Patent ID: 12216056

DETAILED DESCRIPTION OF THE INVENTION

FIG.1schematically illustrates an example of a wavefront analysis device110according to the present description, connected to a fluorescence microscopic imaging system100with optical sectioning.

The fluorescence microscopic imaging system100with optical sectioning comprises an illumination path (not shown inFIG.1) for illuminating an optical section of a volumetric and fluorescent object10.

The microscopic imaging system100also comprises an imaging path103and an analysis path101comprising the wavefront analysis device110.

The imaging path103comprises, in the example ofFIG.1, a microscope objective lens130comprising a pupil131in a pupil plane P4, a focusing optic134, a fluorescence filter132, an imaging detector140comprising a detection plane P6, a processing unit142for processing signals acquired by the detector140.

Depending on the type of microscopic imaging system (for example, light-sheet or multiphoton), the imaging detector140can be a two-dimensional detector, for example, a CCD (Charge Coupled Device) camera or a high-sensitivity CMOS (Complementary Metal Oxide Sensor) camera, such as, for example, sCMOS cameras, or a spot detector (for example, a photomultiplier).

The object10of interest is positioned in the vicinity of a focal plane P1of the microscope objective lens130. The object10is, for example, a transparent or semi-transparent sample, such as a fluorescent biological object, and the focal plane P1of the microscope objective lens130is, for example, located at a non-zero distance from the surface of the object10, so as to produce an image of a plane located in depth.

The microscopic imaging system100is a fluorescence imaging system with optical sectioning, i.e. a microscope allowing, using different techniques, selection of the light originating solely from an optical section perpendicular to the optical axis of the microscope objective lens. The optical section is located in the focal plane of the objective lens, the thickness of which can be less than the depth of field of the microscope objective lens. Several technical approaches exist for producing an optical section within a fluorescence microscope, such as, for example, light-sheet fluorescence microscopy and multiphoton fluorescence microscopy, embodiments of which will be described with reference toFIGS.4and5. For the sake of simplicity, the illumination path of the object comprising means for generating a fluorescence optical section are not shown inFIG.1.

The fluorescence filter132is a spectral filter allowing only the spectral band corresponding to the emission of fluorescence to be selected, in order to remove the excitation light that is possibly backscattered by the object. It can be a high-pass filter, a low-pass filter or a band-pass filter, depending on the relative spectral features between the excitation beam and the fluorescence emission beam.

The focusing optic134allows an image to be formed of an optical section superimposed with the focal plane P1on the imaging detection plane P6. The focusing optic134can comprise one or more lenses, often called “tube lens(es)”, and has features which, combined with the features of the imaging detector140, in particular the size of an elementary detector or pixel of the imaging detector140, allow sampling, by the detector140, of the focal plane P1without a spectrum overlap effect, i.e. meeting the correct sampling conditions defined by the Shannon theorem.

The microscopic imaging system100also comprises a beam splitter element135allowing a portion of the fluorescence light emitted by the object to be taken from the imaging path103in order to send it toward the wavefront analysis device110on the analysis path.

According to one or more embodiments, the beam splitter element135comprises a beamsplitter cube or a beamsplitter plate, allowing transmission (or reflection) of a proportion of the fluorescence light originating from the object toward the imaging detector140and reflection (or transmission) of the remainder of the fluorescence light originating from the object toward the wavefront analysis device110, with this proportion being 50%, for example.

According to one or more embodiments, the beam splitter element135comprises a plate or a dichroic cube, allowing transmission (or reflection) of a first spectral band of the fluorescence light originating from the object toward the imaging detector140and reflection (or transmission) of a second spectral band of the light originating from the object toward the wavefront analysis device110, with these two spectral bands not having any overlap. For example, the use of a dichroic type beam splitter element is implemented concomitantly with the use of one or more fluorescence excitation sources and two types of fluorescent markers on the object, so as to obtain two separate fluorescence emission spectra. This thus avoids removing a signal useful for forming the image by the imaging detector140for the purpose of wavefront measurement. Thus, the photometric balance is optimal both on the imaging path103and on the analysis path101. For example, in the case of a biological object such as the brain of animals models used in neuroimaging (drosophila, zebra fish), an anatomical fluorescent marker can be used for measuring the wavefront by the wavefront analysis device110, and a specific fluorescent marker can be used for the imaging, for example, a marker of the calcium activity associated with the response of the individual neurons, with these 2 markers emitting a fluorescent signal in 2 separate spectral bands.

