Linear scanning microscopy devices and methods

Abstract

The present description relates to a linear scanning optical coherence tomography microscopy device (200). It comprises an interferometric microscope (201) including a reference arm, an object arm configured to receive an object (10), a beam splitter (204) coupling said object and reference arms to a light source (210) and a detector (222), and a first microscope objective (202) arranged on said object arm.It further comprises a one-dimensional confocal spatial filtering device configured to cooperate with said light source to illuminate said object along a focal line located in an object space of the first microscope objective, and a one-way scanning device (235, 237) of said focal line, arranged on said object arm upstream of said first microscope objective, and configured to scan the focal line in a lateral direction (y) substantially perpendicular to an optical axis (z) of said first microscope objective. FIG. 2.

Description

Title of the invention: Linear scanning microscopy devices and methods Technical field

[0001] The present disclosure relates to devices and methods for line scanning microscopy. More specifically, the devices and methods are based on line scanning optical coherence tomographic microscopy. State of the art

[0002] Optical coherence tomography or OCT (abbreviation of the English expression “Optical Coherence Tomography”) is based on the use of a low coherence interferometer.

[0003] This imaging technique, described for example in D. Huang et al. [Ref.l] makes it possible to produce tissue section images, with an axial resolution of a few microns.

[0004] More specifically, scanning OCT techniques are known in particular.

[0005] In one version of Time Domain OCT, As described in [Ref. 1], a beam of white light is divided into two parts, one focused on the tissue to be studied and the other on a reference mirror. The light reflected (backscattered) by the observed object is combined with that reflected by the reference mirror and detected by a photodetector. Interference occurs only when the optical path difference is at most of the order of the coherence length of the radiation; by changing the optical length of the reference arm of the interferometer, different depths in the object can be accessed. A 2- or even 3-dimensional image can be constructed using interferometry (which allows acquisition along the axial dimension, i.e., depth) and scanning (which allows acquisition along one or two lateral dimensions).In frequency domain OCT, the reference arm has a fixed optical length and the interferometric signal is analyzed spectrally. See the article by AF Fercher et al. [Ref. 2]. In practice, scanning OCT hardly allows lateral resolutions better than about a few micrometers to be obtained.

[0006] With the aim in particular of improving lateral resolution, the article by JA Izatt et al. [Ref. 3] describes an improved technique combining optical coherence tomography and confocal microscopy. Confocal microscopy makes it possible to significantly reduce the number of photons backscattered by areas of the sample located outside the coherence volume and perceived by the sensor, which makes it possible to increase the signal to noise ratio.

[0007] More recently, the article by Y. Chen et al. [Ref. 4] proposed an LS-OCM device for linear scanning optical coherence tomographic microscopy. Such a device is shown in Fig. 1.

[0008] The LS-OCM device 100 illustrated in Fig. 1 comprises a Linnik-type interferometric microscope 101 with two microscope objectives 102, 103 respectively in the object and reference arms of the interferometer, said object and reference arms being separated by a beam splitter cube 104. The device 100 further comprises a laser source 110, in this example a femtosecond laser source emitting broad spectral band pulses (80 nm). Linear illumination of the sample 10 is obtained with an arrangement of a cylindrical lens 111 and spherical lenses 112, 113. The optical power in the reference arm is controlled by a neutral density filter 105 and a glass slide 106 is arranged in the object arm to compensate for dispersion.Light from the object and reference arms is recombined by the beam splitter cube 104 and projected by means of a lens 121 onto a line camera 122, thereby obtaining an interferometric signal. The interferometric signal is modulated by sinusoidally operating a reference mirror 107 arranged in a focal plane of the microscope objective 103 of the reference arm, by means of a piezoelectric transducer 108, and an image of a line of the sample is reconstructed from a combination of four interference signals resulting from the modulation.

[0009] The microscopy device described in Fig. 1 has a linear or one-dimensional (in one direction) confocal filtering configuration; in fact, the illumination line is optically conjugated with a linear detector whose detection surface has a width substantially identical to a width of the image of the line, resulting in a spatial filtering of a region of the object to be observed.

[0010] Thus, the microscopy device described in Fig. 1 has advantages linked to confocal filtering but allows for gains in acquisition speed compared to the scanning optical coherence tomographic microscopy described in [Ref. 3] due to the parallel acquisition on a line.

[0011] Furthermore, in [Ref. 4], images in front of the sample can also be generated by scanning the sample 10 in a direction perpendicular to the illumination line, by means of a translation stage 109.

[0012] Although allowing the formation of images in front of the sample with an axial resolution, i.e. in the direction of the optical axis of the microscope objective 102, of approximately 3 pm, and a lateral resolution, i.e. in a direction contained in a plane perpendicular to the optical axis of the microscope objective, of approximately 2 pm, the device described in [Ref. 4] is however not suitable for the mi in vivo sample microscopy, the sample having to be moved to produce en face images. Furthermore, if they were considered, scanning mechanisms for the illumination and detection beams would lead to an increase in the size of the device which we are trying to avoid with a view to industrialization.

[0013] The present description provides devices and methods for linear scanning optical coherence tomographic microscopy for forming images in front of an in vivo sample, with rapid image acquisition and reduced device footprint. Summary of the invention

[0014] In the present description, the term "comprise" means the same as "include", "contain", and is inclusive or open and does not exclude other elements not described or shown. Furthermore, in the present description, the term "approximately" or "substantially" is synonymous with (means the same as) a lower and / or upper margin of 10%, for example 5%, of the respective value.

[0015] According to a first aspect, the present description relates to a linear scanning optical coherence tomographic microscopy device, comprising: - a broad spectral band light source; - a detector with a linear detection surface; - an interferometric microscope comprising a reference arm at the end of which is arranged a reference reflecting surface, an object arm configured to receive an object to be imaged, a beam splitter coupling said object and reference arms to said light source and said detector, and at least a first microscope objective arranged on said object arm; - a one-dimensional confocal spatial filtering device configured to cooperate with said light source to illuminate said object along a line of focus located in an object space of said first microscope objective, and configured to select the light backscattered by said object and coming from said line of focus to form a one-dimensional image of said line of focus on the detection surface; - a unidirectional scanning device of said focus line, arranged on said object arm upstream of said first microscope objective, and configured for scanning the focus line in a lateral direction substantially perpendicular to an optical axis of said first microscope objective; - a processing unit configured to produce at least one two-dimensional tomographic image in front of said object to be observed, arranged in a plane substantially perpendicular to said optical axis of the microscope objective, from a plurality of one-dimensional interferometric images of the object produced by the interferometric microscope for different positions of said focus line during said unidirectional scan and acquired by said detector.

[0016] In the present description, an optical axis of the first microscope objective arranged in the object arm is defined by a line passing through the center of a pupil of said objective and substantially perpendicular to the plane of said pupil. This line may be a broken line if there are in the device deflecting mirrors or other light deflection elements.

[0017] Thus, in general terms, the term "axial direction" in a given space of the device will be used to refer to a direction collinear with the optical axis of said first microscope objective, considered in said space.

[0018] In general, a "lateral direction" in a given space of the device will be called a direction perpendicular to the optical axis of said first microscope objective, considered in said space.

