Method for fluorescence microscopy imaging and wavefront correction device for carrying out such a method - Patents.com

Abstract

The present description relates in particular to a method for fluorescence microscopic imaging of an object with a fluorescence microimaging system (200) using light sheet illumination, comprising generating a focused light beam and scanning the focused light beam to generate a light sheet (100), further comprising performing a wavefront analysis on an analysis field of the object, using spatial filtering of the fluorescent light, which comprises scanning a filtering element with respect to a fluorescence image of the focused light beam formed on a filtering surface (PO5) of the filtering element, the scanning of the filtering element being synchronized with the scanning of the focused light beam so as to obtain a superposition at each instant of time of the filtering element and the fluorescence image of the focused light beam.

Description

[Technical field]

[0001] The present application relates to a method for fluorescence microscopy imaging using light sheet illumination, to a wavefront correction device adapted for carrying out such a method, and to a microscopy imaging system equipped with such a wavefront correction device. [Background technology]

[0002] In microscopic imaging, especially in high-resolution imaging, the quality of the image, in terms of resolution and contrast, is directly related to the incident wavefront on the imaging detector (such as a camera). The wavefront, i.e. the equiphase surface of the wave, is perturbed in a microscopic imaging system by optical defects introduced by the optical components of the imaging system (e.g. manufacturing defects in the optical components, misalignment, mismatch of the refractive index between the immersion medium of the microscope objective and the object, etc.) and by the object itself. This is especially true in the case of transparent or partially transparent living bodies, where the spatial distribution of the refractive index becomes inhomogeneous on a fine scale when generating images corresponding to deep surfaces in the object, indeed deep surfaces in the living body, due to the complexity of the biological structure. Thus, light rays from each point of the deep surface in the object are deflected by multiple successive layers traversing the object. The wavefront from each of these points therefore differs from a reference wavefront, e.g. a plane or spherical wavefront, and the corresponding microscopic image is correspondingly worse. Adaptive optics (AO) is a technique that allows dynamic modification of the wavefront. In particular, when this technique is used in an imaging system, it allows correction of possible defects in the wavefront at each point, thereby restoring the quality of the image generated by said system. According to various specific embodiments, for example as described in Non-Patent Document 1 ([Reference 1]), the use of AO in microscopy imaging can significantly improve the imaging performance by correcting at least some of the optical defects introduced by the optical system and the target object.

[0003] All methods developed to date for implementing AO in microscopic imaging are based on the use of wavefront correction devices that allow the phase of a light wave to be locally modified. Known correction devices include, for example, deformable mirrors, liquid crystal modulators (or SLMs "spatial light modulators"), and also deformable lenses. A deformable mirror, for example, comprises a reflective membrane and a set of actuators that allow the local deformation of this membrane in a controlled way.

[0004] Nevertheless, the implementations of AO in microscopic imaging differ in terms of the method by which optical defects are measured for the purpose of correction. Two main categories of measurement of optical defects can be distinguished: indirect and direct methods. Indirect methods are based on the analysis of the image produced by the microscope by means of a quality criterion that describes the quality of the wavefront that led to the formation of said image. In direct methods, optical defects are measured by means of a wavefront analyzer, e.g. a Shack-Hartmann type analyzer.

[0005] The first approach (indirect measurement) allows for a simple instrument implementation and therefore lower cost since there is no wavefront analyzer. However, it is based on a global optimization that requires the use of an iterative algorithm. Its convergence reliability, execution speed and calibration are limited, especially for dynamic imaging of living organisms.

[0006] Thus, in particular for microscopic imaging of living organisms, methods based on direct measurement of optical imperfections accompanied by wavefront correction have demonstrated that optical imperfections caused by the imaging system and the object can be very well corrected, while at the same time a significant improvement in image contrast and resolution can be obtained. Such methods are described, for example, in [Reference 2].

[0007] However, techniques involving direct measurement of optical defects remain difficult to implement. Indeed, this measurement usually requires a "source point" emitting a single wavefront. Currently, the most effective techniques involve optically guiding or isolating a light-emitting volume (also called an "artificial star" or "guide star") in the image. According to a first example, the artificial star is created using fluorescent beads. Its size is approximately equal to the diffraction limit of the microscope objective lens used and placed on the object of interest, as described in US Pat. No. 5,399,433 ([Reference 3]). This requires significant modification of the object. According to a second example, described in US Pat. No. 5,399,433 ([Reference 4]), the artificial star is created using an ultrafast laser that generates a two-photon fluorescent emitting volume locally in the object of interest.

[0008] Therefore, these techniques require complex implementation in microscopic imaging, whether in the preparation of the target object or in the instrument design. Moreover, only by using a light source point as a wavefront source can the measurement of the optical defects induced by the object be valid, and thus the corresponding AO corrections. These are valid in the field of view limited by the isoplanatic patch of the object, i.e. in the area near the object's imaging plane, where the corresponding images do not show any significant degradation due to the extremely small amount of variation of its optical defects. However, the majority of living organisms of interest in high-resolution microscopy consist of multiple fine structures that correspond to significant phase variations. Isoplanatic patches are usually limited to about 100 square microns, which is a significant limitation in the study of large structures with high spatiotemporal resolution, for example, in the study of neural networks in the field of neuroimaging.

[0009] More recently, an AO method has been proposed in microscopic imaging systems using "light sheet" illumination (or "light sheet microscopy"). This obviates the need for a light source point for the measurement of optical defects. Such a method is described in [Reference 5]. For this purpose, a Shack-Hartmann wavefront analyzer has been proposed. Each microlens of this analyzer forms an image of the extended object. It is therefore named the extended source wavefront analysis device. The wavefront analysis is performed by cross-correlation between the images formed by the various microlenses, allowing the acquisition of a two-dimensional map of the local gradient (or local inclination) of the wavefront.

[0010] More recently, a fluorescence microscope imaging system using light sheet illumination with an improved extended source wavefront analyzer has been proposed. See US Pat. No. 5,399,433 ([Reference 6]). The wavefront analyzer described in US Pat. No. 5,399,433 comprises, inter alia, a two-dimensional detector having a detection surface and a two-dimensional arrangement of microlenses arranged in the analysis surface. When the analyzer is connected to a microscope imaging system, each microlens is configured to form in the detection surface an image of an object located in the focal plane of the microscope objective lens, having a given analysis field of view. The wavefront analyzer further comprises a field stop optically conjugate with the detection surface of the two-dimensional detector. This field stop allows the limitation of the analysis field of view of each microlens.

