Device for wavefront analysis and microscopic imaging systems with such analysis devices

Description

[0001] The present invention contains a description and a set of claims. It also contains figures, an abstract, and an abstract figure. Technical field of the invention

[0002] This description relates to wavefront analysis devices, microscopic imaging systems comprising such analysis devices, and microscopic imaging methods using such analysis devices. More specifically, this description relates to optically sectioned fluorescence microscopy imaging systems and methods, for example, light-sheet or multiphoton fluorescence microscopy imaging systems and methods. State of the art

[0003] In microscopic imaging, particularly at high resolution, image quality in terms of resolution and contrast is directly related to the wavefront incident on the imaging detector, for example, a camera. The wavefront, that is, the surface of equal phase of a wave, is, in a microscopic imaging system, perturbed both by optical defects introduced by optical elements of the imaging system, such as manufacturing defects of the optical elements, alignment defects, variations in refractive index between the immersion medium of the microscope objective and the object, and by the object itself.This is particularly true in the case of transparent or partially transparent biological objects, when imaging a plane deep within the object. Indeed, in biological objects, the spatial distribution of the refractive index is inhomogeneous at the microscopic scale due to the complexity of biological structures. Rays originating from different points within a plane deep within the object are therefore perturbed by the successive layers they pass through. The wavefront from each of these points is thus different from a perfect wavefront, the latter being defined as a planar or spherical wavefront, and the corresponding microscopic image is degraded accordingly.

[0004] Adaptive optics (AO) is a technique for dynamically modifying a wavefront, and, particularly when used in an imaging system, for correcting any wavefront defects at each point in order to restore the quality of the images produced by that system. Typically, an AO system comprises three main elements: a wavefront analysis (or measurement) device, a wavefront correction device, and a control device that transforms a parameter from the wavefront analysis into an optimal correction to be applied by the correction device.When used in microscopic imaging, AO significantly improves imaging performance by correcting optical defects introduced by the optical system and the object of interest, according to various specific implementations, such as those described for example in "Adaptive optics for fluorescence microscopy" by MJ Booth. et al. [Ref 1].

[0005] The methods for implementing optical alignment (AO) in microscopic imaging developed to date all rely on the use of a wavefront correction device that locally modifies the phase of an optical wave. Examples of such correction devices include deformable mirrors, liquid crystal modulators (or SLMs for "Spatial Light Modulator"), and deformable lenses. A deformable mirror, for instance, comprises a reflective membrane and a set of actuators that allow for controlled local deformation of the membrane.

[0006] However, the methods for implementing optical aberration (OA) in microscopic imaging differ in their methods for measuring optical defects for correction purposes. Two main categories can be distinguished for measuring optical defects: indirect measurement methods based on analyzing the image produced by the microscope using quality criteria representative of the wavefront quality that led to the formation of said image, and direct measurement methods for optical defects using wavefront analyzers, for example, Shack-Hartmann type analyzers.

[0007] The first approach (indirect measurement) allows for simplified—and therefore less expensive—instrumental implementation due to the absence of a wavefront analyzer. However, it is based on global optimization requiring the use of iterative algorithms, whose convergence robustness, execution speed, and calibration are limiting factors, particularly in the case of dynamic imaging of living objects.

[0008] Thus, particularly for microscopic imaging of biological objects, methods based on the direct measurement of optical defects coupled with wavefront correction have demonstrated their ability to achieve very good correction of optical defects introduced by the imaging system and the object, and a substantial improvement in image contrast and resolution. Such methods are described, for example, in the review article by N. Ji, “ Adaptive optical fluorescence microscopy » [Ref. 2].

[0009] However, the direct measurement of optical defects remains complex to implement; indeed, it generally requires a "point source" emitting a single wavefront. Currently, the most effective approaches consist of optically inducing or isolating a light-emitting volume (also called an "artificial star" or "guide star") within the image. In one example, the artificial star is created by using fluorescent beads, approximately the size of which is equal to the diffraction limit of the microscope objective used, placed within the object of interest, as described in US patent 855730 B2 [Ref. 3], which necessitates a substantial modification of the object. See also the article by R. Jorand et al. [Ref. 6] or the article by H. Masayuki et al. [Ref. 7]. According to a second example described in published patent application US 2015 / 0362713 [Ref. 4], the artificial star is created using an ultrashort laser that locally generates a 2-photon fluorescence emission volume in the object of interest.

[0010] These approaches require complex implementation in microscopic imaging, both in terms of object preparation and instrument design. Furthermore, using a point source as the wavefront source means that measuring object-induced optical defects—and therefore the corresponding AO correction—is valid only within a field limited by the object's isoplanetary domain. This means the field at the object's imaging plane is where the optical defects vary sufficiently small that the resulting image shows no significant degradation.However, the majority of biological objects of interest in high-resolution microscopy consist of multiple microscopic structures corresponding to significant phase fluctuations: the isoplanetary domain is generally limited to a hundred square microns, which represents an important limitation for the high spatio-temporal resolution study of large structures, such as the study of neural networks in the field of neuroimaging.

[0011] Very recently, a method of optical analysis (OA) has been proposed in a light-sheet microscopy imaging system that eliminates the need for a point source to measure optical defects, as described in the article by K. Lawrence et al., "Scene-based Shack-Hartmann wavefront sensor for light-sheet microscopy" [Ref. 5]. This method employs a Shack-Hartmann wavefront analyzer in which each microlens forms an image of an extended object. Wavefront analysis is performed using cross-correlations between the images formed by the different microlenses, resulting in a two-dimensional map of the local wavefront gradients.

[0012] The applicant, however, highlighted limitations of the method described above. In practice, overlaps are observed between the images formed by the microlenses, which can lead to errors in cross-correlation calculations and inaccuracies in wavefront analysis. Furthermore, the described method only allows for an analysis of the average wavefront over a given field without taking into account the object's isoplanetary domains.

[0013] The purpose of this description is, in particular, to propose a wavefront analysis device that makes it possible to overcome all or part of the aforementioned limitations. Summary of the invention

[0014] According to a first aspect, the present description relates to a fluorescence microscopic imaging system with optical sectioning of a volumetric and fluorescent object according to claim 1.

[0015] A wavefront, as defined in this description, is the surface of equal phase of a light wave.

[0016] According to the present description, a characteristic parameter of the wavefront includes a local gradient (or local slope) along two dimensions of the wavefront in the analysis plane.

[0017] According to this description, determining a two-dimensional map of the local gradient involves determining the variations in the positions of the images formed by the microlenses. These variations are measured relative to reference positions of images formed with a reference wavefront, for example, a plane wavefront. According to this description, the variations in the positions of the images formed by the microlenses are determined by cross-correlation operations between the images.

[0018] According to one or more embodiments, a characteristic parameter of the wavefront further includes a local deviation of the intercepted wavefront in the analysis plane from a reference wavefront corresponding to a light wave that has not undergone optical defects, for example, a planar wavefront. The two-dimensional map of these local deviations of the wavefront from a reference wavefront can be obtained from the two-dimensional map of the local wavefront slopes.

[0019] The applicants demonstrated that the wavefront analysis device described above offers a significantly improved accuracy in analysis compared to known state-of-the-art devices, while eliminating the need for artificial star generation. Indeed, the field diaphragm arranged in the wavefront analysis device allows control of the image size formed by each microlens at the detection plane and limits the overlap between two images formed by two adjacent microlenses, without limiting the size of the field imaged by the microscopic imaging system to which the wavefront analysis device is connected.

