system 1 according to the invention, at three different times: the first two-dimensional slice was acquired 24 hours and 25 minutes after the start of recording, the first and second two-dimensional slices were separated by approximately 4 hours, and the second and third two-dimensional slices were separated by 3 hours and 40 minutes, of a second sample consisting of a second type of bacteria. The temporal evolution of the second sample can be observed from the three two-dimensional slices.

[0191] A second embodiment of process 100 allows obtaining a plurality of three-dimensional images of a plurality of L three-dimensional samples 6i, 62, .... 6L, with L an integer for example between 1 and 4, or 1 and 16 or 1 and 96, positioned along an axis parallel to the second direction X according to the present description and is described here with reference to Figure 19. In this second embodiment, the method 100 can be implemented with an imaging system 1 as described previously.

[0192] In the initial step El 10, the imaging process 100 is initialized, and each sample 6j from the plurality of L samples is placed on the sample holder 5 along an axis parallel to the second direction X. Each individual sample 6j is positioned, for example, centered, at a placement position xechj along the axis. For example, when the sample 6j is placed in a container with a perforated lid as described above, the individual sample 6j is positioned so that the observation hole is positioned at a placement position xechj along the axis. In other words, the individual sample 6j is associated with a placement position xechj.

[0193] This second embodiment of process 100 also includes a first variant and a second variant.

[0194] In the first variant, the imaging process 100 is implemented by the imaging system 1 in which the light emission module 21 is configured to emit an incident beam 71 of linear section.

[0195] In the second variant, the imaging method 100 is implemented by the imaging system 1 in which the imaging device 2 includes an additional translation means configured to move the incident beam 71 along the second direction X and along the third direction Y.

[0196] The 100 imaging process includes: - a step E140 of translation of the imaging device 2 to each placement position xechj by control of the translation stage 31 of the displacement device 3 to move along the second direction X; repetition of the acquisition and analysis step E120 described in the first embodiment of the imaging process 100; The E140 and E120 steps are repeated for each individual sample Ej. The imaging process 100 then ends at step E170.

[0197] Optionally, a calibration step can precede the first translation step El 40 corresponding to the first sample El to be imaged. Such a calibration step allows the translation stage 31 to be freely controlled and continuously imaged with the OCT imaging system in order to determine the optimal placement positions xechj, or those corresponding to structures of interest in each of the samples.

[0198] Advantageously, the use of a sample support 5 such as that shown in figures 21 to 28 makes it possible to do away with this calibration step.

[0199] Optionally, after or at the same time as each step El 40 of translation of the imaging device 2 to each placement position xechj, the imaging method includes a step of translation of at least a part of the imaging device 2 along the first direction Z in order to position it at a predetermined distance from the sample 6j of the placement position xechj.

[0200] Optionally, after the acquisition and analysis step E120, the imaging process includes a step of translating the imaging device 2 along the first Z direction in order to raise the imaging device 2 and thus increase the distance between the sample 6j and the imaging device 2. This is particularly advantageous when the sample 6j is placed in a container and when the predetermined distance between the sample 6j and the imaging device 2 is less than the distance between the sample 6j and the upper rim of the container.

[0201] A third embodiment of method 100 allows obtaining a plurality of three-dimensional images of a plurality of L three-dimensional samples positioned in a set of placement positions (xechj, yechj) on the sample support 5 according to the present description and is described here with reference to Figure 20. In this third embodiment, method 100 can be implemented with an imaging system 1 as described previously.

[0202] During the initial step El 10, the imaging process 100 is initialized and each sample 6j from the plurality of L samples is placed on the sample holder 5 at its placement position (xechj, yechj).

[0203] The 100 imaging process includes: - a step E160 of translation of the imaging device 2 to each placement position (xechj, yechj) by control of the translation stage 31 of the displacement device 3 to move along the second direction X and / or along the third direction Y; repetition of the acquisition and analysis step E120 described in the first embodiment of the imaging process 100; The E160 and E120 steps are repeated for each individual sample every 6 days. The imaging process then ends at step E170. Applications

[0204] The imaging system 1 and the optical coherence tomography (OCT) imaging method 100, according to the various embodiments described above, find applications for imaging biological samples and materials such as bacterial or microbial biofilms, and three-dimensional cell cultures, such as spheroids or organoids. Other samples, such as human cell cultures or bioprinted hydrogels, can be imaged with the imaging system 1 and the OCT imaging method 100 according to the invention. The imaging system 1 could also be used to image industrial materials and to perform non-destructive testing (defect detection, impact detection, quantification of structure and optical properties, etc.) during manufacturing processes. EXAMPLES

[0205] The present invention will be better understood by reading the following examples which illustrate the invention in a non-limiting way.

