EP3850439A1

EP3850439A1 - Off-axis transmission digital holographic imaging method, microscope and assembly

Info

Publication number: EP3850439A1
Application number: EP19774071.5A
Authority: EP
Inventors: Michel Gross; Daniel ALEXANDRE
Original assignee: Centre National de la Recherche Scientifique CNRS; Universite de Montpellier I; Universite de Montpellier
Current assignee: Centre National de la Recherche Scientifique CNRS; Universite de Montpellier I; Universite de Montpellier
Priority date: 2018-09-13
Filing date: 2019-09-13
Publication date: 2021-07-21
Also published as: FR3086071A1; WO2020053433A1; FR3086071B1

Abstract

The invention relates to a method (800) for off-axis transmission digital holographic imaging of an object, comprising the following steps: acquisition (802) of a holographic image of the object (102), comprising the steps of illuminating (804) the object with at least three non-coplanar illumination waves (OI), focussing each object wave from the object towards a digital sensor (126) using a focussing means (122), and detecting (806) an interference figure (126) between the object waves (OI) and a reference wave (OR); and digital reconstruction (808) of a holographic image from the at last one interference figure. The invention also relates to a holographic imaging microscope and assembly implementing such a method.

Description

"Process, microscope and set of off-axis digital holographic imaging in transmission"

The present invention relates to a digital off-axis holographic imaging method in transmission. It also relates to a digital holographic imaging microscope off-axis in transmission, and to an imaging assembly implementing such a microscope.

The field of the invention is the field of off-axis digital holographic imaging in transmission using an imaging lens and more generally a focusing means.

State of the art

There are currently various techniques of digital holographic imaging in transmission in order to produce a three-dimensional holographic image of an object: holographic imagery in the axis, holographic imagery off-axis, holographic imagery with tracer, holographic imagery without tracer, holographic imagery with imaging objective, etc. The present invention relates to off-axis digital holographic imaging in transmission with lens, or imaging objective.

Currently most of the off-axis digital holographic imaging methods in transmission with a objective microscope employ a single illumination wave. These methods do not make it possible to actually produce a 3D holographic image of an object.

We know the document "4P holography microscopy of zebrafish larvae microcirculation" from Donnarumma et al. which describes the use of two simultaneous illumination waves. This process makes it possible to improve the holographic imaging techniques known up to now. However, this process suffers from inaccuracies in the description of the images of the objects, this insufficiency does not allow the elimination of false coincidences between two different objects, which can lead to the detection of non-existing objects. An object of the present invention is to remedy these drawbacks.

Another object of the invention is to propose a digital holographic imaging method off-axis in transmission with a more precise imaging objective than the existing methods.

Yet another object of the invention is to propose a digital off-axis holographic imaging method in transmission with an imaging objective having a faster acquisition speed so as to follow, over time, the three-dimensional movements of objects. discreet.

Finally, another object of the invention is to propose a digital holographic imaging method off-axis in transmission with a more precise and / or faster imaging objective, while being more versatile.

Statement of the invention

The invention makes it possible to achieve at least one of these aims by a method of off-axis digital holographic imaging in transmission of an object, said method comprising:

at least one iteration of a step of acquiring an image of said object comprising the following steps:

- illumination of said object with several light waves, called illumination waves, consistent with a wave, called the reference wave, each illumination wave causing the diffusion by said object of a wave, called object wave,

- focusing of each object wave towards a digital sensor by a focusing means,

- Detection of at least one interference figure between the object waves and said reference wave by at least one digital sensor; and

a step of digital construction of said holographic image from the at least one interference figure;

characterized in that the illumination step comprises an emission of at least three non-coplanar illumination waves for the acquisition of a single image. The method according to the invention therefore proposes to use at least three non-coplanar illumination waves for the acquisition of the same three-dimensional holographic image of an object and to recover the object waves originating from the object by a means of focus.

Thus, the method according to the invention makes it possible to produce a truly three-dimensional holographic image of an object, in a single acquisition, unlike methods using a single illumination wave.

In addition, the method according to the invention is more precise than the methods of the prior art because it does not suffer from false coincidences due to the insufficient description of the images of the objects by two illumination waves. Indeed, the use of at least three non-coplanar illumination waves makes it possible to eliminate, for example by triangulation, the points corresponding to the crossings of the illumination waves.

In addition, the method according to the invention, using an imaging objective, can be implemented by all objective microscopes, which allows a simpler and more versatile implementation.

In addition, the use of a focusing means makes it possible to increase the precision of the image and to adapt the size of the imaged field to the digital sensor and to the resolution.

Advantageously, the focusing means can be a microscope lens, or a microscope objective.

Preferably, the focusing means can be concave.

The digital sensor can be a CCD sensor, a CMOS sensor, a Foveon sensor, or any photosensitive electronic sensor.

The illumination step can transmit at least two illumination waves simultaneously.