[0019] In the present description, the "focusing surface" is the surface formed by the focusing line in the object space of the first microscope objective, during the scanning of said line. The object space of the first microscope objective is defined in the present description by the space in which the object of study is located, in operation. The focusing surface is generally planar, substantially perpendicular to the optical axis of the first microscope objective, and defined in a region corresponding to the depth of field of said microscope objective.

[0020] The “focusing surface” is a surface, in the object space, optically conjugated with the linear detection surface by the first microscope objective and all of the optical elements present between the first microscope objective and the detection surface. The device is adjusted upstream of the image formation so that the focusing surface is substantially merged with the focusing surface.

[0021] The original arrangement of the optical coherence tomographic microscopy device thus described, with a unidirectional scanning device of said focusing line arranged on the object arm of the interferometric microscope, upstream of said first microscope objective, allows the formation of images in front of in vivo samples, while maintaining a reduced size for the device.

[0022] The object of study is, for example and in a non-limiting manner, a region of skin and applies, for example, to the diagnosis of different types and subtypes of skin cancers, in particular melanomas, basal cell carcinomas and spinocellular carcinomas, to the diagnosis of inflammatory and bullous pathologies of the skin, to the evaluation of different morphological parameters of healthy skin, the monitoring of the evolution of skin lesions, the examination of excision margins, etc.

[0023] According to one or more exemplary embodiments, the broadband light source spectral includes a light-emitting diode or a combination of light-emitting diodes, a superluminescent diode or a combination of superluminescent diodes, a halogen filament lamp, an arc lamp, a broad spectral band laser source (source by generation of "supercontinuum" for example). In all cases, the spectral width (at half-maximum) of the source will preferably be greater than or equal to 100 nm; the greater this spectral width, the better the axial resolution of the device; the center-band wavelength can be visible or in the near infrared; in biological and medical applications, the near infrared, between 700 nm and 1500 nm, is generally preferred. The source can be polarized or non-polarized, spatially coherent or incoherent.

[0024] Spatially coherent sources (such as lasers or superluminescent diodes) can be advantageous due to their higher luminance. Furthermore, they allow, in cooperation with a cylindrical lens, to form the focusing line.

[0025] According to one or more exemplary embodiments, said interferometric microscope is a Linnik microscope, further comprising a second microscope objective arranged on said reference arm, said reference and object arms being disjoint. For example, the first microscope objective and the second microscope objective are identical, making it possible to have compensation for the effects of chromatic dispersion between the two arms of the interferometer.

[0026] According to one or more exemplary embodiments, the reference arm does not comprise a microscope objective. In this case in particular, the device may further comprise a chromatic dispersion compensation element arranged on one and / or the other of said object and reference arms.

[0027] According to one or more exemplary embodiments, said first microscope objective and / or said second microscope objective, when present, is an immersion microscope objective, that is to say a microscope objective immersed in a medium whose refractive index is substantially equal to the refractive index of the object of study.

[0028] According to one or more exemplary embodiments, said reference reflective surface comprises a surface configured to reflect approximately 5% or less of the incident light. Such a reference reflective surface comprises, for example, a glass / air interface of a glass slide (e.g., quartz). Of course, other reflective surfaces with higher reflection coefficients may be used, such as mirrors, for example.

[0029] According to one or more exemplary embodiments, the microscopy device further comprises an optical power attenuation element, arranged on the reference arm, making it possible to control the difference in optical power of the beams. light propagating in each of the arms of the interferometric microscope. The attenuating element is for example a neutral density filter. A chromatic dispersion compensating element, for example a glass slide, can then be arranged on the object arm to compensate for the dispersion introduced by the attenuating element.

[0030] According to one or more exemplary embodiments, a detector with a linear detection surface is a linear detector, for example a linear camera, of the CCD or CMOS type, comprising a plurality of elementary detectors (or “pixels”) arranged along one or more lines to form a one-dimensional detection surface of given width. A detector with a linear detection surface also comprises a two-dimensional detector of which only one or more lines of pixels are taken into account, for example of which only one or more lines of pixels are electronically activated.

[0031] According to one or more exemplary embodiments, the selection of the light backscattered by said object and coming from said focus line for one-dimensional confocal spatial filtering is obtained by means of said linear detection surface, optically conjugated with said focus line, for example by means of the first microscope objective and a tube lens arranged downstream of the interferometric microscope, and the width of which is adapted to the width of the image of the focus line on the detection surface. The linear detection surface is therefore part of the one-dimensional confocal spatial filtering device.

[0032] According to one or more exemplary embodiments, the selection of the light backscattered by said object and coming from said focus line for one-dimensional confocal spatial filtering is obtained by means of a spatial filtering element, for example a slit, optically conjugated with said focus line by the first microscope objective and a tube lens arranged downstream of the interferometric microscope. In this example, the dimensions of the spatial filtering element, for example the width of the slit, is adapted to the width of the image of the focus line in a plane of said spatial filtering element. The spatial filtering element is for example optically conjugated with the detection surface of the detector.

[0033] In either of the two cases respectively, the width of the linear detection surface or of the slit may be less than 10 times, advantageously less than 5 times the width of the image of the focusing line. For example, the width of the linear detection surface or of the slit may be substantially equal to the width of the image of the focusing line.

[0034] According to one or more exemplary embodiments, the one-dimensional confocal spatial filtering device comprises a cylindrical lens, arranged upstream of the interferometric microscope, and which cooperates with the broadband light source. spectral to form said line of focus in an object space of the first microscope objective. The broad spectral band light source is for example a spatially coherent light source, for example a supercontinuum laser or a superluminescent diode. Such a one-dimensional confocal spatial filtering device is advantageous in that it allows illumination of the object along a line with a satisfactory optical power, for example of the order of a few milliwatts. However, the one-dimensional confocal spatial filtering device may comprise, in other exemplary embodiments, a linear aperture arranged in a plane optically conjugated with a plane of the source, for example a slit, and an objective upstream of the interferometric microscope which cooperates with said first microscope objective to form said line of focus.

[0035] According to one or more exemplary embodiments, the unidirectional scanning device of the focusing line comprises a reflecting surface, for example a mirror, and means for rotating said reflecting surface along an axis of rotation substantially perpendicular to the optical axis of the first microscope objective.

[0036] The unidirectional focusing line scanning device is arranged on the object arm, upstream of the first microscope objective.

[0037] According to one or more exemplary embodiments, the reflective surface of the scanning device is optically conjugated with an entrance pupil of the first microscope objective to limit any vignetting effect.

[0038] According to other exemplary embodiments, the reflective surface of the scanning device is simply arranged in the vicinity of said first microscope objective to limit possible vignetting of the illumination beam at the entrance pupil of the first microscope objective and to limit vignetting of the light backscattered by the object. In particular, no optical element for the optical conjugation of the reflective surface of the scanning device and the entrance pupil of the first microscope objective is introduced, which makes it possible to promote the compactness of the device.

[0039] According to one or more exemplary embodiments, the processing unit comprises one or more physical entities, for example one or more computers. When in the present description, reference is made to calculation or processing steps for the implementation in particular of method steps, it is understood that each calculation or processing step can be implemented by software, hardware, firmware, microcode or any appropriate combination of these technologies. When software is used, each calculation or processing step can be implemented by computer program instructions or software code. These instructions can be stored or transmitted to a storage medium readable by the processing unit and / or or be executed by the processing unit in order to implement these calculation or processing steps. According to one or more exemplary embodiments, the processing unit is connected to a screen and / or interface for interfacing with a user.