[0206] Example 1: Multiplexed bacterial biofilm imaging

[0207] Materials and methods

[0208] In this example, the imaging method 100 according to the second embodiment is used to image bacterial biofilms in a multiplexed manner, using the imaging system 1 according to the embodiments where the imaging device 2 further comprises an additional translation means.

[0209] In this example, the displacement device 3 is a displacement device of a three-dimensional printer and the sample support 5 is fixed to the bed of the three-dimensional printer.

[0210] Also, in this example, the imaging device 2 further includes an adaptive lens allowing the focal length of the imaging lens of the object arm to be changed along the first Z direction.

[0211] Bacterial biofilms are placed in a set of four identical beakers, L = 4. The beakers are cylindrical, 70 mm high, and 40 mm in diameter. The thickness is adapted for an opaque sample (6) but can be adjusted for other samples with different transmission coefficients (6). A dedicated sample holder (5), a three-dimensional view of which is shown in Figure 21, was designed to hold each beaker in a custom-made position along an axis parallel to the second direction (X). The sample holder (5) and the beakers are stationary. Figures 22, 23, and 24 show a top view, a front view, and a side view of the dedicated sample holder (5), respectively. A circular receptacle 52, visible in figures 21, 22, 23 and 24, is configured to receive each beaker.The dedicated sample support 5, with its fixed locations, allows the reproducibility of the positioning of the beakers on the support and therefore that of the imaging process 100.

[0212] In practice, the dedicated sample holder 5, shown in Figures 21, 22, 23, and 24, is fixed in a dedicated mounting bracket, a three-dimensional view of which is shown in Figure 25. Figures 26, 27, and 28 show top, front, and side views of the dedicated mounting bracket, respectively. The dedicated mounting bracket has cubic openings 51 into which the dedicated sample holder 5 is fitted. The dedicated mounting bracket is fixed to a bed of the three-dimensional printer from which the displacement device 3 is derived, so that the beaker positioned in the dedicated sample holder 5 is always in the same positions (X, Y, Z) for each implementation of the imaging process 100.

[0213] A first calibration step allows the translation stage 31 to be controlled freely and to image continuously with the imaging system 1 in order to determine the optimal placement positions xechj, or corresponding to structures of interest in each of the samples.

[0214] Next, the translation plate 31 is controlled so that it positions itself at each placement position xechj. At each placement position xechj: the object arm is moved along the first direction Z by means of the displacement device 3, in particular the translation plate 31, translated along the first direction Z, so as to be positioned in an initial position Zj inside the beaker no. j; step E120 of the imaging process 100 is implemented; The object arm 82 is moved along the first direction Z using the displacement device 3, in particular the translation plate 31, so as to be repositioned in a final position Z up outside of beaker no. j.

[0215] Results

[0216] Advantageously in this example, with the beakers remaining stationary during the implementation of the imaging process 100, the image capture can be automated without risk of damaging the biofilms, which are very fragile structures.

[0217] Figure 29 shows a two-dimensional cross-section of a beaker containing a third biofilm of a bacterium that has naturally mutated to overproduce the polymer imaged using the Imaging Process 100. The structure of this third bacterial biofilm within the beaker can be observed. In particular, traces of biofilm collapse along the vertical axis can be seen. REFERENCES NUMERIQUES 1 - Optical coherence tomography imaging system / / 2 - Frequency domain optical coherence tomography imaging device / / 21 - Light emission module / / 21a - Fiber source / / 21b - Circular fiber arrangement input tip / / 21c - Linear fiber arrangement output tip / / 22 - Interference device / / 23 - Acquisition device / / 24 - Signal processing unit / / 25 - Dispersive element / / 26 - Collimator / / 27 - Cylindrical lens / / 28 - Collimator / / 3 - Displacement device / / 31 - Translation stage / / 4 - Linking element 42 - First support portion 43 - Secondary support receptacle 44 - Ring mount 45 - Base 5 - Sample holder 51 - Cubic openings 52 - Beaker receptacle / / 6 - Three-dimensional sample / / 6i, 62,....6j,..., 6L - Three-dimensional samples / / 71 - Incident beam / / 72 - Reference beam / / 73 - Object beam / / 74 - Reflected beam / / 75 - Interference beam H 81 - Reference arm / / 82 - Object arm H 91 - Object arm imaging lens / / 92 - Reference arm imaging lens / / 93 - Reference mirror.