In particular, the illumination step can transmit all the illumination waves simultaneously.

Such simultaneous emission of at least two illumination waves makes it possible to carry out the acquisition step more quickly. Alternatively, or in addition, the illumination step can perform the emission of at least two illumination waves non-simultaneously.

In particular, the illumination step can transmit all the illumination waves in a non-simultaneous manner, that is to say in turn.

The emission of the illumination waves in a non-simultaneous manner makes it possible to facilitate the detection of the interference pattern (s), as well as the processing of the interference patterns during the digital processing step.

According to a particular and in no way limitative embodiment, the illumination waves can be emitted in turn, in groups. For example, the illumination step can carry out a transmission of a first group of one or more simultaneous illumination waves, then a transmission of a second group of one or more simultaneous illumination waves.

According to a particularly advantageous characteristic, at least one illumination wave can be generated by reflection, from a wave, called source wave, on a matrix of micro-mirrors (or "DMD" for "Digital Micromirror Device" in English).

Using a DMD simplifies obtaining non-coplanar illumination waves. The architecture of the microscope implementing the method according to the invention is simplified. Indeed, the DMD is a device which is easy to use and which limits the number of components for implementing the method according to the invention.

In addition, the DMD makes it possible to obtain a very high acquisition frequency allowing monitoring over time of the movement of the object and therefore 4D holographic imaging.

According to an exemplary embodiment implementing a DMD, the illumination step can comprise the following operations:

- illumination of the micro-mirror matrix by the source wave; - control of said matrix of micro-mirrors to obtain, in turn, two non-parallel patterns; and

- Selection of at least one diffraction order issued by said matrix for one of said patterns and at least two diffraction orders issued by said matrix the other of patterns.

In this exemplary embodiment, the non-coplanar illumination waves are obtained from two patterns displayed on the DMD in turn. The illumination waves are therefore emitted in two groups, one of the groups being emitted before the other group.

At least one of the groups comprises at least two illumination waves emitted simultaneously, each corresponding to a diffraction order of the source wave by the DMD.

According to another exemplary embodiment implementing a DMD, the illumination step can comprise the following operations:

- illumination of the micro-mirror matrix by the source wave;

- controlling said matrix of micro-mirrors to obtain, in turn, three non-parallel patterns, in particular three patterns forming between them an angle of 45 °;

- selection of at least one diffraction order for each pattern.

In this exemplary embodiment, the non-coplanar illumination waves are obtained in turn. The waves of illumination are therefore emitted in turn. According to one embodiment, at least one illumination wave is generated by reflection of a wave, said source, on at least one mirror.

In particular, several, or even all, of the illumination waves can be generated, each by reflection of the source wave on at least one mirror directed towards the object.

At least one mirror can be a semi-reflecting mirror.

According to an advantageous embodiment example:

- at least two illumination waves can be generated by reflection of a wave, called source, on at least a first and a second semi-reflecting mirrors having different inclinations; and

- At least one illumination wave is generated by reflection of the source wave on a third mirror, aligned with said first and second semi-reflecting mirrors, and having a different inclination from said first and second semi-reflecting mirrors.

The third mirror can be totally, or partially, reflective.

Thus, each mirror emits an illumination wave by reflection from a source wave emitted in the same direction.

The source wave is partially reflected by the first and second mirrors to obtain two illumination waves. Part of the source wave passes through the first and second mirrors to reach the third mirror which reflects it, partially or totally, to obtain a third illumination wave.

Advantageously, the stage of construction of the holographic image can comprise:

^■ several iterations of a phase, called cleaning, comprising the following steps:

- generation, from the interference figure provided by the acquisition step, of at least one series of 2D images, each image corresponding to a different depth relative to the plane of the digital sensor;

- for each series of images, obtaining a three-dimensional intensity matrix from the images of said series; and

- Selection of at least one point, called a bright point, from the at least one three-dimensional intensity matrix;

- memorization of said at least one bright point; and - Removal of the field radiated by said at least one bright point in said interference figure for the next iteration of said cleaning phase;

^■ development of a holographic image from said bright points memorized during said cleaning phase.

Thus, each point of the object can be identified in turn. Consequently, the method according to the invention makes it possible to construct a three-dimensional holographic image of an object point by point.

The purpose of the holographic image construction step is to reconstruct the object in a 3D space of dimension much larger than the dimension of the 2D space in which the interference figure was obtained in step d 'acquisition. The reconstruction is nevertheless possible because the solution describing the object is sparse in 3D space, and because the holographic reconstruction equations allowing to calculate the field for each reconstruction distance are linear equations.

Under these conditions, the measurement of the interference pattern obtained in the acquisition step can be considered as a compressed acquisition of the signal describing the object in 3D space. The reconstruction of the holographic image can be done using one of the numerous reconstruction methods of a compressed acquisition, in particular a reconstruction by pursuit or by orthogonal pursuit, known as such by a person skilled in the art.