[0011] The wavefront analyzer described in Patent Document 3 makes it possible to significantly improve the analysis accuracy compared to known devices of the prior art, while eliminating the need to generate artificial guide stars. Indeed, the field stop thus arranged in the wavefront analyzer makes it possible to control the size of the image formed by each microlens in the vicinity of the detection surface. Furthermore, it makes it possible to limit the overlap of two images formed by two adjacent microlenses, without limiting the size of the field of view imaged by the microscope imaging system to which the wavefront analyzer is connected.

[0012] However, the applicants emphasize a further limitation when using an extended light source wavefront analyzer as described in Patent Document 3. This further limitation will be explained with reference to FIG. 1. FIG. 1 shows very diagrammatically a wavefront analyzer with a matrix 120 of microlenses, some of which (121-125) are shown. The wavefront analyzer is arranged on the exit side of a microscope objective 110 with an exit pupil 111 in a pupil plane PP. Each microlens is arranged in a pupil plane optically conjugate with the pupil plane PP and is configured to generate images 131-135 of an object 100 intercepting an object plane PO of the microscope objective in a detection plane 130 of a two-dimensional detector of the wavefront analyzer. For simplicity, in FIG. 1, the planes of the microlenses are shown to coincide with the pupil plane PP.

[0013] As shown in Fig. 1, most fluorescence microscopy imaging systems using light sheet illumination generate a light sheet 101 that is very thick over a large field of view. In particular, its thickness is greater than the depth of field of the microscope objective over the entire field of view of the microscope objective, said depth of field being identified in Fig. 1 by a strip 115. Indeed, it is known that to obtain a light sheet with an approximately constant thickness over a large field of view, this light sheet must be produced by an optical system with a numerical aperture smaller than the numerical aperture of the imaging objective, as described for example in non-patent document 4 ([Reference 7]). Moreover, in the case of scattering samples, in the case of living organisms, the effective thickness of the light sheet increases to a greater or lesser extent due to scattering.

[0014] However, each microlens has a numerical aperture smaller than the numerical aperture of the microscope objective of the microscope imaging system. For example, in a wavefront measurement device with a two-dimensional arrangement of 100 microlenses blocking the pupil of the microscope objective of the microscope imaging system, if the focal length of each microlens is similar to the focal length of the microscope objective, the numerical aperture of each microlens will be 1 / 10 of the numerical aperture of the microscope objective. As a result, the depth of field of each microlens (shown as strip 140 in FIG. 1) will be significantly larger than the depth of field of the microscope objective, 100 times larger in the above example. The numerical aperture and the depth of field are connected by the following formula:

number

[0015] Thus, as shown in Fig. 1, when an extended light source wavefront measurement device is connected to a fluorescence microscopy imaging system with light sheet illumination, each microlens typically creates an image corresponding to the entire thickness of the light sheet, i.e., projects all the fluorescence photons from the light sheet onto one side. As a result, in most fluorescence microscopy imaging systems with light sheet illumination, when the cross-section of the object is significantly larger than the depth of field of the imaging objective, a cross-correlation operation is performed between each image formed by each microlens of the wavefront measurement device.

[0016] This phenomenon, which the applicants highlight, is the source of two main problems. On the one hand, the measurement of the aberrations in this case corresponds to a measurement of the average of the aberrations present in the effective thickness of the light sheet. This feature can be particularly problematic in the case of very inhomogeneous objects, whose axially isoplanatic patches, i.e. the extent along the imaging optical axis where the aberrations are the same, are very small. In such cases, the measured aberrations do not only represent the aberrations corresponding to the imaging plane, and therefore their use in an adaptive optics system does not allow for the most optimal correction possible. On the other hand, if the object is composed of fluorescent elements distributed in three dimensions in the thickness of the light sheet, each image generated by each microlens of the extended source wavefront analyzer corresponds to a plenoptic imaging system, or "light field imaging system". In such a system, each image corresponds to an image of the object seen from a different viewing angle. For a three-dimensional object such as that shown in FIG. 1, the different viewing angles result in images 131 to 135. The reason these images are not similar is that the projection angle of each photon from the object is different throughout the entire thickness of the light sheet. Such relative differences between images tend to corrupt the results of cross-correlation calculations between images when measuring aberrations, and thus can result in significant measurement errors. [Prior art documents] [Patent documents]

[0017] [Patent Document 1] US Patent No. 855730 B2 [Patent Document 2] US Patent Application Publication No. 2015 / 0362713 [Patent Document 3] International Publication No. 2020 / 157265 [Non-patent literature]

[0018] [Non-Patent Document 1] MJBooth, et al., "Adaptive optics for fluorescence microscopy", excerpt from the following literature A Cornea, et al., "Fluorescence Microscopy: Super-resolution and other Novel Techniques", Academic Press, (USA), 2014 [Non-Patent Document 2] N.Ji, “Adaptive optical fluorescence microscopy”, Nature Methods 14, (English), 2017, p.374-380 [Non-Patent Document 3] K. Lawrence, et al., "Scene-based Shack-Hartmann wavefront sensor for light-sheet microscopy", Adaptive optics and Wavefront Control for Biological Systems IV, International Society for Optical Engineering, 2018, Abstract 10502 [Non-Patent Document 4] OEO Larte, et al., "Light-Sheet microscopy: a tutorial", Advances in optics and photonics, (USA), 2018, Vol. 10, No. 1, pp. 122-125 Summary of the Invention [Means for solving the problem]

[0019] The aim of the present specification is, in particular, to propose a method for fluorescence microscopy imaging with light sheet illumination and a system for fluorescence microscopy imaging with light sheet illumination, equipped with a wavefront analysis device, which overcomes all or part of the limitations mentioned above.

[0020] Throughout this specification, the term "comprise" has the same meaning as "include" or "contain" and is inclusive or open-ended and does not exclude other elements not described or shown.

[0021] Furthermore, throughout this specification, the terms "approximately" or "substantially" are synonymous (meaning the same) with a margin of 10%, e.g., 5%, below and above the respective values ​​at least one of which is the same.