Claims

DEMANDS 1. A method (100) for optical coherence tomography imaging of a plurality of samples (6i, ..., 6j, 6L), each sample (6i, ..., 6j, 6L) comprising an air-liquid interface on which biological material grows, the method comprising: provision of an optical coherence tomography imaging system (1) comprising: o an optical coherence tomography imaging device in the frequency domain (2) comprising: • a light emission module (21), • an interference device (22), said light-emitting module (21) being configured to emit an incident beam (71) comprising a plurality of wavelengths, and to illuminate, in operation, said interference device (22), said interference device (22) comprising an object arm (82) and a reference arm (81), the reference arm (81) being traversed by a reference beam (72) and the object arm (82) being arranged in a first direction (Z) to illuminate, in operation, the air-liquid interface of one of the plurality of three-dimensional samples (6i,.. 6j, 6L); said interference device (22) further comprising recombination means configured to recombine the reference beam (72) and a beam reflected (74) by the illuminated air-liquid interface into an interference beam (75); o an acquisition device (23) configured to acquire an interferometric signal from the interference beam (75), o a signal processing unit (24) configured to analyze said interferometric signal in order to determine a three-dimensional image of the biological material developing at the illuminated air-liquid interface, o a displacement device (3) comprising a translation stage (31) configured to cooperate with at least a portion of the imaging device (2) and configured to move in translation along at least a second direction (X) substantially perpendicular to the first direction (Z), the displacement device (3) being configured to position at least a portion of the imaging device (2) in a plurality of acquisition positions; o a linking element (4) configured to ensure cooperation between at least a part of the imaging device (2) and the displacement device (3), the translation stage (31) comprising a receptacle configured to receive and retain said linking element (4), o a sample holder (5) configured to support the plurality of three-dimensional samples (6i,.. 6j, 6L) in a plane substantially orthogonal to said first direction (Z), placement (El 10) of the plurality of three-dimensional samples (6i,.. 6j, 6L) on the sample support (5) along an axis parallel to the second direction (X); for each three-dimensional sample (6i,.. 6j, 6L), acquisition and analysis of at least one interferometric signal (E120) using the signal processing unit (24) comprising: o translator (E140), by means of the translation stage (31), at least a part of the imaging device (2) to an acquisition position above said three-dimensional sample, o production, by the light-emitting module (21), of at least one interference beam (75) by illumination of the interference device (22), o acquisition, by the acquisition device (23), of at least one interferometric signal from the at least one interference beam (75) produced, o analysis, by the signal processing unit (24), of at least one interferometric signal so as to obtain a three-dimensional image of the biological material developing at the air-liquid interface of said three-dimensional sample.

2. The process (100) according to claim 1 wherein the biological material is a bacterial biofilm.

3. The method (100) according to claim 1 or 2 wherein the step of acquiring and analyzing at least one interferometric signal (El 20) is repeated for a plurality of acquisition positions.

4. The method (100) according to any one of claims 1 to 3 wherein the step of acquiring and analyzing at least one interferometric signal (E120) is repeated for a plurality of times, the imaging method enabling monitoring of the development of biological material.

5. The method (100) according to any one of claims 1 to 4 further comprising, for each three-dimensional sample (6i,.. 6j, 6L), during the step of acquiring and analyzing at least one interferometric signal (E120), a step of translating at least one part of the imaging device (2) along the first direction (Z) in order to position at least one part of the imaging device (2) at a predetermined distance from said three-dimensional sample.

6. The method (100) according to claim 5 further comprising, for each three-dimensional sample (6i,.. 6j, 6L), after the step of acquiring and analyzing at least one interferometric signal (El 20), a step of translating at least one part of the imaging device (2) along the first direction (Z) in order to increase the distance between at least one part of the imaging device (2) and said three-dimensional sample.