Advantageously, the images generated during the step of generating the cleaning phase can be holographic images reconstructed in amplitude and phase (complex images in the mathematical sense). The different images of a series can each correspond to a different depth relative to the plane of the digital sensor.

The generation step can generate a single series of images, from the interference figure corresponding to the sum of all the illuminations, without selecting the illuminations. In this case, a intensity matrix, called global, is generated for the sum of all the illuminations.

Alternatively, or in addition, the generation step can generate a generation of at least three series of images, each of the at least three series being obtained by selecting in the interference figure provided by the acquisition step the holographic signal corresponding to one of the illumination waves, and therefore to one of the directions of illumination. In this case, several intensity matrices, called individual, are generated, each intensity matrix corresponding to an illumination wave, or to an illumination direction.

At each iteration of the cleaning phase, the three-dimensional intensity matrix (s) are used to determine the position and intensity of the brightest point, or diffuser.

Several methods can be used to determine the position and intensity of the brightest point. The brightest point can be determined as:

i) the point presenting the maximum intensity in the global intensity matrix obtained for the sum of all the illuminations;

ii) the point presenting the maximum intensity in the product of the individual intensity matrices corresponding to the different directions of illumination;

iii) the point presenting the maximum intensity according to i) or ii), by adding the neighboring points in the plane corresponding to the same reconstruction depth,

iv) the point presenting the maximum intensity according to i) or ii) by adding points there located at different illumination depths but whose intensity is close to the maximum.

The step of suppressing the field radiated by the bright point or points may comprise a subtraction of the field radiated by this or these from the interference pattern provided by the acquisition step. During the deletion step, it may be necessary to normalize the brightness, or adjust the gain, of the selected bright point (s), and the resulting radiated field, so that after deleting the radiated field the energy remaining in the interference pattern is minimal. Normalization corresponds to the transition from a tracking method to an orthogonal tracking method.

In general, it is possible to find more details on the cleaning phase in the document "4P holography microscopy of zebrafish larvae microcirculation" by Donnarumma et al.

The method according to the invention can comprise several iterations of the acquisition step.

Iterations can be performed at a frequency greater than or equal to 24 images per second.

In addition, the method according to the invention can also comprise a step of constructing a holographic video from the holographic images obtained. Thus, it is possible to carry out a holographic monitoring of an object in 4P.

According to another aspect of the present invention, there is proposed a digital holographic imaging microscope off-axis in transmission, comprising:

means for illuminating an object with several light waves, called illumination waves, which are coherent and identical to a wave, known as a reference wave, each illumination wave causing the diffusion by said object of a wave, known as a wave object;

- at least one means of focusing each object wave towards a digital sensor; and

- at least one digital sensor of at least one interference figure between the object waves and said reference wave; characterized in that said illumination means are arranged to emit at least three non-coplanar illumination waves for the acquisition of a single image.

More generally, the microscope may include elements arranged to implement all the steps of the method according to the invention.

According to a particularly advantageous characteristic, the illumination means can comprise a matrix of micro-mirrors (or "DMD" for "Digital Micromirror Device" in English) configured to display at least two non-parallel patterns for the acquisition of each image .

The DMD can be configured to implement one of the examples described above.

Alternatively, or in addition, the illumination means may comprise reflecting or semi-reflecting mirrors arranged to reflect a wave, said source, partially or totally.

In particular, the mirrors can be non-coplanar.

According to a nonlimiting exemplary embodiment, the illumination means can comprise:

- at least two semi-reflecting mirrors having different inclinations; and

- At least one reflecting mirror, aligned with said semi-reflecting mirrors and having a different inclination from said semi-reflecting mirrors.

In this case, each of the mirrors provides an illumination wave by reflection of part of the source wave.

Of course, the microscope according to the invention can also comprise a laser source providing a wave, called a source, from which the reference wave and the illumination waves are obtained. The microscope according to the invention can be configured to carry out an acquisition at a frequency greater than or equal to 24 images per second.

According to yet another aspect of the present invention, a set of digital holographic imaging is proposed comprising:

- a microscope according to the invention, and

at least one digital module for constructing a holographic image from at least one interference figure supplied by said microscope.

The assembly according to the invention can also comprise a module for generating a holographic video from the holographic images provided by the holographic image construction module.

The generation module can in particular be integrated, or integrate, the image construction module.

The digital image construction module (s), respectively video generation module, can be (are) distant from the microscope, and may not be integral with said microscope.

This module (s) can be found on the same as the microscope, or on a different site.

At least one of the digital image construction and video generation modules can be a hardware module, such as a processor or an electronic chip, or a software / computer module executed in an electronic or computer device, such as a computer. or a server.