[0022] According to a first aspect of the present specification, which relates to a method for fluorescence microscopy imaging of a three-dimensional fluorescent object with a fluorescence microscopy imaging system using light sheet illumination, the system comprises an imaging microscope objective having a pupil at a pupil plane and an optical axis, the method comprising: illuminating the object with a light sheet through an illumination path comprising an illumination device, The irradiating step comprises: generating a focused beam of light; scanning the focused light beam in an illumination plane substantially perpendicular to the optical axis of the imaging microscope objective to generate a light sheet; Including, a focal plane of the imaging microscope objective lens is included in the light sheet; The light sheet causes the emission of fluorescent light by the object. Steps and generating at least one first fluorescent image of an optical section of the object superimposed on the focal plane of the imaging microscope objective through an imaging path including the imaging microscope objective and an imaging detector having an imaging detection plane; the at least one first fluorescent image is generated in a spectral imaging band; Steps and analyzing a wavefront of the fluorescent light emitted by the object through an analysis path including the imaging microscope objective and a wavefront analyzer, The wavefront analyzer includes: a two-dimensional detector having an analytical detection plane conjugate with the focal plane of the imaging microscope objective; a two-dimensional arrangement of a plurality of microlenses arranged on an analysis plane conjugate with the pupil plane; Equipped with Analyzing the wavefront includes: each microlens generating a fluorescence image of a given analysis field of view of the object located in a focal plane of the imaging microscope objective in the analysis detection plane, The fluorescence image is generated in a spectral analysis band. Sub-steps and a sub-step of spatially filtering the fluorescent light emitted by the analysis field of view by a spatial filtering device comprising filtering elements arranged in a filtering plane optically conjugate with the analysis detection plane, said spatial filtering step including scanning said filtering element relative to the fluorescence image of the focused light beam formed on said filtering surface in synchronization with said scanning of said focused light beam to obtain a superposition at each instant of time of said filtering element and said fluorescence image of the focused light beam. Sub-steps and processing the plurality of fluorescence images produced by the plurality of microlenses by a processing unit to determine a two-dimensional map of characteristic parameters of the wavefront in the analysis plane; Contains analyzing the wavefront; correcting at least a portion of optical defects between the optical slice of the object and the imaging detection plane (PP1) based on the two-dimensional map of characteristic parameters of the wavefront by a wavefront modulation device (232) having a correction plane conjugate with the pupil plane (PP1); Fluorescence microscopy imaging method comprising:

[0023] Throughout this specification, fluorescence is understood to be the emission of light from an object due to the absorption of photons from a light excitation in a given spectral absorption band. The emission of fluorescent light can result from a linear mechanism using one photon, or from a non-linear mechanism using two or more photons. This mechanism results from the interaction of the absorbed light with fluorescent elements forming the object, or with fluorescent elements added to the object, such as fluorescent proteins in the case of living organisms.

[0024] Thus, a three-dimensional fluorescent object is any object with a microstructure that has either inherent fluorescent properties or is made fluorescent by the addition of "markers". Three-dimensional fluorescent objects are, for example, living organisms such as cells, cell cultures, living tissues, animals, etc. These living organisms have fluorescent properties, which may be either inherently formed by the organism or induced by the addition of fluorescent elements. Among three-dimensional fluorescent objects of interest, fluorescent neural structures in animal brains are particularly cited in the context of neuroimaging research.

[0025] Thus, a "fluorescence image" of an object or of a portion of an object at a given image plane is an image of the object or of said portion of the object formed in the spectral fluorescence band by all optical components located between the object and the image plane.

[0026] A wavefront in the sense of this specification is a surface of equal phase of a light wave.

[0027] According to one or more embodiments, the characteristic parameters of the wavefront include the local gradient (or local slope) across two dimensions of the intercepted wavefront in the analysis plane.

[0028] According to one or more embodiments, the characteristic parameter of the wavefront comprises a local deviation of the obstructed wavefront in the analysis plane relative to a reference wavefront corresponding to a light wave not affected by the optical defect, e.g. relative to a plane wavefront, where the two-dimensional map of said local deviations of the wavefront relative to the reference wavefront is obtained from a two-dimensional map of the local slopes of the wavefront.

[0029] According to one or more embodiments, determining the two-dimensional map includes determining a positional variation of the multiple fluorescence images formed by the multiple microlenses, where the positional variation of one fluorescence image formed by one microlens is measured relative to a reference position of a reference image, and the positional variation is determined by an operation between the image and the reference image, the operation being selected from a cross-correlation operation, a phase correlation operation, or a sum of squared differences operation.

[0030] Applicants have demonstrated that the method described thus far enables a significant improvement in terms of wavefront analysis accuracy compared to known methods of the prior art, resulting in better imaging quality while eliminating the need for the creation of artificial stars.

[0031] In fact, the imaging method according to the first aspect involves an extended source wavefront analysis, i.e. processing on a fluorescence image generated by a microlens larger than the microlens point spread function. Furthermore, the spatial filtering performed in the wavefront analysis allows confocal filtering of the fluorescence light, i.e. any errors in the processing of the fluorescence image are avoided, since only the fluorescence light from an optical slice of the object thinner than the light sheet is analyzed by the wavefront analyzer.

[0032] In the sense of the present specification, the term "microlens" refers to any optical focusing element with lateral dimensions (i.e. dimensions measured in the analytical plane) 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, comprise a transparent material having two diopters. At least one of the diopters is not flat. The non-flat diopter is, for example, a convex diopter, such as a spherical diopter.

[0033] According to one or more embodiments, the microlenses that make up the microlens matrix are identical microlenses, arranged, for example, in a two-dimensional matrix.

[0034] According to one or more embodiments, the microlenses that make up the microlens array are adjacent and have a square pupil.

[0035] According to one or more embodiments, the filtering element comprises a movable slit, and the spatial filtering of the fluorescent light emitted from the analysis field of view comprises a lateral movement of the slit synchronized with the scanning of the collected light beam.

[0036] Lateral movement is movement in a plane approximately perpendicular to the optical axis of the wavefront analyzer, which is considered to be in the vicinity of the slit.

[0037] According to one or more embodiments, the filtering element comprises a fixed slit. The spatial filtering device further comprises a set of optical components including at least one first rotatable mirror, the set of optical components being configured to generate a fluorescence image of the focused line on the slit. In this case, the spatial filtering of the fluorescence light emitted from the analysis field of view comprises rotating the at least one first movable mirror in synchronism with the scanning of the focused line to superimpose the slit and the fluorescence image of the focused line at each instant of time.

[0038] According to one or more embodiments, the device for illuminating the illumination path comprises a laser emission device for emitting a light beam and a light illumination system having a light illumination axis. The light illumination system is configured to generate from the light beam a focused light beam parallel to the light illumination axis. The light sheet is generated by scanning the focused light beam in a direction perpendicular to the light illumination axis. For example, the illumination device comprises a DSLM (Digital Scanning Laser Light Sheet Fluorescence Microscope) type device as described in Non-Patent Document 4, pages 143-144.