7. Optical coherence tomography (OCT) imaging system (1) of at least one three-dimensional sample (6i, ..., 6j, 6L) configured to implement the method (100) according to any one of claims 1 to 6, the imaging system (1) comprising: - an OCT imaging device in the frequency domain (2), said imaging device (2) comprising: o a light emission module (21), o an interference device (22), o said light emission module (21) being configured to emit an incident beam (71) comprising a plurality of wavelengths, and to illuminate, in operation, said interference device (22), o said interference device (22) comprising an object arm (82) and a reference arm (81), the reference arm (81) being traversed by a reference beam (72) and the object arm (82) being arranged in a first direction (Z) to illuminate, in operation, the air-liquid interface of one of the plurality of three-dimensional samples (6i,.. 6j, 6L); o said interference device (22) further comprising recombination means configured to recombine the reference beam (72) and a beam reflected (74) by the illuminated air-liquid interface into an interference beam (75); - an acquisition device (23) configured to acquire an interferometric signal from the interference beam (75), - a displacement device (3) configured to cooperate with at least a part of the imaging device (2), said displacement device (3) comprising a translation stage (31) configured to move in translation along at least a second direction (X) substantially perpendicular to the first direction (Z), the displacement device (3) being configured to position at least a part of the imaging device (2) in a plurality of current positions; - a linking element (4) configured to ensure cooperation between at least a part of the imaging device (2) and the displacement device (3), the translation stage (31) comprising a receptacle configured to receive and retain said linking element (4), - a sample holder (5) configured to support the plurality of three-dimensional samples (6i, ..., 6j, 6L) along an axis parallel to the second direction (X) in a plane substantially orthogonal to said first direction (Z), - a signal processing unit (24) configured to: to translate the imaging device (2) to said position by means of the translation stage (31), to produce at least one interference beam (75) by illuminating the interference device (22) by the light-emitting module (21), to acquire at least one narrowed interferometric signal from at least one interference beam (75) by the acquisition device (23), to analyze at least one interferometric signal so as to obtain a three-dimensional image of the biological material developing at the air-liquid interface of at least one three-dimensional sample (6i,... 6j, 6L) corresponding to said position by the signal processing unit (24).

8. System (1) according to claim 7, characterized in that the displacement device (3) is a displacement device for a 3D printer.

9. System (1) according to any one of claims 7 or 8, characterized in that the translation plate (31) is further configured to move in translation along the first direction (Z).

10. A system according to any one of claims 7 to 9, characterized in that: said light-emitting module (21) is configured to emit, in operation, a beam of linear cross-section extending along a third direction (Y) perpendicular to the first direction (Z) and the second direction (X), such that said interference beam (75) has a linear cross-section, said imaging device (2) further includes a dispersive element (25) configured to spatially separate the wavelengths of the plurality of wavelengths along the first direction (Z), so as to transform said linear cross-section interference beam (75) into a two-dimensional cross-section beam, said interferometric signal is a two-dimensional signal, said acquisition device (23) is configured to acquire two-dimensional signals parallel to a plane defined by the first direction (Z) and the third direction (Y), the two-dimensional signals together forming said three-dimensional image.

11. System (1) according to claim 10, characterized in that the light emission module (21) comprises a fiber source (21a) including an output tip, and an optical device comprising a collimator (26) followed by a cylindrical lens (27), the output tip being positioned in an object focal plane of said collimator (26).

12. System (1) according to claim 10, characterized in that the light emission module (21) comprises a round-to-linear fiber bundle, including an input tip (21b) with a circular arrangement of fibers and an output tip (21c) with a linear arrangement of fibers, and an optical device including a collimator (28), the output tip (21c) being positioned in a focal plane of said collimator (28).

13. System (1) according to any one of claims 10 to 12, characterized in that the connecting element (4) comprises a mounting plate including fixing fasteners in positions centered with respect to the translation plate (31).

14. System (1) according to any one of claims 7 to 9, characterized in that: said imaging device (2) further includes an additional translation means configured to move the incident beam (71) along a third direction (Y) perpendicular to the first direction (Z) and the second direction (X), said interferometric signal is a one-dimensional signal, and said acquisition device (23) is a spectrometer.

15. System (1) according to claim 14, characterized in that the object arm (81) is mounted in a dedicated mount and in that the linking element (4) comprises a first support portion (42) configured to be fixed on said translation plate (31) and a second support portion configured to retain said mount and to be fixed in the first support portion.

16. System (1) according to claim 14 or 15, wherein the additional translation means is configured to move the incident beam (71) with a lower amplitude than the translation plate of the displacement device and at a higher speed than the translation plate of the displacement device.

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