Description of the figures and embodiments

Other advantages and characteristics will appear on examining the detailed description of an embodiment which is in no way limitative, and the attached drawings in which: - FIGURE 1 is a schematic representation of a first non-limiting embodiment of a microscope according to the invention;

- FIGURES 2a-2d are schematic representations of an example of using the microscope of FIGURE 1;

- FIGURE 3 is a schematic representation of a second non-limiting embodiment of a microscope according to the invention;

- FIGURES 4a-4b are schematic representations of an example of using the microscope of FIGURE 3;

- FIGURE 5 is a schematic representation of a third non-limiting exemplary embodiment of a microscope according to the invention;

- FIGURE 6 is a schematic representation of an example of using the microscope of FIGURE 5;

- FIGURE 7 is a schematic representation of a nonlimiting exemplary embodiment of an imaging assembly according to the invention; and

- FIGURE 8 is a schematic representation in the form of a diagram of a non-limiting exemplary embodiment of a method according to the invention.

It is understood that the embodiments which will be described below are in no way limiting. It is possible in particular to imagine variants of the invention comprising only a selection of characteristics described hereinafter isolated from the other characteristics described, if this selection of characteristics is sufficient to confer a technical advantage or to differentiate the invention from the prior art. This selection comprises at least one preferably functional characteristic without structural details, or with only a part of the structural details if this part is only sufficient to confer a technical advantage or to differentiate the invention from the state of the prior art.

In the figures, the elements common to several figures keep the same reference. FIGURE 1 is a schematic representation of a first non-limiting embodiment of a microscope according to the invention.

The microscope 100, shown in FIGURE 1, makes it possible to produce one or more holographic images of an object 102.

The microscope 100 comprises a laser source 104 emitting a laser wave 106, called the source wave, towards a semi-reflecting mirror 108. The latter separates the source wave 106 into two waves:

- a wave, denoted OR, passing through the semi-reflecting mirror 108, and which will be used as a reference wave for holographic imaging; and

a wave 110, reflected by the semi-reflecting mirror 108, which will be used to generate at least three waves of non-coplanar illuminations to illuminate the object 102.

To generate the non-coplanar illumination waves, the microscope 100 comprises an array of micro-mirrors 112 ("DMD") illuminated by the wave 110.

The DMD 112 is configured to display in turn a first pattern 112i, then a second pattern 112 ₂ , said two patterns 112i and 112 ₂ being non-parallel to each other. In particular, each of the patterns 112 _I -112 ₂ is formed by a series of lines which are mutually parallel, and perpendicular to those forming the other of the patterns 112 _I - 112 ₂ .

For the vertical pattern 112i, the reflection of the wave 110 on the DMD 112 generates several illumination waves among which:

- a wave, denoted OIi °, corresponding to the diffraction order 0 of the DMD 112;

- a wave, noted Olf ¹ , corresponding to the diffraction order -1 of the DMD 112; and

- a wave, denoted OIi ⁺¹ , corresponding to the +1 diffraction order of the DMD 112;

These three waves OIi °, OIi ⁺¹ , and Olf ¹ are used as the illumination wave for object 102. These three illumination waves OIi °, OIi ⁺¹ , and Olf ¹ are emitted simultaneously and are coplanar.

Similarly, for the motif 112 ₂ , the reflection of the wave 110 on the DMD 112 generates several waves among which:

- a wave, denoted OI ₂ °, corresponding to the diffraction order 0 of the DMD 112;

- a wave, denoted OI ₂ ^_1 , corresponding to the diffraction order -1 of the DMD 112; and

- a wave, denoted OI ₂ ⁺¹ , corresponding to the +1 diffraction order of the DMD 112;

These three waves OI ₂ °, OI ₂ ⁺¹ , and OI ₂ ^_1 are also used as the illumination wave for object 102.

The three illumination waves OI ₂ °, OI ₂ ⁺¹ , and OI ₂ ^_1 obtained from the pattern 112 ₂ are coplanar with each other and are emitted simultaneously.

However, the three illumination waves OI ₂ °, OI ₂ ⁺¹ , and OI ₂ ^_1 obtained from the motif 112 ₂ , are not coplanar with the three illumination waves OIi °, OIi ⁺¹ , and Olf ¹ obtained from pattern 112i. In addition, the illumination waves OIi °, OIi ⁺¹ , and Olf ¹ are emitted before the illumination waves OI ₂ °, OI ₂ ⁺¹ , and OI ₂ ^_1 .

Consequently, the object 102 is illuminated with a first and a second group of illumination waves, in turn. The illumination waves of the first group are not coplanar with those of the second group.

In FIGURE 1, the reference OI ⁺¹ designates either the OIi ⁺¹ wave or the OI ₂ ⁺¹ wave according to the pattern displayed by the DMD. The same reasoning applies by analogy to the references OG ¹ and OI ° used in FIGURE 1.