[0040] According to one or more exemplary embodiments, a one-dimensional tomographic image is generated from a plurality of one-dimensional interferometric images by determining a dispersion of the values ​​between successive images of said plurality of images. The dispersion is determined on the plurality of successive one-dimensional interferometric images in parallel for each point. For example, determining said one-dimensional tomographic image comprises calculating the variance of said plurality of successive one-dimensional interferometric images. According to one or more exemplary embodiments, said plurality of one-dimensional interferometric images comprises between 2 and 20, advantageously between 2 and 10 one-dimensional interferometric images.

[0041] According to one or more exemplary embodiments, the processing unit is configured to: - producing a plurality of one-dimensional tomographic images of said object, each image of said plurality of one-dimensional tomographic images being produced from a plurality of one-dimensional interferometric images acquired by said detector for different positions of said line of focus during said one-way scan; and - producing said two-dimensional tomographic image in front of said object from a plurality of said one-dimensional tomographic images.

[0042] In these embodiments, a movement of the reference reflecting surface is not necessary for the acquisition of a one-dimensional tomographic image; in fact, due to the scanning of the focus line, and if the reflecting surface of the focus line scanning device is not optically conjugated with the entrance pupil of the first microscope objective, a plurality of one-dimensional interferometric images (or one-dimensional interferometric signals) can be produced by the interferometric microscope and acquired by the detector for a plurality of successive positions of the focus line in the object, the interferometric images of said plurality of interferometric images having at each point different interference states corresponding to different path differences between the light coming from the reference arm and the light coming from the object arm.From said plurality of one-dimensional interferometric images, it is possible to produce a one-dimensional tomographic image.

[0043] According to one or more exemplary embodiments, the microscopy device according to the first aspect comprises means for unidirectional movement of said surface. reference reflecting in an axial direction.

[0044] According to one or more exemplary embodiments, the processing unit is configured to: - producing a plurality of one-dimensional tomographic images of said object, each image of said plurality of one-dimensional tomographic images being produced from a plurality of one-dimensional interferometric images acquired by said detector for different positions of the reference reflecting surface; and - producing said two-dimensional tomographic image in front of said object from a plurality of said one-dimensional tomographic images.

[0045] In these exemplary embodiments, a one-dimensional tomographic image is produced from a plurality of one-dimensional interferometric images acquired by said detector for different positions of the reference reflecting surface, resulting in different path differences between the light from the reference arm and the light from the object arm, and therefore different interference states for the interferometric images of said plurality of one-dimensional interferometric images. In these exemplary embodiments, the displacement of the focus line during the production of a one-dimensional interferometric image is advantageously substantially zero or sufficiently small so as not to deteriorate the tomographic image thus produced.

[0046] According to one or more exemplary embodiments, said means for moving said reference reflecting surface are configured to further introduce an additional axial movement of said reference reflecting surface making it possible to compensate for a phase shift introduced by the focusing line scanning device.

[0047] The applicant has shown in fact that during unidirectional scanning of the focusing line for the production of a face image, the "coherence surface", that is to say the surface of zero path difference which is the actually observable surface, is not strictly flat but takes the form of a parabolic surface. This can result in a "blurred" face image. The introduction of an additional axial displacement of said reference reflecting surface then makes it possible to introduce a compensating phase shift in order to obtain a better superposition of the coherence surface and the focusing surface, and to produce a substantially flat face image with better sharpness.

[0048] According to one or more exemplary embodiments, the microscopy device according to the first aspect further comprises a reflecting return surface arranged in the reference arm and means for moving said reflecting return surface, in one and / or the other of an axial direction and a lateral direction.

[0049] According to one or more exemplary embodiments, said processing unit is configured to: - producing a plurality of one-dimensional tomographic images of said object, each image of said plurality of one-dimensional tomographic images being produced from a plurality of one-dimensional interferometric images acquired by said detector for different positions of said reflecting return surface; and - producing said two-dimensional tomographic image in front of said object from a plurality of said one-dimensional tomographic images.

[0050] Moving a reflecting return surface arranged in the reference arm of the interferometric microscope makes it possible to further save space in the microscopy device since it is thus possible to avoid moving the reflecting reference surface. Furthermore, in particular when the reflecting reference surface is in a focal plane of a microscope objective, the risk of variation in the luminous intensity of the light coming from the reference arm during a movement of said reflecting reference surface is limited, such a variation being able to result from impurities on the reflecting reference surface and being able to cause artifacts in the image.

[0051] According to one or more exemplary embodiments, for each image of said plurality of one-dimensional tomographic images, said means for moving said reflecting return surface are configured to further introduce an additional movement of said reflecting return surface making it possible to compensate for a phase shift introduced by the focusing line scanning device.

[0052] Of course, compensation for a phase shift introduced by the focusing line scanning device can also be obtained by means of an additional axial displacement of said reference reflecting surface even when the one-dimensional tomographic images are produced by acquiring a plurality of one-dimensional interferometric images for different positions of said reflecting return surface. Conversely, compensation for a phase shift introduced by the focusing line scanning device can be obtained by means of an additional displacement of said reflecting return surface even when the one-dimensional tomographic images are produced by acquiring a plurality of one-dimensional interferometric images for different positions of the reference reflecting surface.

[0053] According to one or more exemplary embodiments, the tomographic microscopy device according to the first aspect further comprises means for axial displacement of said focusing line, in a direction parallel to said optical axis of said first microscope objective for moving said focusing line deep into the object. The focusing surface is then moved deep into the object.

[0054] According to one or more exemplary embodiments, the processing unit is configured to further produce a three-dimensional image of said object to be observed, from a plurality of face images produced for different focusing surfaces.

[0055] According to one or more exemplary embodiments, said means for axial displacement of the focusing line comprise means for displacement integral with an assembly comprising said first microscope objective and said beam splitter, in a direction parallel to said optical axis of said first microscope objective. Such a configuration is advantageous in that it makes it possible to simultaneously and integrally move the coherence surface and the focusing surface, during axial scanning, while minimizing the weight to be moved.

[0056] Of course, other means of axial displacement of the focusing line can be envisaged.

[0057] For example, and in a non-limiting manner, said means for axial displacement of the focusing line may comprise means for displacement integral with an assembly comprising said first microscope objective and said unidirectional scanning device of the focusing line or with an assembly comprising said first microscope objective and said reflective return surface of the reference arm when it is present, or with an assembly comprising all the elements of said interferometric microscope.

[0058] In the examples cited above, it will be possible to choose for said first microscope objective and said second microscope objective if it is present, an immersion microscope objective. Indeed, in these examples, the movements of the coherence surface and the focusing surface are integral; the adaptation of the refractive index of the object of study with that of the medium in which the first microscope objective is immersed makes it possible to keep them merged.

[0059] According to other embodiments, said means for axial displacement of the focusing line may comprise means for independent displacement of the first microscope objective on the one hand and of the assembly formed by the second microscope objective, when it is present, with the reference reflecting surface, on the other hand, said means for axial displacement of the focusing line may also comprise means for independent displacement of the first microscope objective on the one hand and of a return reflecting surface arranged on the reference arm, on the other hand.

[0060] In these examples, the movements of the coherence surface and the focusing surface are separated and the two planes can be moved by a different amount. so as to keep them confused.