[0039] According to one or more embodiments, the device for illuminating the illumination path comprises a laser device for emitting a light beam and a light illumination system configured to generate from the parallel light beam a focused light beam having a light illumination axis and perpendicular to the light illumination axis. The light sheet is generated by scanning the focused light beam. For example, the illumination device comprises an ASLM (Axis-Sweeping Light Sheet Microscopy) type device as described in [Reference 8].

[0040] According to one or more embodiments, the light sheet is generated by scanning a focused light beam in a direction parallel to the light illumination axis. In other embodiments, the light sheet is generated by scanning a focused light beam in an oblique direction relative to the light illumination axis.

[0041] According to one or more embodiments, the spectral imaging band is the same as the spectral analysis band.

[0042] According to one or more embodiments, the spectral imaging band is different from the spectral analysis band, which can be advantageous if the goal is to use the maximum amount of optical power on the imaging path without sending a portion of the optical power useful for imaging on the analysis path.

[0043] According to a second aspect, the present description relates to a wavefront correction device configured to be connected to a fluorescence microscope imaging system using light sheet illumination, the fluorescence microscope imaging system comprising an imaging path with an imaging microscope objective having a pupil in a pupil plane and an optical axis, and an illumination path with an illumination device configured to scan a focused light beam in an illumination plane substantially perpendicular to the optical axis to generate a light sheet.

[0044] In a second aspect, the wavefront correction device comprises: The wavefront analyzer includes: a two-dimensional detector having an analytical detection surface; A two-dimensional arrangement of a plurality of microlenses disposed on an analysis surface, Each of these microlenses is configured to generate a fluorescence image of a given analysis field of view of the object located in a focal plane of the imaging microscope objective at the analysis detection plane when a wavefront correction device is connected to the microscope imaging system; The fluorescence image is generated in a spectral analysis band. A two-dimensional arrangement of a plurality of microlenses; 1. A spatial filtering device, comprising: the spatial filtering device is configured to spatially filter the fluorescent light emitted by the analysis field of view when the wavefront correction device is connected to the microscope imaging system; The spatial filtering device comprises: a filtering element disposed in a filtering plane optically conjugate with the analytical detection plane; a scanning means configured to scan the filtering element relative to the fluorescence image of the focused light beam formed on the filtering surface in synchronization with the scanning of the focused light beam so as to obtain a superposition at each instant of time of the filtering element and the fluorescence image of the focused light beam, A spatial filtering device; a processing unit configured to determine a two-dimensional map of characteristic parameters of the wavefront at the analysis plane based on all of the images formed by the plurality of microlenses; Equipped with The wavefront correction device 1. A wavefront modulation device having a correction surface, comprising: and when the wavefront correction device is coupled to the microscope imaging system, the wavefront correction device is configured to correct at least a portion of the optical imperfections between the optical slice of the object and the imaging detection plane based on the two-dimensional map of characteristic parameters of the wavefront. A wavefront modulator; a first optical relay system configured such that the pupil plane, the correction plane, and the analysis plane are optically conjugate when the wavefront correction device is connected to the microscope imaging system; a second optical relay system configured such that the focal plane, the analysis detection plane, the imaging detection plane, and the filtering plane of the imaging microscope objective are optically conjugate when the wavefront correction device is connected to the microscope imaging system; It further comprises:

[0045] According to one or more embodiments, the filtering element of the confocal filtering device comprises a movable slit, the scanning means being configured to cause a lateral movement of the slit synchronized with the scanning of the focused beam.

[0046] According to one or more embodiments, the movable slit is formed by a movable opto-mechanical element configured to transmit or reflect fluorescent light.

[0047] According to one or more embodiments, the movable slit is formed by addressing a group of one or more rows (or columns) of spatial intensity modulators.

[0048] According to one or more embodiments, the filtering element comprises a fixed slit and the spatial filtering device further comprises a set of optical components including at least one first rotatable mirror, the set of optical components being configured to generate a fluorescence image of the focused line on the slit, the scanning means being configured to cause a rotation of at least one first movable mirror in synchronism with the scanning of the focused line in order to superimpose at each instant of time the slit and the fluorescence image of the focused line.

[0049] According to one or more embodiments, the scanning means are configured to cause a rotation of a second movable mirror of the set of optical components in synchronism with the scanning of the collected beam and the scanning of the first movable mirror, allowing a fluorescence image to be reconstructed in an intermediate focal plane conjugate to the analytical detection plane by a time succession of multiple fluorescence images of the collected beam generated by the scanning of the collected beam.

[0050] In another embodiment, the set of optical components comprises at least one folding mirror configured to enable the first movable mirror to reconstruct a fluorescence image, which is generated in an intermediate focal plane conjugate to the analytical detection plane by a time-succession of multiple fluorescence images of the collected light beam generated by scanning the collected light beam.

[0051] According to one or more embodiments, the width of the slit is between a minimum value equal to twice the diffraction limit of the imaging microscope objective multiplied by the optical magnification between the filtering surface where the slit is located and the focal plane of the imaging microscope objective, and a maximum value equal to the width of the Rayleigh range corresponding to the Gaussian beam generating the fluorescent focused line multiplied by the optical magnification between the focal plane of the imaging microscope objective and the filtering surface where the slit is located.

[0052] According to one or more embodiments, the wavefront modulation device comprises a deformable mirror, which typically consists of a membrane and an actuator allowing local modification of the axial position of said membrane. The wavefront modulation device may further comprise a spatial light modulator (SLM), which typically consists of a two-dimensional arrangement of liquid crystal cells coupled to electrodes, allowing local modification of the refractive index of said cells. The wavefront modulation device may further comprise a deformable lens, which typically consists of moving parts allowing local modification of the shape and / or thickness of said lens.

[0053] Correction of optical defects or correction of a wavefront is understood to mean locally modifying the phase of a wave at a correction surface in order to obtain a reference wavefront. Depending on the intended application of the wavefront correction method, the reference wavefront can be a plane wavefront, as is the case for example for optimizing the performance of an imaging system, or a specific wavefront.

[0054] In accordance with one or more embodiments, the apparatus according to the second aspect further comprises a beam splitter element disposed between the wavefront modulation device and the wavefront analysis device, the beam splitter element configured to split the wavefront analysis device and the imaging path when the wavefront correction device is connected to a microscope imaging system.