For the acquisition of the same image, the DMD 112 is configured to alternate at least once the patterns 112i and 112 ₂ so as to display in total at least once each pattern 112 _I -112 ₂ . In this case, the object 102 is illuminated, in turn, at least once:

- with the OIi °, OIi ⁺¹ and Olf ¹ illumination waves simultaneously; and

- with the OI ₂ °, OI ₂ ⁺¹ and OI ₂ ^_1 illumination waves simultaneously.

Of course, each pattern 112 _I -112 ₂ can be displayed several times, as will be described later with reference to FIGURE 2a.

In the microscope 100 of FIGURE 1, before reaching the object 102, each illumination wave 01 leaving the DMD 112:

- passes through a lens 114 and a zoom 116: the lens 114 and the zoom 116 allow the conditioning of the beam coming from the DMD 112. They allow on the one hand to project the image of the DMD 112 in the plane of the object 102 and in the plane of the digital sensor 126 used. They allow the adjustment and adaptation of the magnifications of the image of the DMD 112 in the plane of the object 102, and the adjustment of the angles of the beams coming from the DMD 112 in the plane of the object 102. The zoom 116 can be placed before or after the lens 114 and the illumination waves can possibly cross between the zoom 116 and the imaging objective 122; other optical elements (objective, lenses, mirrors) can be added or be already present in the microscope before or after the zoom 116 and the imaging objective 122;

- is reflected on a reflecting mirror 118 directed towards the object 102; and

- Can pass through a condenser 120 which allows, by replacing the laser illumination with a white light source, to make the device compatible with the usual operation of a microscope in bright field illumination.

Each illumination wave 01 causes the emission by the object 102 of at least one wave, said object, denoted 00. Each object wave 00 emitted by the object 102 passes through an imaging objective 122 making it possible to enlarge the image and to improve the resolution of the image. The microscope 100 further comprises a separating cube 124, serving as a semi-transparent mirror, receiving on the one hand the reference wave OR and each object wave 00, and directing them towards a photosensitive digital sensor 126.

In particular, the separating cube 124 reflects the reference wave OR towards the digital sensor 126 and transmits each object wave 00.

The function of the digital photosensitive sensor 126 is to detect the interference pattern between the reference wave OR and each object wave 00 emitted by the object 102 for each of the at least three illumination waves. The digital sensor 126 can be, for example, a CCD sensor, a CMOS sensor, a Foveon sensor, etc.

In addition, as is known in holographic imaging, each illumination wave causes three orders of interference at the level of the digital sensor 126: namely an interference of order 0, an interference of order +1 and an interference d 'order -1.

To offset the +1 and -1 interference orders from the center of the Fourier hologram, the separator cube 124 is angularly offset.

FIGURES 2a-2d are schematic representations of an example of use of the device 100 of FIGURE 1.

In FIGURE 2a, arrow 202 represents time.

The references 204 and 206 each designate a capture carried out by the sensor 126.

Thus, for each image 204 and 206, it is understood that the DMD 112 is controlled to display in turn, and alternately, four times each of the patterns 112 _I - 112 ₂ . FIGURE 2b shows the interference figure obtained by the sensor 126 each time the pattern 112i is displayed on the DMD 112.

The three tasks 208 correspond to the interference of order -1 obtained for each of the illumination waves OIi °, OIi ⁺¹ and Olf ¹ . Tasks 210 correspond to the interference order +1 obtained for each of the illumination waves OIi °, OIi ⁺¹ and Olf ¹ , and tasks 212 correspond to the interference order 0 obtained for each of the waves d illumination OIi °, OIi ⁺¹ and Olf ¹ .

Generally the tasks 208 are clear and are used for the generation of the holographic image while the tasks 210 and 212 are not used for the generation of the holographic image of the object 102, and are eliminated.

FIGURE 2c shows the interference figure obtained by the sensor 126 each time the pattern 112 _{2 is} displayed on the DMD 112.

The three tasks 214 correspond to the interference obtained for each of the illumination waves OI ₂ °, OI ₂ ⁺¹ and OI ₂ ^_1 . Tasks 216 correspond to the interference order +1 obtained for each of the illumination waves OI ₂ °, OI ₂ ⁺¹ and OI ₂ \ and tasks 218 correspond to the interference order 0 obtained for each of the OI ₂ °, OI ₂ ⁺¹ and Oΐ illumination waves

Generally the tasks 214 are clear and are used for the generation of the holographic image while the tasks 216 and 218 are not used for the generation of the holographic image of the object 102, and are eliminated.

FIGURE 2d shows the total interference figure obtained for the two patterns 112 _I -112 ₂ for each 204 or 206.

It is this interference figure which is then used to generate an image of the object 102. In particular, as indicated above, only the interferences of order -1, materialized by the tasks 208 and 214 are used. FIGURE 3 is a schematic representation of a second non-limiting exemplary embodiment of a microscope according to the invention.