The wavefront analysis (or measurement) device110shown inFIG.1comprises a two-dimensional detector112with a detection plane P3, configured to detect light signals originating from a matrix114of microlenses115, said matrix being arranged in an analysis plane P5. The wavefront analysis device110also comprises an optical relay system116configured to optically conjugate the analysis plane P5and the pupil plane P4of the microscope objective lens130, a field diaphragm118and a processing unit120for processing signals originating from the detector112.

The two-dimensional detector112is, for example, a CCD or CMOS type two-dimensional camera configured to detect fluorescence light in a given spectral band, originating from the object. Typically, in fluorescence microscopy, fluorescent markers that are used emit in a visible or near infrared spectral band, for example, between 400 and 900 nm. However, it is possible to adapt the sensitivity of the detector to other spectral bands if necessary, by using other detection technologies, such as, for example, other light-sensitive materials on the pixels of the two-dimensional detector. For example, InGaAs is a material that is sensitive between 0.9 μm and 1.7 μm for far infrared, in which spectral range numerous fluorescent markers are being developed due to the greater penetration depth of light, in particular in scattering environments.

The microlenses array114comprises a two-dimensional arrangement of optical focusing elements115arranged in an analysis plane P5, for example, arranged in a two-dimensional matrix. In particular, each optical focusing element115, or microlens, is characterized by the same focal distance fm, as well as the same pupil size dm, with the pupil size being defined as a function of the shape of the microlenses. The microlenses array114can be produced using different techniques, including, but not exclusively, machining a substrate made up of an optical material such as glass, photolithography applied to a light-sensitive resin deposited onto an optical substrate, the iterative deposition of optical material on a substrate allowing an array of Fresnel lenses to be formed, or pressing plastic optical material using a mold. The microlenses typically have a round pupil, with dmin this case corresponding to its diameter, or a square pupil, with dmin this case corresponding to its side. Other pupil geometries are possible depending on the technology that is used, such as, for example, hexagonal pupils. Preferably, a matrix of joined, square microlenses will be used, with the joining aspect of this geometry allowing all the incident light to be used, unlike round, non-joined microlenses. In order to maximize transmission efficiency, an anti-reflection treatment is generally used that corresponds to the spectral usage band of the microlenses array. The microlenses array114is positioned at the distance fmrelative to the two-dimensional detector112, with each microlens forming an image in the detection plane P3.

The beam splitter element135allows an intermediate image plane P2to be defined in the wavefront analysis device110that is conjugated with the object focal plane P1. The field diaphragm118is arranged in the intermediate focal plane P2. The optical relay system116, in combination with the microlenses array114, allows an optical conjugation to be carried out between the plane P2and the detection plane P3, at a magnification that is defined by the ratio of the focal lengths of the optical relay system116and of the microlenses array114. Furthermore, the optical relay system116, in combination with the focusing optic134(tube lens), allows an optical conjugation to be carried out between the pupil plane P4of the microscope objective lens130and the analysis plane P5(plane of the microlenses array), at a magnification that is defined by the ratio of the focal lengths of the optical relay system116and of the focusing optic134.

The field diaphragm118, located in the intermediate image plane P2, allows the size of the field-of-view imaged by each microlens115of the microlenses array114to be limited. According to an advantageous embodiment, the dimensions and the geometry of the field diaphragm118are selected so that no overlap is possible between 2 adjacent images originating from adjacent microlenses of the microlenses array114. For example, for square microlenses, with a side dm, and an optical relay system116with a focal length of fc=3 fm, it is possible to define a field diaphragm118that is square and has a side that is less than or equal to 3 dm. The field diaphragm is part of the wavefront analysis device110and therefore of the analysis path101, but it is not part of the imaging path105, so as to avoid minimizing the field-of-view imaged by the imaging detector140.

The processing unit120is configured to process the signals originating from the detector112, and in particular to carry out all operations on these signals in order to measure the wavefront originating from an optical section of the object, i.e. to determine a parameter characteristic of the wavefront.