[0020] For the purposes of this description, a "microlens" is defined as any focusing optical element with lateral dimensions (i.e., measured in the plane of analysis) of 5 mm or less, a focal length of 20 mm or less, and a pupil size of 5 mm or less. A microlens may, for example, consist of a transparent material with two diopters, at least one of which is not planar; the non-planar diopter could be, for example, a convex diopter, such as a spherical diopter.

[0021] According to one or more embodiment examples, the microlenses of the microlens matrix are identical, for example arranged according to a two-dimensional matrix.

[0022] According to one or more embodiment examples, the microlenses of the microlens matrix are contiguous and have a square pupil.

[0023] According to one or more embodiments, the field diaphragm is a transmission field diaphragm and includes, for example, an aperture of given finite dimensions. According to one or more embodiments, the field diaphragm is a reflection field diaphragm and includes a reflecting element of given finite dimensions. In the following description, the shape and / or dimensions of the field diaphragm may refer, unless otherwise specified, to both the shape and / or dimensions of an aperture for a transmission field diaphragm and the shape and / or dimensions of a reflecting element for a reflection field diaphragm.

[0024] According to one or more embodiment examples, the field diaphragm has a shape similar to the shape of a microlens pupil.

[0025] According to one or more embodiment examples, the field diaphragm includes dimensions determined so that two adjacent images formed by two adjacent microlenses do not overlap.

[0026] In particular, according to one or more embodiment examples, the analysis device further includes one or more optical element(s) enabling optical conjugation between the field diaphragm plane and the detection plane, and the dimensions of the field diaphragm are less than or equal to the dimensions of a microlens divided by the optical magnification defined by the optical element(s).

[0027] According to one or more exemplary embodiments, said optical element(s) comprise said optical relay system.

[0028] Thus, according to one or more embodiment examples, for a matrix of contiguous square microlenses of which each microlens has a side of size dm, if said optical magnification is G, the field diaphragm comprises a square shape and of size dm / G.

[0029] According to one or more embodiment examples, the field diaphragm has dimensions less than or equal to an analysis field corresponding to a given isoplanetic domain.

[0030] An isoplanetary domain is defined as a finite field of the object within which optical defects vary sufficiently small. Thus, wavefront measurements taken from areas at any point within an isoplanetary domain exhibit the same wavefront defects in a given analysis plane, to within a fraction of the analysis wavelength, for example, to within a quarter of the analysis wavelength. In practice, this means that the same correction can be applied to correct the wavefront throughout the entire isoplanetary domain.

[0031] According to one or more embodiments, the field diaphragm has variable dimensions. This makes it possible to vary the size of the analysis field, which allows for the determination of an isoplanatic field of the object. For example, an isoplanatic domain of the object is determined by performing a series of wavefront measurements, each measurement being carried out for a decreasing field diaphragm size from the maximum size. The isoplanatic domain corresponds to the largest size beyond which the measured wavefront does not show a significant change, that is, no change greater than a fraction of the analysis wavelength, for example, no change greater than a quarter of the analysis wavelength.

[0032] According to one or more embodiment examples, the size varies between a size for which two adjacent images formed by two adjacent microlenses have no overlap and a smaller size.

[0033] According to one or more embodiment examples, the field diaphragm is square in shape, and the dimension of the side is variable.

[0034] According to one or more embodiments, the position of the field diaphragm is variable in the plane conjugate to the detection plane of the analysis device. It is thus possible to measure the wavefront as a function of the transverse position of the analysis field relative to the focal plane of the microscope objective, which allows, where necessary, the application of an optimal correction based on this position using an optical defect correction system.

[0035] Indeed, for a complex object, the isoplanetary domain has a limited size. The microscopic imaging system images a field of view of a given size. The analysis field (of the analyzer) is typically smaller, and the isoplanetary domain is possibly even smaller. By moving the diaphragm, it is possible to measure the optical defects of the wavefront at different positions within the imaging field, and even to determine the isoplanetary domain at these different positions.

[0036] In one or more embodiments, the field diaphragm exhibits spatially structured optical transmission (or optical reflection). This structuring of optical transmission (or optical reflection) allows for the emergence of additional intensity patterns in the images formed by the microlenses, which can improve intercorrelations between images during image processing.

[0037] According to one or more embodiments, said optical transmission (respectively, optical reflection) is structured to comprise a regular alternation of transparent and opaque areas along a given direction (respectively, a regular alternation of reflective and non-reflective areas along a given direction), with a spatial frequency greater than or equal to the cutoff frequency of the microlenses in the microlens array. This particular configuration allows additional intensity patterns, resulting from a moiré effect, to appear in the images of the object formed by the microlenses. This configuration is particularly advantageous when the object contains only structures whose spatial frequencies are greater than the cutoff frequency of the microlenses in the microlens array.

[0038] According to one or more embodiments, the two-dimensional detector comprises a two-dimensional arrangement of elementary detectors, and a diffraction spot of a microlens comprises between 0.2 and 5 elementary detectors along a given direction, advantageously between 0.2 and 2 elementary detectors along a given direction. The dimension of a diffraction spot of a microlens along a given direction is defined as the distance separating the first two intensity minima located on either side of the intensity maximum. The applicants have shown that this particular configuration represents a good compromise between wavefront measurement accuracy and the size of the analysis field.

[0039] In this description, fluorescence is understood as the emission of light from an object resulting from light excitation by absorption of photons in a given spectral absorption band. Fluorescence light emission can result from a linear, one-photon mechanism or a non-linear, two- or more-photon mechanism. This mechanism may result from the interaction of absorbed light with a fluorescent element constituting the object or with a fluorescent element added to the object, such as, for example, in the case of a biological object, a fluorescent protein.

[0040] A three-dimensional fluorescent object thus includes any object comprising microscopic structures, possessing intrinsic fluorescence properties or rendered fluorescent by the addition of a "marker." A three-dimensional fluorescent object includes, for example, a biological object such as a cell, a cell culture, or an animal; these biological objects possess fluorescence properties, whether intrinsically constituting the object or induced by the addition of fluorescent elements. Among the three-dimensional fluorescent objects of interest, fluorescent neuronal structures in an animal brain are particularly noteworthy in neuroimaging studies.

[0041] An optical sectioning fluorescence microscopy imaging system, as defined herein, is an imaging system equipped with a microscope objective in which only fluorescence light from an optical section of the object, perpendicular to an optical axis of the microscope objective, is detected. An optical section can be created by various methods of illuminating the object known from the state of the art. An optical section can have a thickness on the order of or less than the depth of field of the microscope objective.

[0042] Wavefront analysis includes the determination of a two-dimensional map of a characteristic parameter of the wavefront measured in the analysis plane.

[0043] The characteristic parameter includes, according to the present description, a local gradient (or local slope) along two dimensions of the wavefront in the analysis plane.

[0044] According to one or more embodiments, the processing unit is configured to determine, from the two-dimensional map of local slopes, a two-dimensional map of the deviation of said wavefront from a reference wavefront corresponding to a flawless light wave, for example, a plane wavefront. The two-dimensional map of the deviation of said wavefront from a reference wavefront can be used to determine a property of the object, for example, the presence and distribution of inhomogeneities within the object.For example, in the case where the object is a cell, the refractive index of the cell nucleus is different from that of the cytoplasm; a two-dimensional map of the wavefront deviation from a reference wavefront can allow visualization of the cell nucleus, and the wavefront values ​​at each point can deduce certain properties of the cell such as its physiological state, whereas intensity information from the single imaging channel of the object does not allow obtaining such information.

[0045] According to one or more embodiments, the imaging channel further includes a signal processing unit for the signals from the imaging detector. Of course, in practice, the processing units of the analysis channel and the imaging channel can be combined into a single unit.