[0055] According to one or more embodiments, the beam splitter element is dichroic, i.e. the beam splitter element 235 allows for splitting the incident light into a first spectral band that is reflected and a second spectral band that is transmitted and is different from the first spectral band.

[0056] According to one or more embodiments, a dichroic beam splitter element allows for splitting two spectral bands, an imaging band and an analysis band, respectively, towards an imaging path and towards an analysis path.

[0057] According to one or more embodiments, the two-dimensional detector comprises a two-dimensional arrangement of elementary detectors, the point spread function of the microlenses comprises between 0.2 and 2 elementary detectors in one direction. The dimension of the point spread function of the microlenses in one direction is defined by the distance separating the first two intensity minima located on either side of the intensity maximum. Applicants have shown that this particular configuration represents a good compromise between the wavefront measurement accuracy and the size of the analysis field.

[0058] According to one or more embodiments, determining the two-dimensional map includes determining a positional variability of each image formed by each microlens, where the positional variability of an image formed by one microlens is measured relative to a reference position of a reference image, the positional variability being determined by a cross-correlation operation between the images and the reference image.

[0059] According to a third aspect, the present application relates to a system for fluorescence microscopy imaging of three-dimensional fluorescent objects using light sheet illumination, the system comprising: an imaging path configured to generate at least one first image of an optical slice of the object in a spectral imaging band, the imaging path comprising: the imaging path includes an imaging microscope objective having a pupil at a pupil plane and an imaging detector having an imaging detector plane; an imaging path, in which the optical section is superimposed on a focal plane of the imaging microscope objective; an illumination path of the object, the illumination device being configured to scan a focused beam in an illumination plane substantially perpendicular to an optical axis of the imaging microscope objective; An analysis and correction path comprising the imaging microscope objective and a wavefront correction device according to a second aspect, the wavefront correction device is configured to correct at least a portion of optical imperfections between the optical slice of the object and the imaging detector plane based on a two-dimensional map of characteristic parameters of a wavefront. Analysis and correction path, Equipped with.

[0060] According to one or more embodiments, the apparatus for illuminating the illumination path comprises a laser emission device for emitting a light beam and an illumination system having a light illumination axis, the illumination system being configured to generate a focused light beam from the light beam parallel to the light illumination axis, and the light sheet being generated by scanning the focused light beam in a direction perpendicular to the light illumination axis.

[0061] According to one or more embodiments, the apparatus for illuminating the illumination path comprises a laser emission device for emitting a light beam and a light illumination system configured to generate from the parallel light beam a focused light beam having a light illumination axis and perpendicular to the light illumination axis, and the light sheet is generated by scanning the focused light beam.

[0062] According to one or more embodiments, the imaging detector comprises a two-dimensional detector having a two-dimensional arrangement of a number of elementary detectors or "pixels". The detector is configured to read out the pixels row by row or column by column simultaneously with the scanning of the collected light beam. Readout row by row or column by column is understood to mean sequential readout of groups of one or more rows or groups of one or more columns. For example, the imaging detector is a rolling shutter camera.

[0063] According to one or more embodiments, the imaging path further comprises a unit for processing the signals from the imaging detector. Of course, in practice the processing units of the analysis path and the imaging path may be combined in the same unit. Further advantages and features of the invention will become apparent on reading the description illustrated by the following figures. [Brief description of the drawings]

[0064] [Figure 1] 1 shows a schematic representation of the formation of multiple fluorescence images by multiple microlenses of a wavefront analyzer according to the prior art, connected to the previously mentioned fluorescence imaging system with light sheet illumination. [Diagram 2]FIG. 1 shows a diagram of an example of a fluorescence imaging system using light sheet illumination as described herein. [Figure 3A] 1 shows a diagram of the illumination path in a first embodiment of a fluorescence imaging system using light sheet illumination as described herein. [Figure 3B] 1 shows a diagram of the illumination path in a second embodiment of a fluorescence imaging system using light sheet illumination as described herein. [Figure 3C] 1 shows a diagram of the illumination path in a third embodiment of a fluorescence imaging system using light sheet illumination as described herein. [Figure 4A] 1 illustrates a first example of a spatial filtering device of a wavefront analyzer as described in the present specification, in which the filtering element of the spatial filtering device is provided with a movable slit formed by a movable optical-mechanical element configured to transmit or reflect fluorescent light.

[0065] [Figure 4B] A second example of a spatial filtering device of a wavefront analysis device described in the present specification is shown, in which the filtering element of the spatial filtering device is provided with a movable slit formed by addressing a group of one or more rows (or columns) of a spatial intensity modulation device. [Figure 5A] 1 shows a first example of a spatial filtering device having a fixed slit and a means for scanning a fluorescence image of a focused line on the slit in a wavefront analyzer as described herein. [Figure 5B] 1 shows a second example of a spatial filtering device having a fixed slit and a means for scanning the fluorescence image of the focused light beam on the slit in a wavefront analyzer as described herein. [Figure 6] FIG. 2 shows a diagram illustrating synchronization between different paths in one embodiment of the method described herein. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0066] FIG. 2 illustrates a schematic diagram of an example microscopic imaging system 200 using light sheet illumination for implementing an example method for microscopic imaging of a three-dimensional fluorescent object 10 as described herein.

[0067] The microscopy imaging system 200 shown in FIG. 2 comprises an illumination path 201 configured to illuminate an object with light sheet illumination, an imaging path 202 configured to generate at least one first image of an optical slice of the object in a spectral imaging band, and an analysis and correction path 203.

[0068] The imaging path 202 comprises an imaging microscope objective 210 having a pupil in a pupil plane PP1 and an imaging detector 220 with an imaging detection plane arranged in a plane PO3 conjugate with the focal plane PO1 of the imaging microscope objective. The optical section is superimposed on the focal plane PO1 of the imaging microscope objective. As shown in FIG. 2, the imaging path may further comprise a processing unit 225 for processing the signal emitted from the imaging detector 220. The imaging detector 220 is, for example, a two-dimensional detector, for example a CCD (charge-coupled device) camera or a high-sensitivity CMOS (complementary metal oxide detector) camera, for example a CMOS camera, for example a rolling shutter camera.

[0069] The illumination path 201 comprises an illumination device 205 configured to scan a focused light beam in an illumination plane substantially perpendicular to the optical axis of an imaging microscope objective to generate a light sheet 100. The focal plane PO1 of the imaging microscope objective is included in the light sheet, which is configured to produce an emission of fluorescent light in one or more spectral bands, including a spectral imaging band. Several examples of illumination devices are described with reference to Figures 3A, 3B, and 3C.