[0061] According to a second aspect, the present description relates to a method of linear scanning optical coherence tomographic microscopy, implemented by a device according to the first aspect.

[0062] According to one or more exemplary embodiments, said method according to the second aspect comprises: - the formation of an illumination beam by means of a broad spectral band light source; - separating the illumination beam, by means of a beam splitter, into a first fraction of light and a second fraction of light, said first fraction of light being sent into a reference arm at the end of which is arranged a reference reflecting surface, said second fraction of light being sent into an object arm receiving an object to be imaged; - focusing said second fraction of light, by means of a first microscope objective arranged on said object arm and cooperating with a confocal spatial filtering device, to illuminate said object to be imaged along a focusing line located in an object space of said microscope objective; - selecting, by means of said confocal spatial filtering device, the light backscattered by the object and coming from said focus line to form a one-dimensional image of said focus line on a linear detection surface of a detector; - combining, by means of said beam splitter, the light backscattered by the object thus selected and the light resulting from the reflection by the reflective surface of the reference arm of said first fraction of light, to generate a one-dimensional interferometric image; - unidirectional scanning of said focusing line in a lateral direction substantially perpendicular to an optical axis of said first microscope objective, by means of a scanning device arranged on said object arm upstream of said first microscope objective; - producing, by means of a processing unit, at least one two-dimensional tomographic image in front of said object to be observed, arranged in a plane substantially perpendicular to said optical axis of the microscope objective, from a plurality of one-dimensional interferometric images acquired by said detector for different positions of said line of focus during said one-way scanning.

[0063] According to one or more exemplary embodiments, the production of said at least one two-dimensional tomographic image in front of said object comprises: - the production of a plurality of one-dimensional tomographic images of said object, each image of said plurality of one-dimensional tomographic images being produced from a plurality of one-dimensional interferometric images acquired by said detector for different positions of said line of focus during said one-way scan; and - producing said two-dimensional tomographic image in front of said object from a plurality of said one-dimensional tomographic images.

[0064] According to one or more exemplary embodiments, the production of said at least one two-dimensional tomographic image in front of said object comprises: - producing a plurality of one-dimensional tomographic images of said object, each image of said plurality of one-dimensional tomographic images being produced from a plurality of one-dimensional interferometric images acquired by said detector for different positions of the reference reflecting surface or for different positions of a return reflecting surface arranged in the reference arm; and - producing said two-dimensional tomographic image in front of said object from a plurality of said one-dimensional tomographic images.

[0065] According to one or more exemplary embodiments, the method further comprises, for each image of said plurality of one-dimensional tomographic images, the introduction of an additional axial displacement of said reference reflecting surface or the introduction of an additional displacement of said return reflecting surface, to compensate for a phase shift introduced by the focusing line scanning device.

[0066] According to one or more exemplary embodiments, the method further comprises: - the axial displacement of said focusing line in depth in the object, in a direction parallel to said optical axis of said first microscope objective; - the production of a three-dimensional image of said object to be observed, from a plurality of face images produced for different depths of the line of focus. Brief description of the figures

[0067] Other advantages and characteristics of the invention will appear on reading the description, illustrated by the following figures:

[0068] [fig. 1], already described, represents a diagram illustrating a linear scanning optical coherence tomographic microscopy (LS-OCM) device for an object, known from the state of the art;

[0069] [fig.2] represents a diagram illustrating an example of a tomo microscopy device linear scanning optical coherence graph, according to the present description;

[0070] [fig.3A] illustrates, according to a first view, the operation of filtering means one-dimensional confocal spatial in a line-scanning optical coherence tomographic microscopy device, according to an example of the present disclosure;

[0071] [fig.3B] illustrates one-dimensional confocal spatial filtering means as illustrated in Fig. 3A, according to a second view;

[0072] [fig.4A] represents a diagram illustrating an example of a linear scanning optical coherence tomographic microscopy device, according to the present description;

[0073] [fig.4B] represents a diagram illustrating an example of a linear scanning optical coherence tomographic microscopy device, according to the present description;

[0074] [fig.4C] represents a diagram illustrating an example of a linear scanning optical coherence tomographic microscopy device, according to the present description;

[0075] [fig.5A] represents a diagram illustrating according to an example the unidirectional scanning of the focusing line, as a function of time;

[0076] [fig.5B] represents a diagram illustrating, according to an example, a section of the coherence surface when the focus line is moved laterally according to the scan illustrated in Fig. 5A and the coherence surface when the focus line is moved laterally according to the scan illustrated in Fig. 5A, with compensation by means of a variation in the axial position of the reference reflecting surface, also represented in Fig.5B;

[0077] [fig.5C] represents a diagram illustrating, according to an example, a section of the coherence surface when the focus line is moved laterally according to the scan illustrated in Fig. 5A and the coherence surface when the focus line is moved laterally according to the scan illustrated in Fig. 5A, with step compensation by means of a variation in the axial position of the reference reflecting surface, also represented in Fig.5C;

[0078] [fig.6A] represents a diagram illustrating according to an example the coherence surface in an example of a linear scanning optical coherence tomographic microscopy device according to the present description, without compensation;

[0079] [fig.6B] represents a diagram illustrating according to an example the coherence surface in an example of a linear scanning optical coherence tomographic microscopy device according to the present description, with compensation;

[0080] [fig.7] represents images of a sample obtained by means of an example of method according to the present description, with and without compensation. Detailed description of the invention

[0081] In the following detailed description, numerous specific details are set forth in order to provide a more thorough understanding of the present disclosure. However, it will be apparent to those skilled in the art that the present disclosure may be implemented without these specific details. In other instances, well-defined features known have not been described in detail to avoid unnecessarily complicating the description.

[0082] Furthermore, in the figures, the elements are not shown to scale for better visibility.

[0083] Fig. 2 represents a diagram illustrating an example of a linear scanning optical coherence tomographic microscopy device, according to the present description.

[0084] The tomographic microscopy device 200 shown in Fig. 2 comprises a broad spectral band light source 210, a detector 222 with a linear detection surface 223, for example a linear camera of the CCD or CMOS type, a processing unit 240 configured in particular to process signals from the detector 222 and an interferometric microscope 201.

[0085] The interferometric microscope 201 comprises in particular a reference arm at the end of which is arranged a reference reflecting surface 207, an object arm configured to receive an object to be imaged 10, a beam splitter 204 coupling the object and reference arms to the light source 210 and to the detector, and at least one first microscope objective 202 arranged on said object arm. The interferometric microscope is for example a Linnik type microscope, as illustrated in Fig. 2, with a second microscope objective 203 identical to the first microscope objective 202, arranged on the reference arm and in the focal plane of which is the reference reflecting surface 207. In the example of Fig. 2, the microscope objectives 202, 203 are immersion objectives, immersed in a liquid of index 205, 206, that is to say a liquid whose refractive index is adapted to the refractive index of the object 10.The interferometric microscope 201 may further comprise, in a known manner, an optical power attenuation element (not shown), arranged on the object arm and / or a chromatic dispersion compensation element (not shown) arranged on one and / or the other of said object and reference arms.