[0046] According to one or more embodiment examples, the beam splitter element is of the dichroic type, that is to say that it allows the incident light to be separated according to a first spectral band in reflection and according to a second spectral band different from the first spectral band in transmission.

[0047] According to one or more embodiment examples, the dichroic type beam splitter element allows the two spectral bands to be separated towards the imaging channel and towards the analysis channel.

[0048] According to one or more exemplary embodiments, the lighting path includes at least two lighting sources, one of the sources being intended for fluorescence excitation for the imaging path, another source being intended for fluorescence excitation for the analysis path, the latter being configured so as to produce structured illumination of the object according to a two-dimensional cross-shaped pattern.

[0049] This implementation allows, in the case of an object producing a uniform or very low contrast fluorescence emission, to obtain in the detection plane of the wavefront analysis device images produced by the microlenses of the wavefront analysis device according to a geometry facilitating the calculation of intercorrelation between said images.

[0050] According to one or more exemplary embodiments, the microscopic imaging system of the second aspect further comprises a wavefront correction device including a correction plane optically conjugate to the pupillary plane of the microscope objective. This correction plane is included in the analysis channel, and the correction device is configured to correct a wavefront originating from the object and analyzed by said analysis device.

[0051] Wavefront correction refers to the local modification of the wave phase within the correction plane to obtain a reference wavefront. Depending on the intended use of the wavefront correction method, the reference wavefront can be a plane wavefront, such as for optimizing the performance of an imaging system, or a specific wavefront.

[0052] A wavefront correction device may include, for example, a deformable mirror, typically consisting of a membrane and actuators that locally modify the membrane's axial position. The wavefront correction device may also include a spatial light modulator (SLM), generally composed of a two-dimensional arrangement of liquid crystal cells coupled to electrodes that locally modify the refractive index of these cells. The wavefront correction device may also include a deformable lens, typically composed of active elements that locally modify the lens's shape and / or thickness.

[0053] According to one or more embodiment examples, the processing unit of the analysis device is configured to control the correction device based on the wavefront analysis in the analysis plane.

[0054] According to one or more exemplary embodiments, the optical sectioning fluorescence microscopy imaging system is of the "light sheet" type. The illumination path is then configured to form said optical section by transverse illumination of the object.

[0055] According to one or more exemplary embodiments, the light-sheet fluorescence microscopy imaging system includes a correction device, and the correction plane of the correction device is common to both the analysis and imaging channels. Wavefront correction using the correction device improves image quality, particularly when the focal plane of the microscope objective is located deep within the object, by compensating for optical defects induced by inhomogeneities in the object between its surface and said focal plane.

[0056] According to one or more embodiment examples, the fluorescence microscopic imaging system is of the multiphoton type.

[0057] According to one or more embodiment examples, the illumination path of the multiphoton imaging system comprises: one or more laser source(s) for the emission of ultrashort pulses, said microscope objective enabling each pulse to be focused at a focal point of a focal plane of said objective to form a multiphoton fluorescence emission, a scanning device configured for transverse scanning of said focal point.

[0058] According to one or more exemplary embodiments, the multiphoton fluorescence microscopy imaging system includes a correction device, and the correction plane is common to both the analysis and illumination channels. Wavefront correction using the correction device improves the focusing quality within the object, and consequently the fluorescence signal at each point in the image, by compensating for optical defects induced by inhomogeneities in the object between its surface and the focal plane at the point where each pulse passes through.

[0059] According to a second aspect, the present description relates to optical sectioning fluorescence microscopy imaging processes implemented by systems according to the first aspect and its various embodiments.

[0060] More specifically, the present description relates to a method for microscopic imaging of an object according to claim 13.

[0061] According to the present description, wavefront analysis includes the determination of a two-dimensional map of local wavefront slopes.

[0062] According to one or more exemplary embodiments, the microscopic imaging process further includes the correction of a wavefront from said optical section of the object in a correction plane conjugate with a pupillary plane of the microscope objective, said correction plane being positioned in the analysis channel.

[0063] According to one or more embodiment examples, the beam splitter element is dichroic, imaging is performed in a first spectral band and analysis is performed in a second spectral band, distinct from the first spectral band.

[0064] This implementation is made possible, for example, by the introduction of two types of fluorescent elements within the object, and makes it possible not to use photons of the fluorescent emission light intended to form the image of the object to perform the wavefront analysis, which is particularly critical in the case of weakly fluorescent objects as is generally the case for biological objects.

[0065] According to the present description, the illumination of the object includes a first illumination for fluorescence excitation for the imaging channel, and a second illumination for fluorescence excitation for the analysis channel, the second illumination producing structured illumination of the object in a two-dimensional cross-shaped pattern.

[0066] According to one or more embodiment examples, the field diaphragm is of variable dimensions and the microscopic imaging procedure further includes wavefront analysis for different dimensions of the field diaphragm and determination of at least one isoplanetism domain from the different wavefront analyses.

[0067] According to one or more embodiment examples, the position of the field diaphragm is variable in the plane of the field diaphragm and the microscopic imaging method further includes the analysis of the wavefront for different positions of the field diaphragm and the determination of a plurality of isoplanetism domains from the different wavefront analyses.

[0068] According to one or more exemplary embodiments, the microscopic imaging process further includes the sequential correction of a wavefront from said optical section of the object in different areas of the field, as a function of said plurality of isoplanetism domains. Brief description of the figures

[0069] Other advantages and features of the invention will become apparent upon reading the description, illustrated by the following figures: [ Fig. 1 [ ] represents a diagram illustrating an example of a wavefront analysis device as described herein, connected to a microscopic imaging system; [ Fig. 2A ] represents images formed by a microlens array in a detection plane of a wavefront analysis device as described herein, by way of example; [ Fig. 2B ] represents an image formed on an imaging detection plane of an imaging system, in a microscopic imaging system as described herein, by way of example; [ Fig. 3 ] represents an image of a biological object formed on an imaging detection plane of an imaging system, as well as images formed in a detection plane by a microlens array of a wavefront analysis device according to an example in this description, for two analysis fields of different sizes and positions defined by the sizes and positions of the field diaphragm; Fig. 4 ] represents a diagram illustrating an example of a "lightsheet" type fluorescence microscopy imaging system, as described herein; Fig. 5 ] represents a diagram illustrating an example of a "multiphoton" type fluorescence microscopy imaging system, as described herein; microscope; the analysis channel being separated from the imaging channel by means of a beam splitter.

[0070] According to one or more embodiment examples, wavefront analysis includes the determination of a two-dimensional map of a wavefront parameter, for example the determination of a two-dimensional map of the local wavefront slopes.

[0071] According to one or more exemplary embodiments, the microscopic imaging process further includes the correction of a wavefront from said optical section of the object in a correction plane conjugate with a pupillary plane of the microscope objective, said correction plane being positioned in the analysis channel.

[0072] According to one or more embodiment examples, the beam splitter element is dichroic, imaging is performed in a first spectral band and analysis is performed in a second spectral band, distinct from the first spectral band.

[0073] This implementation is made possible, for example, by the introduction of two types of fluorescent elements within the object, and makes it possible not to use photons of the fluorescent emission light intended to form the image of the object to perform the wavefront analysis, which is particularly critical in the case of weakly fluorescent objects as is generally the case for biological objects.

[0074] According to one or more exemplary embodiments, the illumination of the object includes a first illumination for fluorescence excitation for the imaging channel, and a second illumination for fluorescence excitation for the analysis channel, the second illumination producing a structured illumination of the object according to a two-dimensional pattern.

[0075] According to one or more examples of implementation, the two-dimensional pattern is in the shape of a cross.

[0076] According to one or more embodiment examples, the field diaphragm is of variable dimensions and the microscopic imaging procedure further includes wavefront analysis for different dimensions of the field diaphragm and determination of at least one isoplanetism domain from the different wavefront analyses.