[0070] The analysis and correction path 203 comprises the imaging microscope objective and a wavefront correction device 230. The wavefront correction device 230 is configured to correct at least a portion of optical imperfections between the optical slice of an object and the imaging detection plane PP1 based on a two-dimensional map of characteristic parameters of a wavefront.

[0071] Thus, in the embodiments described with reference to FIG. 2, the microscope imaging system 200 is modular, consisting of three main modules: a "microscope" module with the illumination device 205 and the imaging microscope objective 210, a detection module with the imaging detector 220, and an analysis and correction module with the wavefront correction device 230. In practice, the wavefront correction device 230 can be configured to be connected to an existing microscope imaging system with a microscope and a detection module. In such an existing microscope imaging system, the detection plane of the imaging detector 220 is usually positioned at an intermediate focal plane PO2 of the objective 212 (or a cylindrical lens). To connect the wavefront correction device 230 to an existing imaging system, it is only necessary to move the imaging detector 220 so that its detection plane is located at a focal plane PO3 conjugate with the focal plane PO1 of the imaging microscope objective, as can be seen in FIG. 2. The wavefront correction device 230 can have a mechanical interface (not shown) for connecting said wavefront correction device 230 to the microscope and to the imaging detector. Such a modular configuration has the advantage that it is compatible with existing microscopy imaging systems that use light sheet illumination.

[0072] In another embodiment, the microscope imaging system is not modular and is designed and optimized as a whole with all paths 201, 202, 203 without attempting to plug the correction device into an existing system.

[0073] In either case, the wavefront correction device 230 comprises a wavefront analyzer 240 comprising a two-dimensional detector 248 having an analysis detection plane PO4 and a two-dimensional arrangement 243 of a number of microlenses 244 arranged in an analysis plane PP3. Each microlens is configured to produce a fluorescence image of a given analysis field of the object in the analysis detection plane in a spectral analysis band. The analysis field is located in the focal plane PO1 of the imaging microscope objective.

[0074] In some embodiments, the spectral imaging band is the same as the spectral analysis band, in which case fluorescent light having the same spectral band is detected in each of the analysis and imaging paths.

[0075] In other embodiments, the spectral imaging band may be different from the spectral analysis band. This may be advantageous if the aim is to use the maximum amount of optical power on the imaging path without sending part of it on the analysis path. For this purpose, it is possible, for example, to illuminate the object with a light sheet comprising two separate excitation spectral bands, such that the object emits fluorescent light in two separate fluorescence spectral bands. One fluorescence spectral band forms the spectral imaging band and the other fluorescence spectral band forms the spectral analysis band. The wavefront correction device 230 further comprises a processing unit 250, which is configured to determine a two-dimensional map of characteristic parameters of the wavefront in the analysis plane based on all the images formed by the multiple microlenses.

[0076] According to one or more embodiments, the two-dimensional detector comprises a two-dimensional arrangement of elementary detectors, the point spread function of the microlenses comprises between 0.2 and 2 elementary detectors in one direction. The dimension of the point spread function of the microlenses in one direction is defined by the distance separating the first two intensity minima located on either side of the intensity maximum. Applicants have shown that this particular configuration represents a good compromise between the wavefront measurement accuracy and the size of the analysis field.

[0077] For example, the characteristic parameters of the wavefront include the local gradient (or local tilt) over two dimensions of the wavefront obstructed in the analysis plane. According to another example, the characteristic parameters of the wavefront comprise the local deviation of the wavefront obstructed in the analysis plane relative to a reference wavefront corresponding to a light wave not affected by the optical defect, e.g. relative to a planar wavefront. A two-dimensional map of said local deviation of the wavefront relative to the reference wavefront is obtained from the two-dimensional map of the local tilt of the wavefront.

[0078] Furthermore, determining the two-dimensional map of the characteristic parameters of the wavefront may include determining a positional variation of each fluorescence image generated by each microlens. The positional variation of one fluorescence image generated by one microlens is measured relative to a reference position of a reference fluorescence image. The positional variation is determined, for example, by a cross-correlation operation between the fluorescence image and the reference fluorescence image. The local slope of the wavefront is determined from the positional variation of the fluorescence images.

[0079] The cross-correlation between two two-dimensional images I1(x,y) and I2(x,y), acquired by a two-dimensional detector and representing the light intensity values ​​of each of the images I1 and I2 at the coordinate point (x,y), is known as the result of the following calculation:

number

[0080] The positional shift between the fluorescent image and the reference fluorescent image can be determined by other operations known to those skilled in the art, such as, but not limited to, a phase correlation operation or a sum of squared differences operation. The phase correlation between two images is known as the result of the following calculation, using the same symbols as before:

number

[0081] The sum of squared differences of two images is known as the result of the following calculation, using the same symbols as before:

number

[0082] In these various examples, the difference in position of one fluorescence image generated by one microlens relative to a reference position of a reference fluorescence image is found, for example, by finding the position of the maximum value across two dimensions of the numbers resulting from the cross-correlation or phase correlation operation, or by finding the position of the minimum value across two dimensions of the numbers resulting from the sum of squared differences operation.

[0083] The processing unit 250 is generally configured to perform the computational and / or processing steps performed in the methods according to the present application.

[0084] In general, when the present specification refers to computational or processing steps, particularly for performing method steps, it is understood that the computational or processing steps, respectively, may be implemented by software, hardware, firmware, microcode, or any suitable combination of these technologies. When using software, the computational or processing steps, respectively, may be implemented by computer program instructions or software code. These instructions may be stored or transmitted to a computer-readable storage medium (or processing unit) or executed by a computer (or processing unit) to perform the computational or processing steps, or a combination thereof. Thus, processing unit 250 and processing unit 225 may be integrated within the same unit, e.g., within a computer.

[0085] The wavefront correction device 230 further comprises a wavefront modulation device 232. The wavefront modulation device 232 comprises a correction plane PP2, which is configured to correct at least a part of the optical defects between the optical section of the object and the imaging detection plane PP1 based on a two-dimensional map of the characteristic parameters of the wavefront. The first optical relay system allows the optical conjugation of the pupil plane PP1 of the imaging microscope objective 210, the correction plane PP2 and the analysis plane PP3. In the example of Fig. 2, which represents a modular microscope imaging system, the first optical relay system comprises, among other things, the objectives 231, 241, 242. Furthermore, the second optical relay system allows the optical conjugation of the object focal plane PO1 of the imaging microscope objective 210 and the detection plane PO3. In the example of Fig. 2, the second optical relay system comprises, among other things, the objectives 231, 236. Of course, other arrangements are possible to ensure optical conjugation between the various pupil planes and between the various focal planes.