The processing unit120is generally configured to implement computation and/or processing steps implemented in methods according to the present application. In general, when the present description refers to computation or processing steps for particularly implementing steps of methods, it is understood that each computation or processing step can be implemented by software, hardware, firmware, microcode or any suitable combination of these technologies. When software is used, each computation or processing step can be implemented by computer program instructions or by the software code. These instructions can be stored or transmitted to a computer-readable storage medium (or processing unit) and/or can be executed by a computer (or processing unit) in order to implement these computation or processing steps.

Of course, the processing unit120and the processing unit140can be consolidated within the same unit, for example, a computer.

Each microlens115of the microlenses array114thus forms an image on the detection plane P3of the detector112of an optical section of a fluorescent, in a field-of-view defined by the field diaphragm118, as previously described. When the object and/or the optical imaging system do not have any aberration, each microlens forms an image on the detection plane of an optical section of the object centered on the optical axis of the considered microlens. When the plane P1is located deep in a heterogeneous object, optical aberrations are present, particularly in the pupil131, corresponding to a non-flat wavefront. In this case, a transverse shift of each image formed by each microlens of the matrix115is observed on the detection plane P3, with this shift being proportional to the local shift of the wavefront on the corresponding microlens, i.e. to the slope of the wavefront. Since the planes P5and P4are conjugated, each of these images corresponds to the image formed by the detector140of the imaging path103, but through a portion of the pupil131, the portion corresponding to the image of a microlens of the matrix P3by the optics116and134. This shift can be viewed in a similar way to a Shack-Hartmann wavefront analyzer, for which the shift of a diffraction spot is observed that originates from each microlens and not from an image, with this type of sensor being used with a point source.

The geometrical effect produced on the detection plane of a wavefront analysis system by an imperfect wavefront at the scale of a microlens115of the microlenses array114, as well as an example of advantageous dimensioning of the microlenses, will be described in further detail with reference toFIG.8.

By way of an illustration,FIG.2Ashows an image202showing the diffraction spots formed by a microlenses array in a Shack-Hartmann type wavefront analyzer and an image204showing the images formed by the microlenses of a microlenses array in a wavefront analyzer according to the present description. The image206illustrates a magnified image formed by a microlens.FIG.2Bshows the image of the same object formed on the detector of the imaging path. In these examples, the object is a HeLa type fluorescent cell including a fluorescent marker of tubulin and the imaging system is of the light-sheet type. In this example, the microlenses are joined together and square and have a ratio of 15 between their focal distance and their side, which corresponds to a numerical aperture that is substantially less than that of the microscope objective lens, which explains the loss of resolution of the images formed by the microlenses, of which the image206is an example. For this reason, the images formed by the microlenses carry out low-pass filtering of the spatial frequencies of the object through a comparison with the image formed on the detector of the imaging path. In this example, the surface of the analysis field-of-view defined by the field diaphragm approximately corresponds to a quarter of the surface of the field-of-view imaged by the microscope.

Measuring all the shifts of the images formed by the set of microlenses allows, using a processing unit120, a two-dimensional map of slopes of the wavefront in the pupil plane P4to be deduced. Using numerical integration it is possible to deduce a map of the wavefront therefrom. The measurement of the shifts of the images formed by the microlenses is typically carried out by cross-correlation operations of each image relative to a reference image. This reference image is defined, for example, as the image formed by a reference microlens, for example, a central microlens of the microlenses array114. By limiting the field-of-view imaged by each microlens on the detection plane P3, the field diaphragm118avoids overlapping images originating from adjacent microlenses likely to introduce errors during the cross-correlation computation.

The wavefront analysis device110, when it is implemented within a fluorescence microscope with optical sectioning, as illustrated inFIG.1, for example, allows a wavefront measurement to be carried out without requiring the presence of a point source within the object, as is generally the case, for example, with the use of a Shack-Hartmann type wavefront analyzer.

When it is implemented within a fluorescence microscope with optical sectioning, the wavefront analysis device110allows a measurement to be carried out of the optical defects on an analysis field-of-view defined by the field diaphragm118. When the analysis field-of-view has similar dimensions, or is smaller than an isoplanetic patch, the wavefront measurement is valid irrespective of the point of the field-of-view. When the dimensions of the analysis field-of-view are greater than an isoplanetic patch, the wavefront measurement corresponds to the measurement of an average wavefront on the field-of-view defined by the diaphragm118.

According to one or more embodiments, the field diaphragm118is referred to as “active”, i.e. its dimensions and/or its position in the plane P2are variable.