[0077] According to one or more embodiment examples, the position of the field diaphragm is variable in the plane of the field diaphragm and the microscopic imaging method further includes the analysis of the wavefront for different positions of the field diaphragm and the determination of a plurality of isoplanetism domains from the different wavefront analyses.

[0078] According to one or more exemplary embodiments, the microscopic imaging process further includes the sequential correction of a wavefront from said optical section of the object in different areas of the field, as a function of said plurality of isoplanetism domains. Brief description of the figures

[0079] Other advantages and features of the invention will become apparent upon reading the description, illustrated by the following figures: [ Fig. 1 [ ] represents a diagram illustrating an example of a wavefront analysis device as described herein, connected to a microscopic imaging system; [ Fig. 2A ] represents images formed by a microlens array in a detection plane of a wavefront analysis device as described herein, by way of example; [ Fig. 2B ] represents an image formed on an imaging detection plane of an imaging system, in a microscopic imaging system as described herein, by way of example; [ Fig. 3 ] represents an image of a biological object formed on an imaging detection plane of an imaging system, as well as images formed in a detection plane by a microlens array of a wavefront analysis device according to an example in this description, for two analysis fields of different sizes and positions defined by the sizes and positions of the field diaphragm; Fig. 4 ] represents a diagram illustrating an example of a "lightsheet" type fluorescence microscopy imaging system, as described herein; Fig. 5 ] represents a diagram illustrating an example of a "multiphoton" type fluorescence microscopy imaging system, as described herein; Fig. 6 ] represents a diagram illustrating an example of the appearance of intensity patterns by the Moiré phenomenon resulting from the superposition of two intensity patterns comprising different spatial frequencies corresponding respectively to a spatial frequency of microstructures of an object and to a spatial frequency of structuring of the field diaphragm; [ Fig. 7A ] represents a diagram illustrating an example of structured illumination of an object, according to a two-dimensional cross-shaped pattern, in an example of a "lightsheet" type fluorescence microscopy imaging system as described herein; Fig. 7B ] represents a diagram illustrating an example of structured illumination of an object, according to a two-dimensional cross-shaped pattern, in an example of a "multiphoton" fluorescence microscopy imaging system as described herein; Fig. 8 ] represents a diagram illustrating the geometric effect produced at the detection plane by a non-zero wavefront gradient at the scale of a microlens of a microlens array of a wavefront analysis device, according to the present description. Detailed description of the invention

[0080] There Fig. 1 schematically illustrates an example of a wavefront analysis device 110 according to this description, connected to an optical sectioning fluorescence microscopy imaging system 100.

[0081] The optical sectioning fluorescence microscopy imaging system 100 includes one illumination channel (not shown in the Fig. 1 ) for the illumination of an optical section of a volumetric and fluorescent object 10.

[0082] The microscopic imaging system 100 also includes an imaging channel 103 and an analysis channel 101 comprising the wavefront analysis device 110.

[0083] Imaging pathway 103 includes, in the example of the Fig. 1 a microscope objective 130 including a pupil 131 in a pupillary plane P 4, a focusing optic 134, a fluorescence filter 132, an imaging detector 140 including a detection plane P 6, a processing unit 142 for processing the signals acquired by the detector 140.

[0084] Depending on the type of microscopic imaging system (e.g. light sheet or multiphoton), the 140 imaging detector can be a two-dimensional detector, e.g. a CCD (Charge Coupled Device) camera or a highly sensitive CMOS (Complementary Metal Oxide Sensor) camera, such as sCMOS cameras, or a point detector (e.g. a photomultiplier).

[0085] The object 10 of interest is positioned in the vicinity of a focal plane P 1 of the microscope objective 130. The object 10 is, for example, a transparent or semi-transparent sample, such as a fluorescent biological object, and the focal plane P 1 of the microscope objective 130 is, for example, located at a non-zero distance from the surface of the object 10, so as to make an image of a plane located in depth.

[0086] The Microscopic Imaging System 100 is an optical sectioning fluorescence imaging system, meaning a microscope that, using various techniques, selects light coming only from an optical section perpendicular to the optical axis of the microscope objective. The optical section is located in the focal plane of the objective, and its thickness can be less than the depth of field of the microscope objective. Several technical approaches exist for creating an optical section within a fluorescence microscope, such as light-sheet fluorescence microscopy and multiphoton fluorescence microscopy, examples of which will be described with reference to [reference to relevant documentation]. Figs. 4 And 5 For the sake of simplicity, the object illumination path, including means for generating a fluorescence optical section, is not shown on the Fig. 1 .

[0087] The fluorescence filter 132 is a spectral filter that selects only the spectral band corresponding to fluorescence emission, thereby suppressing any excitation light that may be backscattered by the object. It can be a high-pass, low-pass, or band-pass filter, depending on the relative spectral characteristics of the excitation beam and the fluorescence emission beam.

[0088] The focusing optics 134 allows an image of an optical section superimposed on the focal plane P 1 on the imaging detection plane P 6 to be formed. The focusing optics 134 may include one or more lenses, often called "tube lens(es)," and has characteristics which, combined with the characteristics of the imaging detector 140, in particular the size of an elementary detector or pixel of the imaging detector 140, allow sampling by the detector 140 of the focal plane P 1 without spectral overlap effect, i.e. satisfying the correct sampling conditions defined by the Shannon theorem.

[0089] The microscopic imaging system 100 also includes a beam splitter element 135 which allows a portion of the fluorescence light emitted by the object to be taken from the imaging channel 103 and sent to the wavefront analysis device 110 of the analysis channel.

[0090] According to one or more exemplary embodiments, the beam splitter element 135 comprises a splitter cube or a splitter blade, allowing the transmission (or reflection) of a proportion of the fluorescence light from the object to the imaging detector 140 and the reflection (or transmission) of the remainder of the fluorescence light from the object to the wavefront analysis device 110, this proportion being for example 50%.

[0091] According to one or more embodiments, the beam splitter 135 comprises a dichroic plate or cube, enabling the transmission (or reflection) of a first spectral band of fluorescence light from the object to the imaging detector 140 and the reflection (or transmission) of a second spectral band of light from the object to the wavefront analysis device 110, these two spectral bands not overlapping. For example, the use of a dichroic beam splitter is carried out concurrently with the use of one or more fluorescence excitation sources and two types of fluorescent markers on the object, so as to obtain two distinct fluorescence emission spectra. This avoids the need for the imaging detector 140 to sample signal useful for image formation for wavefront measurement purposes.The photometric balance is thus optimal on both the imaging channel 103 and the analysis channel 101. For example, in the case of a biological object such as the brain of model animals in neuroimaging (Drosophila, Zebra Fish), an anatomical fluorescent marker can be used for wavefront measurement by the wavefront analysis device 110, and a specific fluorescent marker for imaging, for example a marker of calcium activity associated with the response of individual neurons, these 2 markers emitting a fluorescent signal following 2 distinct spectral bands.

[0092] The wavefront analysis (or measurement) device 110 shown on the fig. 1 includes a two-dimensional detector 112 with a detection plane P3, configured for the detection of light signals from a microlens array 114 115, said array being arranged in an analysis plane P5. The wavefront analysis device 110 also includes an optical relay system 116 configured to optically conjugate the analysis plane P5 and the pupil plane P4 of the microscope objective 130, a field diaphragm 118 and a processing unit 120 for processing the signals from the detector 112.