[0086] The wavefront modulator 232 may, in a known manner, comprise, but is not limited to, a deformable mirror, which typically consists of a membrane and an actuator allowing local modification of the axial position of said membrane. The wavefront modulator may further comprise a spatial light modulator (SLM), which typically consists of a two-dimensional arrangement of liquid crystal cells coupled to electrodes, allowing local modification of the refractive index of said cells.

[0087] 2, the wavefront correction device 230 may further comprise a beam splitter element 235 disposed between the wavefront modulation device 232 and the wavefront analysis device 240. The beam splitter element 235 is configured to split the wavefront analysis path 203 and the imaging path 202 of the wavefront analysis device 240 when the wavefront correction device is connected to a microscope imaging system.

[0088] In some embodiments, the beam splitter element 235 is dichroic, i.e., the beam splitter element 235 allows the incident light to be split into a first spectral band that is reflected and a second spectral band that is different from the first spectral band and is transmitted. The dichroic nature of the splitter element is noted when the spectral imaging band is different from the spectral analysis band.

[0089] According to the present specification, the wavefront analysis device 240 further comprises a spatial filtering device 245 configured for spatial filtering of the fluorescent light emitted by the analysis field of view when connected to a microscope imaging system.

[0090] Several examples of spatial filtering devices are described in detail with reference to Fig. 4A, Fig. 4B, Fig. 5A and Fig. 5B. The spatial filtering device comprises a filtering element arranged in a filtering plane optically conjugate with the analysis detection plane PO4. The spatial filtering device further comprises scanning means arranged to scan the filtering element with respect to the fluorescence image of the focused light beam formed in said filtering plane. The scanning of the filtering element with respect to the fluorescence image of the focused light beam is synchronized with the scanning of the focused light beam so that a superposition of the filtering element and the fluorescence image of the focused light beam at each instant of time is obtained. The applicants have demonstrated that the synchronization of the scanning of the filtering element with respect to the fluorescence image of the focused light beam with the scanning of the focused light beam allows confocal filtering of the fluorescence light, i.e. only the fluorescence light from an optical slice of the object thinner than the light sheet is analyzed by the wavefront analyzer. Such confocal filtering avoids any errors in the processing of the fluorescence image generated by the microlenses. This results in a significantly improved wavefront analysis accuracy as well as a better imaging quality compared to known methods of the prior art.

[0091] FIG. 3A shows a schematic diagram of a first embodiment of an illumination device 301 of a fluorescence imaging system using light sheet illumination as described herein.

[0092] In this example, the illumination device comprises a laser emission device 310 for emitting a light beam, more specifically, a collimated light beam in this example. The laser emission device 310 comprises at least one spatially coherent first light source 312 (laser light source, laser diode, etc.) that emits a fluorescent marker of the object at an excitation wavelength, and a collimator lens 314. A diaphragm 316, such as an iris diaphragm, may further be provided to control the diameter of the light beam output from the laser emission device 310. The illumination device 301 further comprises a light illumination system having a light illumination axis Δ1. The light illumination system is configured to generate a focused light beam 341 from the light beam that is parallel to the light illumination axis Δ1. The illumination device 301 further comprises an angle scanning device 320 (such as a galvanometric mirror) configured to scan the focused light beam 341 in a direction perpendicular to the light illumination axis Δ1 to generate a light sheet 340.

[0093] In this example, the illumination system comprises a focusing optic 335 (such as a microscope objective lens) that defines an illumination axis Δ1.

[0094] In this example, the light illumination system further includes an optical relay system with optical elements 331 and 332. The optical relay system is configured to relay the light beam from the laser emission device 310 to the focusing optical element 335 by the angle scanning device 320. The optical elements 331 and 332 form an optical conjugate between the surface where the angle scanning device 320 performs the angle scanning (such as the reflecting surface of a galvanometric mirror) and the back focal plane 336 of the focusing optical element 335 (such as a microscope objective). The back focal plane may coincide with or be close to the pupil of the microscope objective. The optical elements 331 and 332 preferably form an afocal system so that the collimated beam from the laser emission device 310 is collimated at the input side of the focusing optical element 335.

[0095] Optical elements 331 and 332 are typically achromatic lenses, such as doublet lenses, which allow minimization of both optical aberrations, as well as minimization of chromatic aberration.

[0096] The achromatic optical elements 331, 332 allow the use of a laser emitting device 310 which emits a light beam whose dominant wavelength can be tuned to a wide (typically several hundred nanometers) spectral band or which emits a light beam with several dominant wavelengths.

[0097] The dominant wavelength of the light beam output from the laser emitter 310 can be adjusted by changing the light source 312 or by using several laser sources (not shown in FIG. 3A) with different wavelengths selected using a beam splitter.

[0098] The use of several light sources with different dominant wavelengths makes it possible, for example, to excite the fluorescence of a sample provided with several fluorescent labels with different characteristics. For example, in the case of imaging of fluorescent cells, it is possible to specifically prepare the cells by providing them with several fluorescent proteins located across several specific structures. According to the same example, using suitable excitation light, it is possible to obtain from such cells a fluorescent signal originating specifically from the nucleus, for example when the mCherry fluorescent protein is present, particularly in the nuclear structure, and a signal originating specifically from the cytoplasm, for example when the GFP protein is present, particularly in the tubulin. When excited by a laser, for example with a wavelength of 561 nm, the mCherry protein emits red fluorescent light. When excited by a laser, for example with a wavelength of 488 nm, the GFP protein emits green fluorescent light. Thus, for example, two laser light sources with different dominant wavelengths may be provided to view these various cellular structures using a camera that collects the fluorescent signals. In some embodiments, these two laser light sources, when combined by a suitable dichroic beam splitter, allow simultaneous excitation of these fluorescent proteins. The wavefront measurement and correction device as described in the present invention can also benefit from the use of several laser sources for exciting several fluorescent labels of the sample. In practice, the fluorescent signal from a living body, for example benefiting from a particular preparation, as described above, is usually a low amplitude signal. Therefore, in most cases, it is worth minimizing the loss of the fluorescent signal used to generate the image of the object of interest. In the case of the wavefront correction device described herein, for example, as shown in FIG. 2, when the fluorescent signal is split by a conventional beam splitter 235, i.e., the proportion of the light to be transmitted and reflected is selected according to the intensity of the incident light, and not according to its wavelength or other characteristics such as its polarization, the part of the fluorescent signal useful for generating the image of the object of interest is lost from the point of view of generating the image, since it is used for the analysis of the wavefront using the subset 240.To eliminate these losses, the object can be specifically prepared by adding a fluorescent label that emits fluorescent light in a spectrum that is shifted from the spectrum of the fluorescent light useful for generating an image. In this case, if a laser source that specifically excites fluorescence in a spectral analysis band that is shifted from the spectral imaging band is added to the laser emission device 310, the dichroic beam splitter 235 can be used to provide a fluorescent signal that can be conveniently directed to the wavefront analysis device 240 without inducing or minimizing losses of the fluorescent signal useful for generating an image of the object. In these various examples using several light sources with different wavelengths, the selection of achromatic optical elements 331 and 332 ensures that the focusing of the beam 341 from the optical element 335 is approximately the same.