[0086] The tomographic microscopy device 200 further comprises a one-dimensional confocal spatial filtering device, comprising in particular a cylindrical lens 212. The confocal filtering device is configured to cooperate with the light source to illuminate the object 10 along a line of focus located in an object space of the first microscope objective (here 1 “object space” is defined from the imaging point of view, i.e. the space of the microscope objective in which, in operation, the object of study is located). It is also configured to select the light backscattered by the object and coming from said line of focus to form a one-dimensional image of the line of focus on the detection surface 223. Examples of one-dimensional confocal spatial filtering device will be described in more detail with reference to Figs 3A and 3B.

[0087] In the example of Fig. 2, the optical axis of the first microscope objective 202, defined by a line passing through the center of a pupil of the objective and substantially perpendicular to the plane of said pupil, is referenced by a z axis in the space of the object 10. The x and y axes correspond, in the example of Fig. 2, to two lateral directions in the space of the object 10, that is to say two directions orthogonal to each other and perpendicular to the optical axis z of the first microscope objective 202. In the space of the reference reflecting surface, zr denotes the axial direction, that is to say the direction collinear with the optical axis of the first microscope objective, considered in this space, and xr, yr, the lateral directions.

[0088] In an interferometric microscope of a device according to the present description, due to the low temporal coherence length of the broad spectral band source, interference between the light reflected by the reference reflecting surface 207 and that backscattered by the object 10 only occurs when the optical paths in the object and reference arms are equal, with a tolerance equal to the coherence length of the source. In the present description, the virtual slice in the object for which interference can occur is called a "coherence surface" (or "zero path difference surface"). The coherence surface is the surface that can actually be observed. The interference signal resulting from the acquisition by the detector of the interference formed on the detection surface is called an "interferometric image".Thus, each point of an interferometric image has a given "interference state", i.e. a path difference between the light coming from the object and reference arms for said point less than the coherence length. In the present description, the interferometric image is one-dimensional due to the linear confocal illumination and detection. The light backscattered by the object on either side of the coherence surface contributes to creating a constant light background in the interference signal. By modifying the path difference between the two arms of the interferometric microscope, according to means which will be described below, the interference states of the points can be modulated between successive images. A combination of the one-dimensional interferometric images acquired by the detector then makes it possible to form a one-dimensional tomographic image.

[0089] In practice, when the path difference between successive one-dimensional interferometric images is generated by displacement according to a known law of the reference reflecting surface for example, the one-dimensional tomographic image can be determined from said interferometric images based on this law.

[0090] For example, a simple approach for the formation of the one-dimensional tomographic image is based on the use of a method called interferometry at phase shift, consisting of digitally combining several one-dimensional interferometric images that are out of phase. For example, four one-dimensional interferometric images corresponding to positions of the line of focus spaced X / 8n in the axial direction can be combined, / being the central wavelength of the illuminating light in the object and n the index in which the first microscope objective is immersed. This corresponds to a phase shift of ir / 2 between two adjacent images. If we denote by Eb E2, E3, E4 these images, (Er E3)2 + (E2- E4)2 corresponds to the amplitude of the interference signal - that is to say to the amplitude of the reconstructed image - and (Er E3) / (E2- E4) corresponds to the phase of the interference signal. This phase can provide other information than structural and tomographic information on the object.It should be noted that there is no contradiction between the notion of phase shift, or phase shift, and the fact, mentioned above, that the line of focus always corresponds to a difference in optical path between the object and reference arms equal to zero. Indeed, any structure of the object, likely to backscatter light, is not observed only when it coincides with the line of focus, but also before and after (because the coherence "gate" and that introduced by confocal filtering have a width greater than X). There is therefore a phase shift between the contributions of this structure to the images acquired successively during the axial scan.

[0091] Alternatively, an algorithm suitable for sinusoidal phase shift between successive images may also be used (in the case where the reference surface is sinusoidally modulated). For example, a plurality of one-dimensional interferometric images may be processed by Fourier analysis in order to extract the envelope of the interference fringes (the amplitude of the interference signal) and eliminate the unmodulated part of the signal (non-interferometric signal).

[0092] Generally speaking, the applicant has shown that it is possible to generate a one-dimensional tomographic image from a plurality of one-dimensional interferometric images by determining the dispersion between the successive images of said plurality of one-dimensional interferometric images. It is then not necessary to know precisely the law of variation of the path difference between the successive one-dimensional interferometric images. For example, to generate a one-dimensional tomographic image, it is possible to calculate the variance of N successive one-dimensional interferometric images, N being for example between 2 and 20, advantageously between 2 and 10, for example around 5.

[0093] The broad spectral band source 210 comprises for example a supercontinuum laser, the coherence length typically being between 1 and 5 pm.

[0094] The tomographic microscopy device 200 further comprises a device 235, 237 for unidirectional scanning of said focusing line, arranged on the arm object upstream of the first microscope objective 202. The unidirectional scanning device comprises, for example, and in a non-limiting manner, a reflective surface 235 and means 237 for rotating the reflective surface, along an axis of rotation perpendicular to the optical axis of the first microscope objective. The scanning device is, for example, formed of a galvanometric mirror. The unidirectional scanning device of the focus line is configured for scanning the focus line along a lateral direction (referenced y in the example of Fig. 2) substantially perpendicular to the optical axis z of said first microscope objective.

[0095] It is then possible, according to the present description, to produce, by means of the processing unit 240, at least one two-dimensional tomographic image in front of the object 10, that is to say a two-dimensional tomographic image arranged in an x, y plane substantially perpendicular to the optical axis of the microscope objective, from a plurality of one-dimensional interferometric images of the object acquired by the detector for different positions of said line of focus during the unidirectional scanning.

[0096] Due to the positioning of the unidirectional scanning device of the focusing line in the lower object of the interferometric microscope 201, the size of the device 200 for the acquisition of the face image is reduced.

[0097] According to an exemplary embodiment, the reflective surface of the focusing line scanning device is optically conjugated with the entrance pupil of the first microscope objective 202, for example by means of an optical lens. This makes it possible to avoid vignetting of the illumination beam at the entrance pupil of the first microscope objective as well as vignetting of the light backscattered by the object. However, this configuration complicates the assembly and increases the size of the device. It is therefore preferred, as illustrated in FIG. 2, to position the reflective surface of the scanning device upstream of the first microscope objective 202, close to the first microscope objective to limit vignetting, i.e. as close as possible to the pupil of the objective as is accessible given the mechanical constraints of integration of the scanning device.

[0098] The processing unit 240 is generally configured for implementing steps of the method according to the present description. The processing unit 240 may be connected to a screen and / or interface (not shown in Fig. 2) for interfacing with a user. The processing unit may also comprise means for controlling the rotation means 237 of the reflective surface and / or means for controlling other moving elements of the tomographic microscopy device according to the present description.

[0099] Figs. 3A and 3B illustrate according to two perpendicular sectional planes (respectively the yz and xz planes containing the optical axis z of the microscope objective 202), the one-dimensional confocal spatial filtering in a linear scanning optical coherence tomographic microscopy device, according to an example of the present description. In these figures, only the elements of the tomographic microscopy device useful for understanding the one-dimensional confocal spatial filtering are shown.