[0093] The two-dimensional detector 112 is, for example, a two-dimensional CCD or CMOS camera configured to detect fluorescence light emitted from the object within a specific spectral band. Typically, fluorescence microscopy uses fluorescent markers emitting in the visible or near-infrared spectral band, for example, between 400 and 900 nm. However, it is possible to adapt the detector's sensitivity to other spectral bands if necessary, by using other detection technologies, such as other photosensitive materials at the pixel level of the two-dimensional detector. For example, InGaAs is a sensitive material between 0.9 µm and 1.7 µm for the far-infrared, a spectral range in which many fluorescent markers are under development due to the greater depth of light penetration, particularly in scattering media.

[0094] The microlens array 114 comprises a two-dimensional arrangement of focusing optical elements 115 arranged in an analysis plane P5, for example, arranged according to a two-dimensional matrix. In particular, each focusing optical element 115—or microlens—is characterized by the same focal length fm, as well as the same pupil size dm, the pupil size being defined according to the shape of the microlenses. The microlens array 114 can be fabricated using various techniques, including, but not limited to, machining a substrate made of an optical material such as glass, photolithography applied to a photosensitive resin deposited on an optical substrate, iterative deposition of optical material onto a substrate to form a Fresnel lens array, and pressing plastic optical material using a mold.Microlenses typically have a round pupil, where dm corresponds to its diameter, or a square pupil, where dm corresponds to its side. Other pupil geometries are possible depending on the technology used, such as hexagonal pupils. Preferably, a square microlens array with contiguous edges is used, as the contiguous nature of this geometry allows for the use of all incident light, unlike round microlenses, which are not contiguous. To maximize transmission efficiency, an anti-reflective coating corresponding to the spectral band of the microlens array is generally applied. The microlens array 114 is positioned at a distance fm from the two-dimensional detector 112, with each microlens forming an image in the detection plane P3.

[0095] The beam splitter element 135 allows an intermediate image plane P2 conjugate to the object focal plane P1 to be defined in the wavefront analysis device 110. The field diaphragm 118 is arranged in the intermediate focal plane P2. The relay optical system 116, in combination with the microlens array 114, allows optical conjugation between the plane P2 and the detection plane P3, according to a magnification defined by the ratio of the focal lengths of the relay optical system 116 and the microlens array 114. Furthermore, the relay optical system 116, in combination with the focusing optics 134 (tube lens), allows optical conjugation between the pupillary plane P4 of the microscope objective 130 and the analysis plane P5 (plane of the microlens array), according to a magnification defined by the ratio of the focal lengths of the relay optical system 116 and the focusing optics 134.

[0096] The field diaphragm 118, located in the intermediate image plane P2, allows limiting the size of the imaged field by each microlens 115 of the microlens array 114. According to an advantageous implementation, the dimensions and geometry of the field diaphragm 118 are chosen such that no overlap is possible between 2 adjacent images from adjacent microlenses of the microlens array 114. For example, for square-shaped microlenses, of side dm, and a relay optical system 116 of focal length fc = 3fm, a square-shaped field diaphragm 118 with a side less than or equal to 3 dm can be defined. The field diaphragm is part of the wavefront analysis device 110 and therefore of the analysis channel 101 but it is not part of the imaging channel 105 so as not to minimize the field imaged by the imaging detector 140.

[0097] The processing unit 120 is configured to process the signals from the detector 112, and in particular to perform all operations on these signals to perform a measurement of the wavefront from an optical section of the object, that is to say to determine a characteristic parameter of the wavefront.

[0098] The processing unit 120 is generally configured for the implementation of calculation and / or processing steps carried out in processes according to this application. Generally, when reference is made in this description to calculation or processing steps for the implementation of process steps, it is understood that each calculation or processing step can be implemented by software, hardware, firmware, microcode, or any suitable 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 a computer (or processing unit) and / or executed by a computer (or processing unit) to implement these calculation or processing steps.

[0099] Of course, the processing unit 120 and the processing unit 140 can be grouped together in the same unit, for example a computer.

[0100] Each microlens 115 of the microlens array 114 thus forms an image on the detection plane P3 of the detector 112 of an optical section of a fluorescent, according to a field defined by the field diaphragm 118 as described previously. When the object and / or the optical imaging system exhibits no aberrations, each microlens forms, at the detection plane, an image of an optical section of the object centered on the optical axis of the microlens in question. When the plane P1 is located deep within a heterogeneous object, optical aberrations are present, particularly in the pupil 131, corresponding to a non-planar wavefront. In this case, a transverse shift is observed in each image formed by each microlens of the array 115 on the detection plane P3, this shift being proportional to the local derivative of the wavefront at the corresponding microlens, that is, to the slope of the wavefront.Since planes P 5 and P 4 are conjugate, each of these images corresponds to the image formed by detector 140 of imaging channel 103 but through a part of pupil 131, part corresponding to the image of a microlens of matrix P 3 by optics 116 and 134. This shift can be seen in a similar way to a Shack-Hartmann wavefront analyzer, for which we observe the shift of a diffraction spot from each microlens and not of an image, this type of sensor being used with a point source.

[0101] The geometric effect produced at the detection plane of a wavefront analysis system by a non-perfect wavefront at the scale of a microlens 115 of the microlens array 114, as well as an example of advantageous microlens sizing, will be described in more detail with reference to the FIG. 8 .

[0102] For example, the Fig. 2A Figure 202 shows the diffraction spots formed by a microlens array in a Shack-Hartmann wavefront analyzer, and Figure 204 shows the images formed by the microlenses of a microlens array in a wavefront analyzer as described herein. Figure 206 illustrates an image formed by a magnified microlens. Fig. 2B This represents the image of the same object formed on the detector of the imaging channel. In these examples, the object is a HeLa fluorescent cell containing a fluorescent tubulin marker, and the imaging system is a light-sheet type. In this example, the microlenses are contiguous and square-shaped, with a focal length-to-side ratio of 15, corresponding to a numerical aperture significantly smaller than that of the microscope objective. This explains the loss of resolution in the images formed by the microlenses, of which image 206 is an example. Consequently, the images formed by the microlenses perform low-pass filtering of the object's spatial frequencies compared to the image formed on the detector of the imaging channel. In this example, the area of ​​the analysis field defined by the field diaphragm is approximately one-quarter of the area of ​​the field imaged by the microscope.

[0103] Measuring the image shifts of all the microlenses allows us to deduce, using the processing unit 120, a two-dimensional wavefront slope map in the pupillary plane P4. By integration, a wavefront map can be derived from this. The image shifts of the microlenses are typically measured by cross-correlation operations of each image with respect to a reference image. This reference image is, for example, defined as the image formed by a reference microlens, such as a central microlens of the microlens array 114. By limiting the imaged field of each microlens to the detection plane P3, the field diaphragm 118 prevents the overlap of images from adjacent microlenses that could introduce errors during the cross-correlation calculation.

[0104] The wavefront analysis device 110, when implemented within an optically sectioning fluorescence microscope as illustrated for example on the Fig. 1 , allows for front measurement 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.

[0105] When implemented within an optically sectioning fluorescence microscope, the wavefront analysis device 110 enables the measurement of optical defects over an analysis field defined by the field diaphragm 118. When the analysis field is of dimensions similar to or smaller than an isoplanetic domain, the wavefront measurement is valid regardless of the point within the field. When the analysis field is larger than an isoplanetic domain, the wavefront measurement corresponds to the measurement of an average wavefront over the field defined by the diaphragm 118.

[0106] According to one or more examples of implementations, the field diaphragm 118 is said to be "active", that is to say that its dimensions and / or its position in the plane P 2 are variable.