Thus, the field diaphragm can, according to one example, have variable dimensions. For example, for a square transmission field diaphragm made up of 4 opaque plates arranged facing each other in pairs so as to form a square transparent zone, it is possible to arrange these plates on movable elements, which may or may not be motorized, allowing the relative distance between 2 plates located facing each other to be adjusted. A variable aperture of the field diaphragm allows an isoplanetic patch of an object to be determined, in a zone of the imaging field-of-view, for example, by carrying out a series of wavefront measurements for a steadily decreasing size of the field diaphragm. When the wavefront between 2 successive measurements stops varying, the size of the field diaphragm corresponding to the first one of the 2 measurements corresponds to the size of the isoplanetic patch of the object in the considered zone.

The field diaphragm118can also, according to one example, have a variable position in the plane P2. For example, the variable position is obtained by motorizing the transverse position, for example, by means of piezoelectric motors or stepper motors.

By combining a variable position of the field diaphragm118with variable dimensions, it is possible to determine the dimensions of the isoplanetic patch at different zones of the field-of-view and to determine the optical defects of the wavefront in the analysis plane, for the different zones of the imaging field-of-view. Advantageously, dimensions are selected for the field diaphragm118that are less than those of the isoplanetic patch determined at each zone.

FIG.3thus shows an image302of a biological object formed on an imaging detection plane P6of an imaging system, as well as images304,306formed in the detection plane P3of a wavefront analysis device according to one example of the present description, by a microlenses array, for two analysis field-of-views with different sizes and positions that are defined by the sizes and positions of the field diaphragm.

For biological objects in neuroimaging (drosophila, mouse, zebra fish brain), the typical imaging field-of-view is 400 μm to 500 μm on the side. A corresponding “average” isoplanetic patch is approximately 150 μm on the side (reference available if required).

FIG.4shows a diagram illustrating an example of a “light-sheet” type fluorescence microscopic imaging system200, according to the present description.

The fluorescence microscopic imaging system200comprises elements similar to those described with reference toFIG.1, referenced inFIG.4with identical reference signs, and not reproduced here to avoid overcomplicating the description.

In light-sheet fluorescence microscopy, the illumination path105is configured to form an optical section by transverse illumination of the object. The illumination path can comprise one or more light sources (not shown inFIG.4) for emitting one or more excitation beams that can be emitted in different spectral bands. A fluorescence excitation beam forms a thin, the thickness of which is generally substantially similar to the depth of field of the microscope objective lens, and incident light plane on the object in a direction perpendicular to the optical axis of the microscope objective lens. The excitation beam does not pass through the sample outside the focal plane P1of the objective lens130, thus avoiding the transmission of a spurious fluorescence signal.

In the example illustrated inFIG.4, the “light-sheet” type fluorescence microscopic imaging system comprises a correction device145with a correction plane P7common to the analysis path101and to the imaging path103. Correcting the wavefront by means of the correction device allows the quality of the image formed on the detection plane P6of the detector140to be improved, in particular when the focal plane of the microscope objective lens is located deep in the object, thanks to the compensation of the optical defects induced by the inhomogeneities of the object between its surface and said focal plane.

The microscopic imaging system200illustrated inFIG.4further comprises, in a common part of the analysis and imaging paths, an afocal optical system136,137. It also comprises, on the imaging path103, the tube lens134, allowing the image to be formed on the imaging detection plane P6and, on the analysis path101, a lens146for forming the intermediate image plane P2, in which the field diaphragm118of the wavefront analysis device110is arranged.

FIG.5shows a diagram illustrating an example of a “multiphoton” type fluorescence microscopic imaging system300according to the present description.

The fluorescence microscopic imaging system300comprises elements similar to those described with reference toFIG.1, referenced inFIG.5with identical reference signs, and not reproduced here to avoid overcomplicating the description.

In multiphoton fluorescence microscopy, a fluorescence emission is used that is characterized by a non-linear relationship with respect to the excitation beam: the fluorescence signal of interest is only emitted when a minimum power density is achieved locally. This condition is typically fulfilled only when the excitation beam is focused, namely in the focusing plane of the microscope objective lens, which intrinsically prevents the emission of a spurious fluorescence signal.