[0100] In this example, the one-dimensional confocal spatial filtering device comprises a cylindrical lens 212, arranged upstream of the interferometric microscope, and which cooperates with the broad spectral band light source to form a focus line 301 in the object space of the first microscope objective 202. When scanning the focus line, the focus line moves on a focus surface, substantially merged with a focusing surface. Such a focus surface is generally planar, substantially merged with a focal plane of the first microscope objective 202 if the beam incident on said first microscope objective is collimated. The broad spectral band light source is for example a spatially coherent light source, for example a supercontinuum laser.Such a one-dimensional confocal spatial filtering device is advantageous in that it allows illumination of the object with satisfactory optical power.

[0101] The selection of the light backscattered by the object 10 and coming from said focus line 301 for the one-dimensional confocal spatial filtering is further achieved in this example by means of a linear detection surface 323 of the detector 322, optically conjugated with the focus line by means of the first microscope objective 202 and a tube lens 221 arranged downstream of the interferometric microscope. The width of the detection surface 323 is determined as a function of the width of the image of the focus line on the detection surface. The linear detection surface thus behaves like a filtering slit of given width, and is therefore part of the one-dimensional confocal spatial filtering device.It should be noted that the selection of the light backscattered by the object 10 and coming from said focusing line 301 can also be obtained by a filtering slit separate from the detection surface (not shown in the example of Figs 3A, 3B), optically conjugated with the focusing line 301 and the detection surface 323.

[0102] Thus, in the yz plane (Fig. 3A) perpendicular to the filtering slit formed by the detection surface 323, only the light coming from the region of the object where the focusing line 301 is formed by the cylindrical lens 212 and the first microscope objective 202 (beam shown in a continuous line) is detected by the detector 222 while the light coming from other regions 302, 303 of the object (beams in dotted lines for example) is very strongly attenuated. In a plane parallel to the filtering slot (xz plane in Fig. 3B), such filtering does not occur.

[0103] Confocal spatial filtering is complementary to the “coherent” filtering resulting from low coherence length interferometry with a broad spectral band source, the principle of which is recalled above. In particular, confocal filtering improves the performance of interferometric imaging by eliminating the “background” produced by the scattering of light from areas of the sample located outside the focusing plane and by parasitic reflections. The entire dynamic range of the detector is thus used to detect the useful interferometric signal and the signal-to-noise ratio is improved. Furthermore, interferometry – even at low coherence length – does not make it possible to distinguish between a ballistic photon coming from the imaged region in the object and a photon coming from other regions of the object and having traveled an optical path of the same length due to the scattering undergone.This results in a spurious interferometric signal that is added to the useful interferometric signal, creating artifacts in the images and limiting the accessible imaging depth. In the device according to the present description, the confocal filtering only allows ballistic photons to pass, eliminating this background.

[0104] Compared to confocal filtering alone, the use of interferometric detection allows a considerable amplification of the useful signal (in the case of "pure" confocal microscopy, it is the low signal-to-noise ratio which limits the acquisition depth). In the device according to the present description, there is therefore synergy - and not simple juxtaposition - between the two principles involved: low coherence length interferometric microscopy and confocal slit microscopy.

[0105] Figs 4A, 4B, 4C illustrate three examples of tomographic microscopy devices for implementing methods according to the present description.

[0106] In these diagrams, only the elements of the device necessary for understanding the method according to the present description are illustrated. In particular, the lighting means and the confocal spatial filtering device as well as the processing unit are not illustrated. Although certain exemplary embodiments are only illustrated in some of the diagrams, the different embodiments described above can be combined.

[0107] In these figures, elements identical to those illustrated in the example of Fig. 2 are indicated with the same references and are not described again.

[0108] FIG.4A illustrates a first example of a device in which the interferometric microscope comprises only one microscope objective, namely the first microscope objective 202. The object arm comprises an element 403 for compensating for chromatic dispersion, for example a glass slide.

[0109] In this example, the reference reflective surface 207 is fixed.

[0110] Each one-dimensional tomographic image is produced in this example at from a plurality of one-dimensional interferometric images acquired by the detector for different positions of said focus line during the unidirectional scanning. This is made possible by the fact that the reflective surface of the focus line scanning device is not optically conjugated with the entrance pupil of the first microscope objective. Thus, for a plurality of different successive positions of the focus line resulting from the scanning of observation lines, the points of successive interferometric images acquired by the detector have different interference states corresponding to different path differences (or phase shifts) between the light coming from the reference arm and the light coming from the object arm. From the plurality of one-dimensional interferometric images, it is possible to produce a one-dimensional tomographic image.

[0111] In this example, the law of variation of path difference between two interferometric image acquisitions is not known in as deterministic a manner as when, for example, the reference reflecting surface is moved.

[0112] The applicant has shown, however, that one-dimensional tomographic images can be generated from a plurality of one-dimensional interferometric images by determining the dispersion between successive images, as described above.

[0113] FIG.4B illustrates a second example of a device in which the interferometric microscope is of the Linnik type and comprises, in addition to the first microscope objective 202 in the object arm, a second microscope objective 203 in the reference arm. Advantageously, the two microscope objectives are identical.

[0114] In this example, the reference reflecting surface 207 is movable in translation in an axial direction zr thanks to unidirectional displacement means of the reference reflecting surface shown diagrammatically in Fig. 4B by a double arrow 405, these means comprising for example a piezoelectric motor.

[0115] In this example, each one-dimensional tomographic image may be produced from a plurality of one-dimensional interferometric images acquired by the detector for different positions of the reference reflecting surface, resulting in different path differences between the light from the reference arm and the light from the object arm, and therefore different interference states for the different one-dimensional interferometric images. In these exemplary embodiments, the displacement of the focus line during the production of a one-dimensional interferometric image is advantageously substantially or sufficiently small so as not to degrade the one-dimensional tomographic image thus generated.

[0116] FIG.4C again illustrates an example of a device in which the microscope in- terferometric is of the Linnik type.

[0117] In this example, the reference reflective surface 207 is fixed.

[0118] The interferometric microscope comprises in this example a reflecting return surface 402 arranged in the reference arm and means 406, 408 for moving said reflecting return surface, in one and / or the other of an axial direction zr and a lateral direction (yr, in the example of Fig. 4C). The moving means 406, 408 comprise for example one or two piezoelectric actuators.

[0119] It is possible by means of this particular arrangement to obtain each one-dimensional tomographic image from a plurality of one-dimensional interferometric images acquired by the detector for different positions of the reflecting return surface.

[0120] Moving the reflecting return surface 402 makes it possible to acquire a one-dimensional tomographic image without having to oscillate the reference surface 207. This has, for example, the advantage of limiting the risk of variation in the light intensity of the light coming from the reference arm during a movement of the reflecting reference surface, in particular when the reference surface is in a focal plane of the second microscope objective 203, as illustrated in Fig. 4C, such a variation possibly resulting from impurities on the reflecting reference surface.

[0121] Furthermore, by eliminating the movement of the reference reflecting surface 207, the size of the device can be further limited.

[0122] In the examples illustrated in Figs 4A to 4C, the tomographic microscopy device further comprises means for axially moving the focusing line, in a direction parallel to the optical axis z of said first microscope objective 202. This involves moving the focusing surface to image a plurality of images in front of the object 10, according to a method according to the present description, at different depths. A three-dimensional image of the object to be observed can then be produced from the plurality of facing images.