[0107] Thus, the field diaphragm can, for example, have variable dimensions. For instance, a square transmission field diaphragm made of four opaque blades arranged in pairs to form a square transparent area can be mounted on movable elements, motorized or not, allowing adjustment of the relative distance between two opposing blades. A variable field diaphragm opening makes it possible to determine the isoplanetary domain of an object within a region of the imaging field, for example, by performing a series of wavefront measurements for a progressively decreasing field diaphragm size. When the wavefront between two successive measurements ceases to vary, the field diaphragm size corresponding to the first of the two measurements corresponds to the size of the object's isoplanetary domain within the considered region.

[0108] The field diaphragm 118 can also, as an example, have a variable position in the plane P 2. For example, the variable position is obtained by motorizing the transverse position, for example by means of piezoelectric motors or stepper motors.

[0109] By combining a variable position of the field diaphragm 118 with variable dimensions, it is possible to determine the dimensions of the isoplanetary domain in different areas of the field and to determine the optical defects of the wavefront in the analysis plane for the different areas of the imaging field. Advantageously, the dimensions of the field diaphragm 118 are chosen to be smaller than those of the isoplanetary domain determined in each of the areas.

[0110] There Fig. 3 represents an image 302 of a biological object formed on an imaging detection plane P 6 of an imaging system, as well as images 304, 306, formed in the detection plane P 3 of a wavefront analysis device according to an example of the present description, by a microlens array, for two analysis fields of different sizes and positions defined by the sizes and positions of the field diaphragm.

[0111] For biological objects in neuroimaging (Drosophila, mouse, and Zebrafish brains), the typical imaging field is 400 µm to 500 µm on each side. A corresponding "average" isoplanetary domain is approximately 150 µm on each side (reference available if needed).

[0112] There Fig. 4 represents a diagram illustrating an example of a 200-type "light sheet" fluorescence microscopy imaging system, as described herein.

[0113] The 200 fluorescence microscopy imaging system includes elements similar to those described in reference to the Fig. 1 , referenced on the Fig. 4 with identical references, and not repeated here so as not to make the description too long.

[0114] In light-sheet fluorescence microscopy, the illumination path 105 is configured to form an optical section by transverse illumination of the object. The illumination path may include one or more light sources (not shown in the diagram). Fig. 4 This is for the emission of one or more excitation beams, which can emit in different spectral bands. A fluorescence excitation beam forms a thin plane of light, generally of a thickness approximately similar to the depth of field of the microscope objective, and is incident on the object in a direction perpendicular to the optical axis of the microscope objective. The excitation beam does not pass through the sample outside the focal plane P1 of the objective, thus avoiding the emission of spurious fluorescence signals.

[0115] In the example shown on the Fig. 4 The "light sheet" type fluorescence microscopy imaging system includes a correction device 145 with a correction plane P 7 common to the analysis channel 101 and the imaging channel 103. Wavefront correction by means of the correction device improves the quality of the image formed on the detection plane P 6 of the detector 140, particularly when the focal plane of the microscope objective is located deep within the object, by compensating for optical defects induced by inhomogeneities of the object between its surface and said focal plane.

[0116] The 200 microscopic imaging system illustrated on the Fig. 4 It also includes, on the common part of the analysis and imaging channels, an afocal optical system 136, 137. It further includes, on the imaging channel 103, the tube lens 134 for forming the image on the imaging detection plane P 6, and, on the analysis channel 101, a lens 146 for forming the intermediate image plane P 2 in which the field diaphragm 118 of the wavefront analysis device 110 is arranged.

[0117] There Fig. 5 represents a diagram illustrating an example of a 300-type "multiphoton" fluorescence microscopy imaging system, as described herein.

[0118] The 300 fluorescence microscopy imaging system includes elements similar to those described in reference to the Fig. 1 , referenced on the Fig. 5 with identical references, and not repeated here so as not to make the description too long.

[0119] In multiphoton fluorescence microscopy, fluorescence emission is characterized by a non-linear relationship with the excitation beam: the fluorescence signal of interest is emitted only when a minimum power density is locally reached. This condition is typically met only when the excitation beam is focused, i.e., in the focal plane of the microscope objective, which inherently prevents the emission of a spurious fluorescence signal.

[0120] Thus, in the illustrated example that the Fig. 5 The illumination path 105 includes one or more laser sources 150 for emitting ultrashort pulses, optionally with one or more collecting optics 151. The microscope objective 130 focuses each pulse onto a focal point in the focal plane P1 of the objective to form a multiphoton fluorescence emission. The illumination path 105 further includes a scanning device 152 configured for transverse scanning of the focal point in the focal plane P1. In the example of the microscopic imaging system illustrated in the Fig. 5 The detector 140 includes, for example, a one-dimensional detector, such as a photomultiplier, configured to detect the light energy emitted by the object 10 and sent by the beam splitter 135 for each position of the focal point. Thus, the detector 140 cooperates with the scanning device 152 to form a two-dimensional image of the object.

[0121] According to one or more embodiments, the 300 multiphoton fluorescence microscopy imaging system includes a correction device 145 with a correction plane P 7. In this example, the correction plane P 7 is common to the analysis channel 101 and the illumination channel 105. Wavefront correction by means of the correction device improves the quality of the focus in the object, and consequently the fluorescence signal at each point of the image, by compensating for optical defects induced by inhomogeneities of the object between its surface and said focal plane at the passage of each pulse.

[0122] The 300 microscopic imaging system illustrated on the Fig. 5 It also includes, on the common part of the analysis and illumination channels, an afocal optical system 157, 158. It further includes, on the imaging channel 103, the tube lens 134 for forming the image on the imaging detection plane P 6, and, on the analysis channel 101, a lens 146 for forming the intermediate image plane P 2 in which the field diaphragm 118 of the wavefront analysis device 110 is arranged.

[0123] Regardless of the optical sectioning fluorescence microscopy imaging system implemented in this description, the applicant has shown that the use of cross-correlation calculations to determine the relative positions of the images formed by the microlenses results in a dependence of the measurement accuracy of these relative positions on certain characteristics of said images, in particular the size and contrast of the intensity patterns constituting these images.

[0124] Indeed, for example, a set of uniform images formed by microlenses does not allow for a precise cross-correlation calculation, as the correlation operation lacks any structure that would allow for the formation of a correlation peak whose position can be accurately determined. This situation can arise, for instance, for a homogeneous object, or for an object composed solely of details whose characteristic sizes are smaller than the minimum size that can be imaged by the microlenses of the microlens array.

[0125] THE Fig. 6 And Fig. 7 illustrate two examples of implementation of a microscopic imaging process according to the present description allowing to improve the processing of images formed in the detection plane P 3 of the wavefront analysis device 110 according to the present description.

[0126] There Fig. 6 This illustrates a first example in which the field diaphragm 118 is structured. For example, the field diaphragm is a transmission field diaphragm, and the transmission is structured, along a given direction, to form a regular alternation, with a given spatial frequency, of transmission and opaque areas. In another example, the field diaphragm is a reflection field diaphragm, and the reflection is structured, along a given direction, to form a regular alternation, with a given spatial frequency, of reflective and non-reflective areas.

[0127] Thus, image 602 on the Fig. 6 represents an example of structured transmission of a field diaphragm according to a regular alternation of transparent and opaque areas along a direction, defining a spatial frequency k 0 .

[0128] Image 601 represents an image, in the plane of the field diaphragm (P 2 , Fig. 1 ), of a theoretical fluorescent object that would consist of a regular alternation of fluorescent and non-fluorescent structures along a direction, with a spatial frequency k.

[0129] Image 603 shows the superposition of the two intensity patterns 601 and 602. When the two patterns of periodic intensity and different spatial frequencies overlap in the plane of the field diaphragm, a moiré effect is visible in a plane conjugate to said plane of the field diaphragm. As shown in image 603, the moiré effect creates an additional periodic pattern within a plane conjugate to the plane of the field diaphragm, the spatial frequency of which corresponds to the vector difference of the two spatial frequencies of the initial patterns, that is, a spatial frequency significantly lower than the spatial frequencies of the two initial patterns.