Thus, in the example illustrated inFIG.5, the illumination path105comprises one or more laser sources150for emitting ultrashort pulses, optionally with one or more collection optics151, with the microscope objective lens130allowing each pulse to be focused on a focusing point of the focal plane P1of the objective lens, in order to form a multiphoton fluorescence emission. The illumination path105further comprises a scanning device152configured to transversely scan the focusing point in the focal plane P1. In the example of the microscopic imaging system illustrated inFIG.5, the detector140comprises, for example, a one-dimensional detector, for example, a photomultiplier, configured to detect the light energy emitted by the object10and sent by the splitter element135for each position of the focusing point. Thus, the detector140cooperates with the scanning device152in order to form a two-dimensional image of the object.

According to one or more embodiments, the multiphoton fluorescence microscopic imaging system300comprises a correction device145with a correction plane P7. In this example, the correction plane P7is common to the analysis path101and to the illumination path105. Correcting the wavefront by means of the correction device allows the quality of the focusing in the object to be improved, and consequently allows the fluorescence signal at each point of the image to be increased, thanks to the compensation of the optical defects induced by the inhomogeneities of the object between its surface and said focal plane as each pulse passes through.

The microscopic imaging system300illustrated inFIG.5further comprises, on the common portion of the analysis and illumination paths, an afocal optical system157,158. It also comprises, on the imaging path103, the tube lens134that allows the image to be formed on the imaging detection plane P6and, on the analysis path101, a lens146for forming the intermediate image plane P2, in which the field diaphragm118of the wavefront analysis device110is arranged.

Irrespective of the fluorescence microscopic imaging system with optical sectioning implemented in the present description, the applicant has shown that the use of cross-correlation computations for determining the relative positions of the images formed by the microlenses leads to the measurement precision of these positions becoming dependent on certain features of said images, in particular the size and the contrast of the intensity patterns forming these images.

Indeed, by way of an example, a set of uniform images formed by the microlenses does not allow a precise intercorrelation computation to be carried out, with the correlation operation not having any structure that allows a precise determination of the position of the correlation peak. This example can, for example, occur for a homogenous object, or even for an object only made up of details, the characteristic sizes of which are less than the minimum size that can be imaged by the microlenses of the microlenses array.

FIG.6andFIG.7illustrate two embodiments of a microscopic imaging method according to the present description, allowing the processing of the images formed in the detection plane P3of the wavefront analysis device110according to the present description to be improved.

FIG.6illustrates a first example, in which the field diaphragm118is structured. For example, the field diaphragm is a transmission field diaphragm and the transmission is structured, in one direction, to form a regular alternation with a given spatial frequency of transmission zones and of opaque zones. According to another example, the field diaphragm is a reflection field diaphragm and the reflection is structured, in one direction, to form a regular alternation with a given spatial frequency of reflective zones and of non-reflective zones.

Thus, the image602inFIG.6shows an example of structured transmission of a field diaphragm according to a regular alternation of transparent zones and of opaque zones in one direction, defining a spatial frequency k0.

The image601shows an image, in the plane of the field diaphragm (P2,FIG.1), of a theoretical fluorescent object that will be made up of a regular alternation of fluorescent structures and of non-fluorescent structures in one direction, with a spatial frequency k.

The image603shows the superimposition of two intensity patterns601and602. When the two periodic intensity patterns and different spatial frequencies are superimposed in the plane of the field diaphragm, a Moiré phenomenon occurs that is visible in a plane conjugated with said plane of the field diaphragm. As shown on the image603, the Moiré phenomenon reveals an additional periodic pattern within a plane conjugated with the field diaphragm, for which additional pattern the spatial frequency corresponds to the vector difference of the two spatial frequencies of the initial patterns, that is a spatial frequency that is substantially less than the spatial frequencies of the two initial patterns.