The microscope 300, shown in FIGURE 3, includes all of the elements of the microscope 100 in FIGURE 1 except the lens 114, the zoom 116 and the condenser 120.

Unlike the microscope 100 of FIGURE 1, in the microscope 300 of FIGURE 3, the DMD 112 is programmed to display in turn three patterns, namely the pattern 112i, then the pattern 112 ₂ and finally a pattern 112 ₃ which is not parallel to the patterns 112i and 112 ₂ . In particular, the pattern 112 ₃ is formed by lines, parallel to each other, and forming an angle of 45 ° with the lines forming the pattern 112i and with the lines forming the pattern 112 ₂ .

In addition, the microscope 300 includes three mirrors 302-306, having different inclinations, and positioned to reflect a single illumination wave 01 for each pattern displayed by the DMD 112. In other words, for each pattern, only one order of diffraction returned by DMD 112 is reflected towards object 102.

For example, the mirror 302 can be positioned to reflect only the wave, denoted Of ¹ , coming from the DMD 112 and corresponding to order “-1” reflected by the DMD 112 for the pattern 112i. The waves of order 0, noted Oi °, and of order +1, noted Oi ⁺¹ , reflected by the DMD 112 are not directed towards the object 102 and do not illuminate the object 102.

Similarly, the mirror 304 can be positioned to select only the order wave "-1", denoted 0 ₂ \ reflected by the DMD 112 for the pattern 112 ₂ , and the mirror 306 can be positioned to select only the 'order wave' -1 ', noted 0 ₃ ^_1 , reflected by the DMD 112 for the motif 112 ₃ .

Thus, in the microscope 300 of FIGURE 3, the object 102 is illuminated by three illumination waves emitted in turn, and which are not coplanar. FIGURES 4a-4b are schematic representations of an example of use of the device 300 of FIGURE 3.

In FIGURE 4a, the arrow 402 represents time.

The references 404 and 406 each designate a capture carried out by the sensor 126.

Thus, for each capture 404 and 406, it is understood that the DMD 112 is controlled to display in turn three times an alternation of the three patterns 112 _I -112 ₃ so that said patterns are displayed in turn and that each pattern 112 _I -112 ₃ is displayed three times.

FIGURE 4b shows the total interference figure obtained by the sensor 126 for each capture 404 or 406.

The three tasks 408 correspond to the interference of order -1 obtained for the three illumination waves Olf ¹ , OI2 ^'1 and OI ₃ ^_1 . These tasks are generally sharp and are used for the generation of the holographic image.

The three tasks 410 correspond to the +1 interference order obtained for the three illumination waves Olf ¹ , OI2 ^'1 and OI ₃ ^_1 . These tasks 410 are generally not clear and are not used for the generation of the holographic image.

Task 412 corresponds to the interference order 0 obtained for the three illumination waves Olf ¹ , OI2 ^'1 and OI ₃ ^_1 . This task is generally not clear and is not used for the generation of the holographic image.

FIGURE 5 is a schematic representation of a third non-limiting exemplary embodiment of a microscope according to the invention.

The microscope 500, shown in FIGURE 4, includes all of the elements of the microscope 300 in FIGURE 3, except the DMD 112.

Instead of the DMD, the microscope 500 comprises a series of three mirrors 502-506 aligned in the direction of the wave 110 and having different inclinations between them.

The mirror 502 is a semi-reflecting mirror. It reflects part of the 110 wave, like a first illumination wave, denoted OIi, towards the mirror 302 which directs it towards the object 102. The mirror 502 lets part of the incident wave 110 pass to the mirror 504.

The mirror 504 is a semi-reflecting mirror. It reflects part of the wave coming from the mirror 502, like a second illumination wave, denoted OI ₂ , towards the mirror 304 which directs it towards the object 102. The mirror 504 lets through part of the incident wave 110 to the mirror 506.

The mirror 506 is a fully reflecting mirror. It reflects all the wave coming from the mirror 504, like a third illumination wave, denoted OI ₃ , towards the mirror 306, which directs it towards the object 102.

Thus, one obtains three waves of illumination OIi-OI ₃ not coplanar, and simultaneous.

FIGURE 6 shows the figure of total interference obtained by the sensor 126 with the microscope 500 of FIGURE 5.

The three tasks 602 correspond to the interferences of order -1 obtained for the three illumination waves OIi, OI ₂ and OI ₃ . These tasks 602 are generally clear and are used for the generation of the holographic image.

Tasks 604 correspond to the +1 interference order obtained for the three illumination waves OIi, OI ₂ and OI ₃ . These tasks 604 are generally not clear and are not used for the generation of the holographic image.

The seven tasks 606 correspond to the interference order 0 obtained for the three illumination waves OIi, OI ₂ and OI ₃ . These tasks 606 are generally not clear and are not used for the generation of the holographic image.