[0130] In practice, for any object, the spatial frequency content of the image of a plane of the object is complex and consists of a multitude of spatial frequencies. Some complex microscopic objects, particularly biological objects such as microtubule networks, may consist solely of structures with very small characteristic sizes, and therefore very high spatial frequencies. The wavefront analysis device described herein produces, in the detection plane, a set of conjugate images of the field diaphragm plane, specifically through a microlens array.This microlens array consists of individual microlenses with a numerical aperture significantly smaller than the numerical aperture of the microscope objective. This design achieves a compromise between the field of view imaged by each microlens and the sensitivity required for measuring displacement in the images produced by the microlenses through cross-correlation calculations. Consequently, the resulting images of the field diaphragm plane produced by the microlenses have a significantly reduced spatial frequency content at higher spatial frequencies, directly proportional to the numerical aperture of the microlenses. Therefore, for objects such as those described above, the microlens images may no longer contain sufficient detail for accurate cross-correlation calculations.In this case, by positioning a structured field diaphragm, for example according to a pattern such as illustrated in 602, the images produced by the microlenses reveal an additional intensity pattern following a Moiré phenomenon as illustrated by an example in image 603, at least one spatial frequency of which is likely to be transmitted in the detection plane P3. This additional pattern, characteristic of the object, advantageously allows for a significantly more precise cross-correlation calculation than in the absence of any Moiré pattern, particularly for objects such as those previously described.

[0131] When the image of the object does not correspond to an intensity pattern defining a unique spatial frequency such as represented by the Fig. 6 According to 601, the additional intensity pattern created by the Moiré phenomenon is a more complex pattern than the example shown in image 603.

[0132] For example, a transmission (or reflection) pattern of the field diaphragm with a specific spatial frequency can be chosen so that the resulting Moiré pattern (image 603) has a frequency lower than a maximum spatial frequency Fmax transmitted by the microlenses. This makes it possible to improve the wavefront measurement accuracy for objects where the majority of spatial frequencies lie between the maximum spatial frequency transmitted by the microlenses and twice this maximum spatial frequency. For square microlenses with side length dm, focal length fm, and central imaging wavelength λ, this maximum spatial frequency Fmax is given by Fmax = dm / λ fm.

[0133] THE Fig. 7A et 7B illustrate a second example in which the illumination of the object is structured. The Fig. 7A describes a structured illumination in an imaging system according to the present description, of the light sheet type and The Fig. 7B describes a structured illumination in an imaging system according to the present description, of the multiphoton type.

[0134] There Fig. 7A represents a diagram illustrating an example of a frontal representation, i.e., perpendicular to the optical axis, of an optical section 701 of an object in the focal plane P1 of a microscope objective of a light-sheet type optical sectioning fluorescence microscopy imaging system, as illustrated for example on the Fig. 4 .

[0135] Surface 702 schematically represents the analysis field, the size of which is defined by the field diaphragm of a wavefront analysis device as described herein. As previously explained, when the analysis field 702 corresponds to a homogeneous area in terms of fluorescence intensity emitted by the object, it is difficult to obtain a precise cross-correlation calculation between the images produced by the microlenses of the wavefront analysis device to determine the relative positions of said images, since a cross-correlation between two patterns of homogeneous intensity does not result in a correlation peak that can be precisely localized spatially.The applicant showed that structured illumination of the object at the optical section could then be used advantageously, allowing a set of images formed by the microlenses for which the intensity pattern to the illumination is present and allows the realization of an intercorrelation calculation defining a two-dimensional correlation peak.

[0136] Following the example of the Fig. 7A , corresponding to a simplified implementation, at least one additional source is used at the level of the lighting path 105 of the "light sheet" type, configured to illuminate the object according to two beams 703, arranged for example in 2 perpendicular directions and whose point of intersection is located in the surface 702. Any other lighting pattern comprising at least two non-parallel directions meets the need for structuring the lighting as presented.Advantageously, the additional source provides illumination according to a specific spectral band, enabling fluorescence emission from a specific spectral band different from the fluorescence emission produced by the illumination from the first source. This, combined with the use of a dichroic beam splitter between the imaging channel and the wavefront analysis device, makes it possible to avoid using light intended for the imaging channel for wavefront analysis and thus maximize the contrast of the images formed by the microlenses.

[0137] There Fig. 7B represents a diagram illustrating an example of a frontal representation, i.e., perpendicular to the optical axis, of an optical section 701 of an object in the focal plane P1 of a microscope objective of a multiphoton optical sectioning fluorescence microscopy imaging system, such as, for example, illustrated by the Fig. 5 .

[0138] Surface 702 again represents the analysis field, the size of which is defined by the field diaphragm of a wavefront analysis device according to the present invention. As previously explained, when the analysis field 702 corresponds to a homogeneous area in terms of fluorescence intensity emitted by the object, it is not possible to use an intercorrelation calculation between the images produced by the microlenses of the wavefront analysis device to determine the relative positions of said images, since an intercorrelation between two patterns of homogeneous intensity does not result in a correlation peak that can be precisely localized spatially.The applicant showed that in this type of microscopic imaging system, structured illumination of the object at the optical section level could also be used to obtain a set of images formed by the microlenses for which the intensity pattern to the illumination is present and allows the realization of an intercorrelation calculation defining a two-dimensional correlation peak.

[0139] Following the example of the Fig. 7B In a simplified implementation, at least one additional light source is used in the multiphoton illumination path, configured to illuminate the object according to a pattern 704 comprising, in this example, two perpendicular directions whose point of intersection is located in the surface 702. Practically, for a multiphoton microscope, this pattern is achieved by sequentially scanning the focal point of the additional source in the focal plane of the objective, typically using a pair of galvanometers. Any other illumination pattern comprising at least two non-parallel directions meets the structuring requirement as described.Advantageously, the additional source provides illumination according to a specific spectral band, enabling fluorescence emission from a specific spectral band different from the fluorescence emission produced by the illumination from the first source. This, combined with the use of a dichroic beam splitter between the imaging channel and the wavefront analysis device, makes it possible to avoid using light intended for the imaging channel for wavefront analysis and thus maximize the contrast of the images formed by the microlenses.

[0140] There Fig. 8 represents a diagram illustrating the geometric effect produced at the detection plane of a wavefront analysis system as described by the present invention by a non-perfect wavefront at the scale of a microlens 115 of the microlens array.

[0141] Let us suppose, for example, a microlens array 114 comprising a set of adjacent microlenses with square pupils of side dm and focal length fm as represented on the Fig. 8 in one dimension, for the sake of simplicity. Let us assume a complex wavefront 801 incident in the analysis plane P 5 corresponding to the microlens plane, such that at the level of a microlens the deviation of the wavefront from a perfect plane wavefront corresponds to a wavelength λ of the incident beam, as represented on the Fig. 8 .