In practice, for any object, the spatial frequency content of the image of a plane of the object is complex and is made up of many spatial frequencies. Some complex microscopic objects, in particular biological objects such as, for example, arrays of micro-tubules, can be solely made up of structures with very small characteristic sizes, and therefore with very high spatial frequencies. The wavefront analysis device as described in the present description produces, in the detection plane, a set of conjugated images of the plane of the field diaphragm, in particular through a microlenses array. This microlenses array is made up of individual microlenses, the numerical aperture of which is substantially less than the numerical aperture of the microscope objective lens, so as to provide a compromise between the field-of-view imaged by each microlens and the sensitivity of the movement measurement of the images formed by the microlenses by cross-correlation computation. This results in images of the plane of the field diaphragm produced by the microlenses, the spatial frequency content of which is substantially reduced on the high spatial frequencies, in a manner that is directly proportional to the numerical aperture of the microlenses. Thus, for objects as previously described, it is possible that the images produced by the microlenses no longer contain enough details to obtain precise cross-correlation computations. In this case, by positioning a structured field diaphragm, for example, in a pattern as illustrated in602, the images produced by the microlenses reveal an additional intensity pattern according to a Moiré phenomenon, as illustrated according to an example on the image603, for which at least one spatial frequency is likely to be transmitted in the detection plane P3. This additional pattern, which is characteristic of the object, advantageously allows an intercorrelation computation to be carried out that is substantially more precise than in the absence of any Moiré pattern, in particular for objects such as those previously described.

When the image of the object does not correspond to an intensity pattern defining a unique spatial frequency, as shown inFIG.6per601, the additional intensity pattern created by the Moiré phenomenon is a more complex pattern than the example shown in image603.

For example, a transmission pattern (or reflection) of the field diaphragm can be selected with a determined spatial frequency so that the resulting Moiré pattern (image603) has a frequency that is below a maximum spatial frequency Fmaxtransmitted by the microlenses. It is thus possible to improve the wavefront measurement precision for objects for which the majority of the spatial frequencies range between the maximum spatial frequency transmitted by the microlenses and two times this maximum spatial frequency. For square microlenses, with a side dm, and a focal distance fm, and for a central imaging wavelength λ, this maximum spatial frequency Fmaxis given by Fmax=dm/λ fm.

FIGS.7A and7Billustrate a second example, in which an illumination of the object is structured.FIG.7Adescribes a structured illumination in an imaging system according to the present description, of the light-sheet type andFIG.7Bdescribes a structured illumination in an imaging system according to the present description, of the multiphoton type.

FIG.7Ashows a diagram illustrating an example of a frontal representation, i.e. perpendicular to the optical axis, of an optical section701of an object in the focal plane P1of a microscope objective lens of a “light-sheet” type fluorescence microscopic imaging system with optical sectioning, as illustrated inFIG.4, for example.

The surface702schematically shows the analysis field-of-view, the size of which is defined by the field diaphragm of a wavefront analysis device according to the present description. As previously disclosed, when the analysis field-of-view702corresponds to a homogeneous zone in terms of fluorescence intensity emitted by the object, it is difficult to obtain a precise intercorrelation computation between the images produced by the microlenses of the wavefront analysis device for determining the relative positions of said images, with a cross-correlation between two homogenous intensity patterns not resulting in a correlation peak that can be precisely spatially located. The applicant has shown that it is then possible to advantageously use structured lighting of the object at the optical section allowing a set of images to be obtained, which images are formed by the microlenses for which the lighting intensity pattern is present and allows an intercorrelation computation to be carried out that defines a two-dimensional correlation peak.

According to the example ofFIG.7A, corresponding to a simplified implementation, at least one additional source is used on the “light-sheet” type illumination path105, which source is configured to illuminate the object using two beams703, which are disposed, for example, in 2 perpendicular directions and the point of intersection of which is located in the surface702. Any other lighting pattern comprising at least two non-parallel directions meets the lighting structure requirement as shown. Advantageously, the additional source implements illumination in accordance with a specific spectral band allowing fluorescence emission from a specific spectral band different from the fluorescence emission produced by illumination by the first source, which, combined with the use of a dichroic beam splitter between the imaging path and the wavefront analysis device, allows no light to be used that is intended for the imaging path for analyzing the wavefront and thus allows the contrast of the images formed by the microlenses to be maximized.

FIG.7Bshows a diagram illustrating an example of a frontal representation, i.e. perpendicular to the optical axis, of an optical section701of an object in the focal plane P1of a microscope objective lens of a “multiphoton” type fluorescence microscopic imaging system with optical sectioning, as illustrated inFIG.5, for example.

Here again, the surface702represents the analysis field-of-view, the size of which is defined by the field diaphragm of a wavefront analysis device according to the present invention. As previously disclosed, when the analysis field-of-view702corresponds to a homogeneous zone in terms of fluorescence intensity emitted by the object, it is not possible to use an intercorrelation computation between the images produced by the microlenses of the wavefront analysis device to determine the relative positions of said images, with an intercorrelation between two homogeneous intensity patterns not resulting in a correlation peak that can be precisely spatially located. The applicant has shown that in this type of microscopic imaging system, it is also possible to use structured lighting of the object on the optical section in order to obtain a set of images formed by the microlenses for which the lighting intensity pattern is present and allows an intercorrelation computation to be carried out defining a two-dimensional correlation peak.