FIGURE 7 is a schematic representation of a nonlimiting exemplary embodiment of an imaging assembly according to the invention. The imaging assembly 700 comprises a microscope according to the invention 702, which can be for example any one of the microscopes 100, 300 or 500 of FIGURES 1, 3 and 5.

The imaging assembly 700 includes a module 704 for generating holographic images. This module 704 is configured to construct a three-dimensional holographic image of the object from the total interference figure acquired by the digital sensor for each acquisition. This interference figure is for example that shown in FIGURE 2d, or in FIGURE 4b or even in FIGURE 6, for each image.

The imaging assembly 700 can also comprise a module 706 for generating a holographic video. This module 706 is configured to generate a video from the images supplied by the image generation module 704.

Each of the image generation modules 704 and video generation modules 706 can be produced by a physical electronic component, of the processor or electronic chip type, or by a computer program.

The image generation modules 704 and video generation 706 can be integrated into the same component / program, or into independent components / programs.

At least one of the image generation modules 704 and video generation modules 706 can be local or remote from the microscope 702.

FIGURE 8 is a schematic representation of a nonlimiting exemplary embodiment of a method according to the invention.

The method 800, shown in FIGURE 8, comprises a step 802 of acquiring an interference fig of the object.

Step 802 includes a step 804 of illuminating the object with at least three non-coplanar illumination waves. This illumination step 804 can use more than three wave of illuminations, as long as at least one of these waves is not coplanar with the other waves.

In addition, the illumination step 804 can be repeated several times, as described for example with reference to FIGURES 2a and 4a. The acquisition step 802 comprises a step 806 of detecting and memorizing an interference pattern obtained for all the illuminations produced during the illumination step 804.

The acquisition step 802 can be carried out once and only once when it is desired to produce a single holographic image of the object.

Alternatively, the acquisition step 802 can be repeated at a given frequency when it is desired to produce several holographic images, in particular for producing a holographic video of the object.

The iteration frequency of the acquisition step 802 can be greater than or equal to 24 images per second.

Following the acquisition step 802, the method 800 comprises a step 808 of digital construction of a holographic image of the object.

This construction step 808 is performed, in turn, for each interference figure memorized during each acquisition step.

The construction step 808 comprises several iterations of a phase 810, called cleaning. Each iteration of the cleaning phase 810 includes the following steps.

A step 812 generates one or more image series from the interference figure, namely:

a single series of images, from the interference figure supplied by the acquisition step 802 and corresponding to the sum of all the illuminations, without selection of the illuminations; and or

at least three series of images, each of the at least three series being obtained by selecting in the interference figure supplied by the acquisition step 802 the holographic signal corresponding to one of the illumination waves, and therefore to one of the directions of illumination

In each series of images, each image corresponding to a different depth relative to the plane of the digital sensor. Each series of images obtained in step 812 is converted into a three-dimensional intensity matrix during a step 814. Step 814 therefore provides as many intensity matrices as there are series of images generated during the step 812.

A step 816 then performs a selection of at least one bright point from the three-dimensional intensity matrix or matrixes, according to one of the techniques described above.

During a step 818, the field (s) radiated by the point or points selected during the step 816 is (are) deleted from the interference figure, for a new iteration of the phase cleaning 810. Optionally, the removal step 818 can comprise a normalization of the brightness, or an adjustment of gain, of the selected bright point or points, and of the radiated field or fields which result therefrom, so that after suppression of the radiated fields, the energy remaining in the interference figure is minimal.

The cleaning phase 810 is carried out as many times as necessary, and in particular until the energy of the interference figure has been sufficiently reduced.

According to a first example, the cleaning phase can be carried out until the total energy remaining in the interference figure is less than or equal to a fraction, for example 20%, of the initial total energy in the figure. interference before the start of the cleaning phase.

According to a second example, the cleaning phase can be carried out until the energy of the brightest point in the interference figure is less than or equal to a fraction, for example 1%, of the initial total energy. in the interference figure before the start of the cleaning phase.

Following all the iterations of the cleaning phase 810, the stored points are used to generate the holographic image during a step 820. Step 808 is repeated for each iteration of acquisition step 802 so as to generate a holographic image for each acquisition step 802.

Step 808 can also comprise a step of memorizing the holographic image obtained.

The method 800 can also comprise a step 822 generating a holographic video of the object from the holographic images produced by the different iterations of the step 808. Thus, it is possible to follow the movements of the object over time .

Of course, the invention is not limited to the examples detailed above.