[0123] As illustrated in Figs. 4A to 4C, the means for axially moving the focusing line may comprise means for moving integrally with an assembly 209 comprising the first microscope objective 202 and the beam splitter 204, in a direction parallel to the optical axis z of the first microscope objective 202. Such a configuration is advantageous in that it makes it possible to simultaneously and integrally move the coherence surface and the focusing surface, during the axial scanning, while minimizing the weight to be moved. This makes it possible to maximize the speed of movement and thus the acquisition.

[0124] Of course, other means of axial displacement of the focusing line can be envisaged, not shown in the figures.

[0125] For example, and in a non-limiting manner, the means for axial displacement of the focusing line may comprise means for displacement integral with an assembly comprising the first microscope objective 202 and the scanning device 235, 237 of the focusing line or with an assembly comprising the first microscope objective 202 and the reflecting return surface 402 of the reference arm when it is present (Fig. 4C), or with an assembly comprising all the elements of the interferometric microscope 201.

[0126] In the examples cited above, an immersion microscope objective will advantageously be chosen for the first microscope objective 202 and the second microscope objective 203 if present. Indeed, in these examples, the movements of the coherence surface and the focusing surface are integral; the adaptation of the refractive index of the study object 10 with that of the medium in which the first microscope objective is immersed makes it possible to keep them coincident.

[0127] According to other examples (not shown in the figures), the means for axially moving the focus line may comprise means for moving independently of the first microscope objective 202 on the one hand and of the assembly formed by the second microscope objective 203, when present, with the reference reflecting surface 207, on the other hand. The means for axially moving the focus line may also comprise means for moving independently of the first microscope objective 202 on the one hand and of the return reflecting surface 402 arranged on the reference arm, on the other hand (Fig. 4C).

[0128] In these examples, the movements of the coherence surface and the focusing surface are separate and the two planes can be moved by a different amount so as to keep them merged.

[0129] The applicant has also shown that, in the case in particular where, as illustrated in Figs. 4A to 4C, the reflective surface 235 of the unidirectional scanning device of the focusing line is not optically conjugated with the entrance pupil of the microscope objective 202, a path difference (or phase shift) can be introduced during scanning, which results in a deformation of the surface of zero path difference (coherence surface) compared to a perfectly flat surface. As a result, the face image formed cannot be perfectly sharp over the entire field, because the focusing surface is itself flat, and does not have a large depth of field due to the use of objectives with a large numerical aperture (typically between 0.3 and 0.8).

[0130] To compensate for the phase shift introduced by the focusing line scanning device and to make the coherence surface and the focusing surface coincide, the microscopy method according to the present description may further comprise, for each image of the plurality of one-dimensional tomographic images, the introduction of a additional axial displacement of the reference reflecting surface 207 (Fig. 2) or of said return reflecting surface 402 (Fig. 4C).

[0131] Figs. 5A to 5C thus represent diagrams showing, according to one example, the unidirectional scanning of the focusing line, as a function of time (Fig. 5A), and diagrams (Figs. 5B, 5C) showing, according to two examples, sections of the coherence surface when the focusing line is moved laterally according to the scanning illustrated in Fig. 5A, with or without compensation.

[0132] Figs. 6A and 6B show diagrams illustrating, according to an example, the coherence (610) and focusing (612) planes in the sample, with and without compensation. In the example of Figs. 6A, 6B, the reference reflecting surface 207 is moved, but the desired effect would be the same if the return reflecting surface were moved.

[0133] More specifically, in the example of Fig. 5B, the coherence surface, when the focus line is moved laterally according to the scan illustrated in Fig. 5A, is represented by curve 510. The zero path difference position is represented at 100 pm in depth for a focus line position y=0. As illustrated in Fig. 5B, the coherence surface is substantially parabolic when no compensating phase shift is applied. With compensation, for example by means of a variation in the axial position of the reference reflecting surface represented in Fig.5B by curve 512, the coherence surface, illustrated by curve 514, is substantially planar and coincides with the focus surface.

[0134] As illustrated in Figs. 6A, 6B, the reference reflecting surface 207 is dynamically controlled, for example by means of a piezoelectric transducer 405, so as to introduce a phase shift during the movement of the scanning mirror 235 making it possible to maintain the position of the zero path difference of the interferometer at the level of the object (curve 610) in a plane 612 substantially perpendicular to the optical axis of the microscope objective 202. Figs. 6A, 6B represent in particular three positions (1, 2, 3) of the scanning mirror, associated with three positions of the reference surface for the correction of the flatness of the coherence surface (zero path difference surface).

[0135] The piezoelectric transducer 405 can follow a law whose frequency is equal to that of the scanning mirror 235, and whose amplitude and shape are calibrated according to the amplitude and shape of the deviation of the zero path difference position with respect to the plane perpendicular to the optical axis of the object objective, as a function of the position of the focusing line. This deviation can be evaluated beforehand in order to apply a law adapted to the piezoelectric transducer. This deviation is independent of the sample, and only dependent on the position of the scanning mirror 235 with respect to the microscope objective 202. Once the adapted law has been determined, theoretically or empirically, it therefore no longer needs to be adjusted.

[0136] As illustrated in Fig. 5C, in order to simplify the control and adjustment of the piezoelectric transducer 207, it is also possible to introduce a phase shift 520 varying step by step (control of the piezoelectric transducer by step), with several operating points during the movement of the scanning mirror 237. These operating points are chosen so as to maintain the interferometric probing in a parallelepiped 524 whose height (along the optical axis of the microscope objective 202) is considered acceptable from the point of view of the sharpness of the images (therefore in relation to the depth of field of the microscope objective).

[0137] Fig. 7 shows an in vivo image 710 of a sample, the image being obtained by means of an exemplary method according to the present description, without compensation. The image 710 is obtained with a sample formed from the back of the hand of a 25-year-old man, on a field of 1.2 x 1.2 mm2, with scanning of the scanning mirror 237 at a frequency of 7 Hz.

[0138] In regions 712 and 714 of image 710, the stratum granulosum (with cells) can be observed, and in region 716, the stratum corneum (without cells) can be observed, although these layers are physically at different depths. This image clearly illustrates that the coherence surface, which is the surface that can actually be observed, is not necessarily perfectly flat.

[0139] Compensation makes it possible to isolate these regions.

[0140] Thus, images 720 and 730 correspond to images of the same field, obtained with compensation to make the coherence surface coincide with the focusing surface at two depths respectively. The two imaged areas are thus the stratum corneum 720 and the stratum granulosum 730.

[0141] Compensation is obtained with oscillation of the reference surface (glass slide) at 7 Hz, according to a law similar to that shown in Fig. 5B (512). One-dimensional tomographic images are obtained by adding a high-frequency sinusoidal oscillation (10 kHz) to the compensation signal.