According to the example ofFIG.7B, corresponding to a simplified implementation, at least one additional source is used on the “multiphoton” type illumination path, which source is configured to illuminate the object in a pattern704comprising, in this example, two perpendicular directions and the point of intersection of which is located in the surface702. Practically, for a “multiphoton” type microscope, this pattern is produced by sequential scanning of the focusing point of the additional source in the focal plane of the objective lens, typically using a pair of galvanometers. Any other lighting pattern comprising at least two non-parallel directions meets the structuring requirement of the lighting as shown. Advantageously, the additional source implements illumination in a specific spectral band allowing fluorescence emission of a specific spectral band different from the fluorescence emission produced by the illumination by the first source, which, combined with the use of a dichroic beam splitter between the imaging path and the wavefront analysis device, allows no light to be used that is intended for the imaging path for analyzing the wavefront and thus allows the contrast of the images formed by the microlenses to be maximized.

FIG.8shows a diagram illustrating the geometrical effect produced on the detection plane of a wavefront analysis system as described by the present invention using an imperfect wavefront at the scale of a microlens115of the microlenses array.

Assuming, for example, a microlenses array114comprising a set of adjacent microlenses with square pupils with a side dmand a focal distance fm, as shown in one dimension inFIG.8, for the sake of simplicity. Assuming a complex wavefront801incident in the analysis plane P5corresponding to the plane of the microlenses, such that on a microlens the deviation of the wavefront relative to a perfect flat wavefront corresponds to a wavelength λ of the incident beam, as shown inFIG.8.

For a wavefront analysis device as described in the present description, the spatial resolution of the analysis of the wavefront corresponds to a microlens. The local wavefront that can be measured by a microlens thus corresponds to an elementary wavefront forming an angle α with the analysis plane, such that α=δ/dm, with δ being the local deviation of the wavefront relative to a reference wavefront, in this example a flat wavefront. Thus, for a local deviation of the wavefront relative to a flat wavefront equaling δ=λ, the angle α equals α=λ/dm, with λ being significantly lower than dm. The diffraction spot formed at the focal point of the microlens on the detection plane P3is thus transversely shifted by a distance s, with the shift s being provided by:

s=λ⁢fd.

For a microlens115, the size t of a diffraction spot, measured between the two first intensity minima located on either side of the maximum intensity, is given by:

t=2⁢λ⁢fd.

It is known from the prior art that, for a two-dimensional detector, for example, a camera, made up of a two-dimensional arrangement of elementary detectors or pixels, it is possible to measure the position of an intensity pattern, such as a diffraction spot or a complex figure with a localization precision of up to one hundredth of a pixel for high contrast intensity patterns that are correctly sampled by the detector. In the case of the wavefront analysis device according to the present description, when the two-dimensional detector is designed so that a diffraction spot corresponds to two pixels in one direction, and for localization precision for an intensity pattern of one hundredth of a pixel, it is thus possible to measure the deviation of an incident wavefront on a microlens with a maximum precision of λ/100. Similarly, when the two-dimensional detector is designed so that a diffraction spot corresponds to two tenths of a pixel in one direction, and for localization precision of an intensity pattern of one hundredth of a pixel, it is thus possible to measure the deviation of an incident wavefront on a microlens with maximum precision of λ/10. In practice, measuring a wavefront with precision of less than λ/10 does not allow efficient use of said measurement for the purpose of characterizing an object or for imaging.

Thus, the applicant has shown that, advantageously, the wavefront analysis device according to the present invention can be designed so that a diffraction spot of a microlens of the microlenses array has a dimension, in one direction, ranging between 0.2 and 2 times the size of a pixel of the two-dimensional detector in the detection plane.

Even though it has been described using a certain number of embodiments, the wavefront analysis device and the microscopic imaging systems and methods using the wavefront analysis device include different variants, modifications and improvements that will be obvious to a person skilled in the art, with it being understood that these different variants, modifications and improvements form part of the scope of the invention as defined by the following claims.

BIBLIOGRAPHICAL REFERENCES

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