Claims

1. A method (800) of off-axis digital holographic imaging in transmission of an object (102), said method (800) comprising:

at least one iteration of a step (802) of acquiring a holographic image of said object (102) comprising the following steps:

- Illumination (804) of said object (102) with several light waves (OI), called illumination waves, consistent with a wave (OR), called a reference wave, each illumination wave (OI) causing the diffusion by said object of a wave (OO), called object wave,

- focusing of each object wave (00) to at least one digital sensor (126) by a focusing means (122),

- Detection (806) of at least one interference figure between the object waves (00) and said reference wave (OR) by the at least one digital sensor (126); and

- a step (808) of digital construction of said holographic image from the at least one interference figure;

the illumination step (804) comprising an emission of at least three non-coplanar illumination waves (OI) for the acquisition of a single holographic image;

characterized in that the illumination step (804) transmits at least two illumination waves (OIi ^ -OIs ¹ ) non-simultaneously for the acquisition of a single holographic image.

2. Method (800) according to the preceding claim, characterized in that the illumination step (804) transmits at least two illumination waves (OI) simultaneously (OI ₁ -OI ₃ ).

3. Method (800) according to any one of the preceding claims, characterized in that at least one illumination wave (OI) is generated by reflection, from a wave (110), called source wave, on a matrix micro-mirrors (112).

4. Method (800) according to claim 3, characterized in that the illumination step (804) comprises the following operations:

- illumination of the matrix of micro-mirrors (112) by the source wave (110);

- control of the micro-mirror array (112) to obtain, in turn, two non-parallel patterns (112 _I -112 ₂ ); and

selection of at least one diffraction order emitted by said matrix of micro-mirrors (112) for one of said patterns (112 _I - 112 ₂ ) and at least two diffraction orders emitted by said matrix of micro- mirrors (112) the other of the patterns (112 _I - 112 ₂ ).

5. Method (800) according to claim 3, characterized in that the illumination step (804) comprises the following operations:

- illumination of the matrix of micro-mirrors (112) by the source wave (110);

- control of the micro-mirror array (112) to obtain, in turn, three non-parallel patterns (112 _I -112 ₃ ); and

- selection of at least one diffraction order for each pattern (112 ₁ -112 ₃ ).

6. Method (800) according to any one of claims 1 to 2, characterized in that at least one illumination wave (01) is generated by reflection of a wave (110), said source, on at least a mirror (502-506).

7. Method (800) according to any one of the preceding claims, characterized in that the stage of construction (808) of the holographic image comprises:

^■ several iterations of a phase (810), called cleaning, comprising the following steps:

generation (812), from the interference figure supplied by the acquisition step (802), of at least one series of 2D images, each 2D image corresponding to a different depth relative to the plane of the digital sensor (126); - for each series of 2D images, obtaining (814) a three-dimensional intensity matrix from the 2D images of said series; and

- selection (816) of at least one point, called a bright point, from the at least one three-dimensional intensity matrix;

- storage (816) of said at least one bright point; and

- deletion (818) of the field radiated by said at least one bright point in said interference figure for the next iteration of said cleaning phase (810);

- Elaboration (820) of a holographic image from said bright points memorized during said cleaning phase.

8. Method (800) according to any one of the preceding claims, characterized in that it comprises several iterations of the acquisition step (802) at a frequency greater than or equal to 24 images per second, said method (800 ) further comprising a step (822) of constructing a video from said holographic images.

9. Microscope (100; 300; 500) digital off-axis holographic imaging in transmission, comprising:

- Means (112; 502-506) of illumination of an object (102) with several light waves (01), called illumination waves, coherent to a wave (OR), called reference, each wave of illumination causing the diffusion by said object (102) of a wave (00), said object wave;

- at least one means (122) for focusing each object wave (00) towards a digital sensor (126); and

- at least one digital sensor (126) of at least one interference figure between the object waves (00) and said reference wave (OR);

said illumination means (112; 502-106) being arranged to emit at least three non-coplanar illumination waves for the acquisition of a single holographic image. characterized in that said illumination means (112; 502-106) are arranged to emit at least two illumination waves (OIi ^ -OIs ¹ ) non-simultaneously.

10. Microscope (100; 300) according to the preceding claim, characterized in that the illumination means comprise an array of micro-mirrors (112) configured to display at least two non-parallel patterns (112 _I - 112 ₃ ) for the acquisition of each holographic image.

11. Microscope (500) according to claim 9, characterized in that the illumination means comprise:

- at least two semi-reflecting mirrors (502,504) having different inclinations; and

- at least one reflecting mirror (506), aligned with said semi-reflecting mirrors (502,504) and having a different inclination from said semi-reflecting mirrors (502,504).

12. Microscope (100; 300; 500) according to any one of claims 9 to 11, characterized in that it comprises a laser source (104) supplying a wave (106,110), said source, from which are obtained the reference wave (OR) and the illumination waves (01).

13. Set (700) of digital holographic imaging comprising:

- a microscope (702) according to any one of claims 9 to 12, and

- at least one digital module (704) for constructing a holographic image from at least one interference figure supplied by said microscope (702).

14. An assembly (700) according to the preceding claim, characterized in that it further comprises a module (706) for generating a video holographic from holographic images provided by the holographic image construction module (704).