[0142] Although described through a certain number of exemplary embodiments, the method and device for linear scanning optical coherence tomographic microscopy according to the present description includes various variants, modifications and improvements which will be obvious to those skilled in the art, it being understood that these various variants, modifications and improvements are part of the scope of the invention as defined by the following claims. References

[0143] Ref L: D. Huang et al., “Optical coherence tomography” Science 254(5035), 1178-1181(1991)

[0144] Ref. 2: AF Fercher et al. “Optical coherence tomography - principles and applications”, Reports on Progress in Physics 66 (2003) 239 - 303

[0145] Ref. 3: JA Izatt, et al., “Optical coherence microscopy in scattering media”, OPTICS LETTERS / Vol. 19, No. 8 / April 15 (1994)

[0146] Ref 4.: Y. Chen et al. “High-resolution line-scanning optical coherence microscopy”, Optics Letters, Vol. 32, No. 14, 1971 - 1973 (2007)

Claims

Claims

1. Device (200) for linear scanning optical coherence tomographic microscopy, comprising: - a broad spectral band light source (210); - a detector (222) with a linear detection surface (223); - an interferometric microscope (201) comprising a reference arm at the end of which is arranged a reference reflecting surface (207), an object arm configured to receive an object to be imaged (10), a beam splitter (204) coupling said object and reference arms to said light source and said detector, and at least one first microscope objective (202) arranged on said object arm; - a one-dimensional confocal spatial filtering device (212, 223) configured to cooperate with said light source to illuminate said object along a line of focus located in an object space of said first microscope objective, and configured to select the light backscattered by said object and coming from said line of focus to form a one-dimensional image of said line of focus on the detection surface; - a unidirectional scanning device (235, 237) of said focus line, arranged on said object arm upstream of said first microscope objective, and configured for scanning the focus line in a lateral direction (y) substantially perpendicular to an optical axis (z) of said first microscope objective; - a processing unit (240) configured to produce at least one two-dimensional tomographic image in front of said object to be observed, arranged in a plane (x, y) substantially perpendicular to said optical axis of the microscope objective, from a plurality of one-dimensional interferometric images of the object produced by the interferometric microscope for different positions of said line of focus during said unidirectional scan and acquired by said detector.

2. A microscopy device according to claim 1, wherein: - said processing unit is configured to: - producing a plurality of one-dimensional tomographic images of said object, each image of said plurality of one-dimensional tomographic images being produced from a plurality of one-dimensional interferometric images acquired by said detector for dif- different positions of said focus line during said unidirectional scan; and - producing said two-dimensional tomographic image in front of said object from said plurality of one-dimensional tomographic images.

3. Microscopy device according to claim 1, further comprising: - unidirectional displacement means (401) of said reference reflecting surface in an axial direction (zr); and wherein: - said processing unit is configured to: - produce a plurality of one-dimensional tomographic images of said object, each image of said plurality of one-dimensional tomographic images being produced from a plurality of one-dimensional interferometric images acquired by said detector for different positions of the reference reflecting surface; and - produce said two-dimensional tomographic image in front of said object from said plurality of one-dimensional tomographic images.

4. Microscopy device according to claim 3, wherein: - for each image of said plurality of one-dimensional tomographic images, said means for moving said reference reflecting surface are configured to further introduce an additional axial displacement of said reference reflecting surface making it possible to compensate for a phase shift introduced by the focusing line scanning device.

5. A microscopy device according to claim 1, further comprising: - a reflecting return surface (402) arranged in the reference arm and means (406, 408) for moving said reflecting return surface, in one and / or the other of an axial direction (zr) and a lateral direction (yr); and wherein: - said processing unit is configured to: - produce a plurality of one-dimensional tomographic images of said object, each image of said plurality of one-dimensional tomographic images being produced from a plurality of one-dimensional interferometric images acquired by said detector for different positions of said reflecting return surface; and - produce said two-dimensional tomographic image opposite said object from said plurality of one-dimensional tomographic images.

6. Microscopy device according to claim 5, wherein: - for each image of said plurality of one-dimensional tomographic images, said means for moving said reflecting return surface are configured to further introduce an additional movement of said reflecting return surface making it possible to compensate for a phase shift introduced by the focusing line scanning device.

7. A microscopy device according to any preceding claim, further comprising a chromatic dispersion compensation device (403) arranged on one and / or the other of said object and reference arms.

8. A microscopy device according to any preceding claim, wherein said interferometric microscope is a Linnik microscope, further comprising a second microscope objective (203), arranged on said reference arm, said reference and object arms being disjoint.

9. Microscopy device according to any one of the preceding claims, further comprising: - means for axial displacement of said line of focus, in a direction parallel to said optical axis of said first microscope objective for displacement of said line of focus in depth in the object; and wherein: - the processing unit is configured to further produce a three-dimensional image of said object to be observed, from a plurality of face images produced for different depths of the line of focus.

10. Microscopy device according to claim 9, wherein: - said means for axial displacement of the focusing line comprise means for integral displacement (208) of an assembly (209) comprising said first microscope objective and said beam splitter, in a direction parallel to said optical axis of said first microscope objective.

11. A method of linear scanning optical coherence tomographic microscopy, comprising: - forming an illumination beam by means of a broad spectral band light source (210); - separating the illumination beam, by means of a beam splitter (204), into a first fraction of light and a second fraction of light, said first fraction of light being sent into a reference arm at the end of which is arranged a reference reflecting surface (207), said second fraction of light being sent into an object arm receiving an object to be imaged (10); - focusing said second fraction of light, by means of a first microscope objective (202) arranged on said object arm and cooperating with a confocal spatial filtering device, to illuminate said object to be imaged along a focusing line located in an object space of said microscope objective; - selecting, by means of said confocal spatial filtering device, the light backscattered by the object and coming from said focus line to form a one-dimensional image of said focus line on a linear detection surface (223) of a detector (222); - combining, by means of said beam splitter, the light backscattered by the object thus selected and the light resulting from the reflection by the reflective surface of the reference arm of said first fraction of light, to generate a one-dimensional interferometric image; - unidirectional scanning of said focus line in a lateral direction (y) substantially perpendicular to an optical axis (z) of said first microscope objective, by means of a scanning device (235, 237) arranged on said object arm upstream of said first microscope objective; - producing, by means of a processing unit (240), at least one two-dimensional tomographic image in front of said object to be observed, arranged in a plane (x, y) substantially perpendicular to said optical axis of the microscope objective, from a plurality of one-dimensional interferometric images acquired by said detector for different positions of said line of focus during said one-way scanning.

12. A microscopy method according to claim 11, wherein producing said at least one two-dimensional tomographic image in front of said object comprises: - producing a plurality of one-dimensional tomographic images of said object, each image of said plurality of tomo- one-dimensional graphics being produced from a plurality of one-dimensional interferometric images acquired by said detector for different positions of said line of focus during said one-way scan; and - producing said two-dimensional tomographic image in front of said object from said plurality of one-dimensional tomographic images.

13. A microscopy method according to claim 11, wherein producing said at least one two-dimensional tomographic image in front of said object comprises: - producing a plurality of one-dimensional tomographic images of said object, each image of said plurality of one-dimensional tomographic images being produced from a plurality of one-dimensional interferometric images acquired by said detector for different positions of the reference reflecting surface (207) or for different positions of a return reflecting surface (402) arranged in the reference arm; and - producing said two-dimensional tomographic image in front of said object from said plurality of one-dimensional tomographic images.

14. A microscopy method according to claim 13, further comprising: - for each image of said plurality of one-dimensional tomographic images, introducing an additional axial displacement of said reference reflecting surface or said return reflecting surface, making it possible to compensate for a phase shift introduced by the focusing line scanning device.

15. A microscopy method according to any one of claims 11 to 14, further comprising: - axially moving said line of focus in depth into the object, in a direction parallel to said optical axis of said first microscope objective; - producing a three-dimensional image of said object to be observed, from a plurality of face images produced for different depths of the line of focus.

Images