[0142] For a wavefront analysis device such as the one described herein, the spatial resolution of the wavefront analysis corresponds to a microlens. The local wavefront measurable by a microlens thus corresponds to an elementary wavefront forming an angle α with the analysis plane such that α = δ / dm, where δ is the local deviation of the wavefront from a reference wavefront, in this example a plane wavefront. Thus, for a local deviation of the wavefront from a plane wavefront of δ = λ, the angle α is α = λ / dm, where λ is significantly smaller than dm. The diffraction spot formed at the focus of the microlens at the detection plane P3 is thus shifted transversely by a distance s, the shift s being given by: s = λf d

[0143] For a 115 microlens, the size tThe intensity of a diffraction spot measured between the first two minima of intensity located on either side of the maximum of intensity is given by: t = 2 λf d

[0144] It is known from the state of the art that, for a two-dimensional detector, such as a camera, composed 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 accuracy 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 described here, when the two-dimensional detector is dimensioned such that a diffraction spot corresponds to two pixels in one direction, and with a localization accuracy of one-hundredth of a pixel for an intensity pattern, it is thus possible to measure the deviation of a wavefront incident on a microlens with a maximum accuracy of λ / 100.Similarly, when the two-dimensional detector is sized so that a diffraction spot corresponds to two-tenths of a pixel in one direction, and for a localization accuracy of an intensity pattern of one-hundredth of a pixel, it is thus possible to measure the deviation of a wavefront incident on a microlens with a maximum accuracy of λ / 10. In practice, measuring a wavefront with an accuracy lower than λ / 10 does not allow this measurement to be used effectively for object characterization or imaging.

[0145] Thus, the applicant has shown that advantageously, the wavefront analysis device according to the present invention can be dimensioned such that a diffraction spot of a microlens of the microlens array has a dimension, in one direction, between 0.2 and 2 times the size of a pixel of the two-dimensional detector in the detection plane.

[0146] Although described through a number of embodiment examples, the wavefront analysis device and the microscopic imaging systems and methods using the wavefront analysis device include 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 form part of the scope of the invention as defined by the following claims. REFERENCES BIBLIOGRAPHIQUES

[0147] 1. M. J. Booth et al. « Adaptive optics for fluorescence microscopy », extrait de l'ouvrage « Fluorescence Microscopy : Super-resolution and other Novel Techniques », A. Cornea et al., Academic Press, 2014. 2. N. Ji « Adaptive optical fluorescence microscopy », Nature Methods 14, 374-380, 2017. 3. Azucena et al. Brevet US855730 B2 4. Betzig et al. Demande de brevet publiée US 2015 / 0362713 5. K. Lawrence et al. « Scene-based Shack-Hartmann wavefront sensor for light-sheet microscopy », Proc. SPIE 10502, Adaptive Optics and Wavefront Control for Biological Systems IV, 2018 6. R. Jorand et al. "Deep and Clear Optical Imaging of Thick Inhomogeneous Samples", PLOS ONE, vol. 7, no. 4, 25 avril 2012 7. H. Masayuki et al. "The development of an adaptive optics system and its application to biological microscope" Proceedings of SPIE, SPIE, US, vol. 10021, 31 octobre 2016

Claims

1. A fluorescence microscopic imaging system (100) with optical sectioning of a volumetric and fluorescent object (10) comprising: - an illumination path (105) for the illumination of the object; - an imaging path (103) for imaging an optical section of the object, comprising a microscope objective lens (130) with a pupil in a pupil plane (P4) and an imaging detector (140) comprising an imaging detection plane (P6), said optical section being superimposed on a focal plane (P1) of said microscope objective lens; - an analysis path (101) comprising said microscope objective lens and a wavefront analysis device (110) configured to analyze a wavefront originating from the object; - a beam splitter element (135) for splitting said analysis path and said imaging path; the analysis device comprising: - a two-dimensional detector (112) comprising a detection plane (P3) conjugated with said focal plane (P1) of the microscope objective lens; - a two-dimensional arrangement (114) of microlenses (115) arranged in an analysis plane (P5), each microlens being configured to form on the detection plane (P3) an image of the object located in the focal plane (P1) of the microscope objective lens, with a given analysis field-of-view; - an optical relay system (116) configured to optically conjugate the analysis plane and the pupil plane (P4); - a processing unit (120) configured to determine, based on the set of images formed by the microlenses, a two-dimensional map of a characteristic parameter of the wavefront in said analysis plane, wherein: - the characteristic parameter of the wavefront comprises a local slope in two dimensions of the wavefront in the analysis plane; and - determining said two-dimensional map comprises determining variations of the positions of the images formed by the microlenses, the variation of position of an image formed by a microlens being measured in relation to a reference position of a reference image, the variation of position being determined by a cross-correlation operation between said image and the reference image; the microscopic imaging system being characterized in that: - the wavefront analysis device comprises a field diaphragm (118) positioned in a plane (P2) optically conjugated with the detection plane (P3) of the analysis device and configured to define said analysis field-of-view; and in that: - the illumination path is configured for a first illumination of the object for the fluorescence excitation for the imaging path, and a second illumination of the object for the fluorescence excitation for the analysis path, the second illumination producing a structured illumination of the object according to a cross-shaped two-dimensional pattern.

2. The microscopic imaging system as claimed in claim 1, wherein said microlenses (115) have a square pupil and are joined together.

3. The microscopic imaging system as claimed in any one of the preceding claims, wherein the microlenses have a pupil of a given shape, the field diaphragm (118) having an identical shape to the shape of said pupil of the microlenses.

4. The microscopic imaging system as claimed in claim 3, further comprising one or more optical element(s) allowing optical conjugation to be carried out between the plane of the field diaphragm and the detection plane and wherein the dimensions of the field diaphragm are less than or equal to the dimensions of the pupil of a microlens, divided by the optical magnification (G) defined by the one or more optical element(s).

5. The microscopic imaging system as claimed in any one of the preceding claims, wherein the field diaphragm (118) has variable dimensions.

6. The microscopic imaging system as claimed in any one of the preceding claims, wherein the field diaphragm has a variable position in said plane (P2) optically conjugated with the detection plane (P3).

7. The microscopic imaging system as claimed in any one of the preceding claims, wherein the field diaphragm is spatially structured with a given spatial frequency in one direction.

8. The microscopic imaging system as claimed in any one of the preceding claims, wherein the two-dimensional detector comprises a two-dimensional arrangement of elementary detectors and a diffraction spot of a microlens comprises, in one direction, between 0.2 and 2 elementary detectors.

9. The microscopic imaging system as claimed in any one of the preceding claims, wherein said beam splitter element (135) is a dichroic element allowing light to be reflected in a first spectral band and light to be transmitted in a second spectral band, separate from the first spectral band.

10. The microscopic imaging system as claimed in any one of the preceding claims, further comprising a wavefront correction device (145) comprising a correction plane (P7) optically conjugated with the pupil plane (P4), said correction plane being included in the analysis path, said correction device being configured to correct a wavefront originating from the object and analyzed by said analysis device.

11. A light-sheet type fluorescence microscopic imaging system (200) as claimed in any one of the preceding claims, wherein the illumination path (105) is configured to form said optical section by transverse illumination of the object.

12. A multiphoton type fluorescence microscopic imaging system (300) as claimed in any one of claims 1 to 10, wherein the illumination path (105) comprises: - one or more laser source(s) (150) for emitting ultrashort pulses, said microscope objective lens (130) allowing each pulse to be focused at a focusing point of a focal plane of said objective lens in order to form a multiphoton fluorescence emission; - a scanning device (152) configured to transversely scan said focusing point.

13. A method for microscopic imaging of an object by means of a fluorescence microscopic imaging system with optical sectioning as claimed in any one of the preceding claims, comprising: - illuminating the object along an optical section by means of said illumination path; - imaging said optical section of the object in said detection plane of said imaging detector (140) of the imaging path; - analyzing a wavefront originating from said optical section of the object by means of said analysis path comprising said microscope objective lens and said wavefront analysis device; wherein: - the illumination of the object comprises a first illumination for the fluorescence excitation for the imaging path, and a second illumination for the fluorescence excitation for the analysis path, the second illumination producing a structured illumination of the object according to a cross-shaped two-dimensional pattern.