[0099] The angle scanning device 320 is typically a galvanometric mirror, which allows the rotation of the reflective surface of the mirror along a rotation axis perpendicular to the axis Δ1. This rotation axis is located, for example, on the surface of the mirror. The angle scanning performed by the angle scanning device 320 thus moves the focused beam 341 in a direction perpendicular to Δ1. This direction is represented by the bold arrow in FIG. 3A.

[0100] According to some embodiments, the light sheet 340 has a width defined by the amplitude of the angular scan performed by the angular scanning device 320 and a length defined by the distance along Δ1. The thickness of the focused line is approximately constant, for example approximately double. The center of the light sheet 340 is therefore generally defined along the first axis for the position of the focused line corresponding to the center of amplitude of the scanning device 320, and along the second axis for the position of the waist of the focused line 341. In fact, when the light source 312 is a laser light source, the propagation characteristics of the focused line 341 are those of a Gaussian beam. The numerical aperture of the beam 341 is therefore defined in particular by the diameter of the beam at the input side of the optical element 335. This diameter is adjustable by the diaphragm 316. Adjusting the size of the diaphragm 316 therefore allows the control of some characteristics of the light sheet generated by the illumination path 301, such as the thickness of the light sheet as well as the size of the light sheet 340 when the thickness of the light sheet is approximately constant.

[0101] Specific implementations of the illumination path 301 are possible, which allow for improvements, for example, in terms of image resolution. For example, the ultrafast laser source 312 can be used to generate a nonlinear fluorescent signal, such as a two- or three-photon signal, thus allowing the thickness of the focused beam emitting the fluorescent signal to be minimized. Specific spatial shaping of the focused beam 341 can also be implemented, for example, by the use of Bessel or Airy type beams, which allow the generation of a focused beam with a constant thickness, typically larger than the waist diameter of a Gaussian beam, over significant propagation distances.

[0102] The general operating principle of the illumination path 301 corresponding to this specification, as well as details regarding its operation in linear or non-linear fluorescence regimes, and various properties of the focused beam 341 associated with specific shaping operations, are described in Non-Patent Document 4, e.g., pp. 143-153. The illumination approach described herein is commonly referred to as digital scanning laser light sheet fluorescence microscopy or DSLM.

[0103] Fig. 3B shows a schematic of a second embodiment of an illumination device 302 of a fluorescence imaging system with light sheet illumination as described herein. The illumination device described with reference to Fig. 3B is an axially swept light sheet fluorescence microscopy illumination device or an ASLM type illumination device. Details for implementing this illumination approach are given e.g. in [Reference 8], together with several examples of fluorescence images generated when this illumination approach is implemented in a light sheet microscope.

[0104] As in the case of FIG. 3A, the illumination device comprises a laser emitting device 310 emitting a beam, such as a parallel light beam. The laser emitting device 310 comprises one or more light sources 312, such as laser sources of the laser diode type and collimator lenses 314, as explained above. In FIG. 3B, a single light source 312 is shown. As explained with reference to FIG. 3A, it is possible to use several different dominant wavelength light sources, which are combined using a dichroic splitter, in order to excite several fluorescent signals. Similarly, it is possible to use different types of laser light sources 312, such as ultrafast laser sources, which allow the generation of nonlinear fluorescent signals. In the example of FIG. 3B, the emitted light beam is, for example, polarized. The laser emitting device may therefore comprise a half-wave plate or polarizer 318, which is arranged at the output side of the light source 312 so as to define the polarization axis of the emitted light beam.

[0105] In the example of Fig. 3B, the illumination device 302 further comprises a cylindrical lens 351 for generating an astigmatic beam, i.e. a beam in which light is focused along a line at the focal plane of the lens 351. The polarizing beam splitter 352 reflects the astigmatic beam towards a quarter wave plate 361 and towards a lens 362, allowing conjugation of the focused light beams at the back focal plane of a first microscope objective lens 363. The first microscope objective lens 363, which defines an optical axis Δ2, forms a focused light beam on the reflecting surface of a mirror 366. The mirror 366 is movable along the optical axis Δ2 by a linear translation system 364, typically by a motor-driven controllable translation system, such as a piezoelectric actuator or a stepper motor. The remote focusing optical relay system, which in this example comprises a first microscope objective 363, a second microscope objective 375 with an optical axis collinear with that of the first microscope objective 363, and further lenses 362 and 371, can relay the light beam reflected on the mirror 366 at the exit of the objective 375. In this optical system, the lenses 362 and 371 generally form an afocal and telecentric system. The image focal point of the lens 362 coincides with the object focal point of the lens 371. The lenses 362 and 371 are arranged such that the pupil planes of the objectives 363 and 375 are optically conjugate with each other, for example by positioning the pupil plane of the objective 363 in the object focal plane of the lens 362 and the pupil plane of the objective 375 in the image focal plane of the lens 371. The two microscope objectives 363 and 375 are positioned in opposite directions along the optical axis Δ2. The unique feature of this remote focusing optical system is its ability, when these optical elements are specifically selected, to generate an image of an object located in front of one microscope objective in front of another microscope objective in a practically aberration-free manner, even if the object is not located in the focal plane of the objective. Thus, in the case of FIG. 3B, the focused light beam created by the objective 363 on the reflecting surface of the mirror 366 is imaged downstream of the second microscope objective 375 along the focused light beam 341.In addition, when the mirror 366 is moved along the optical axis Δ2, for example by the linear translation device 364, the properties of the remote focusing relay system allow the image of the collected beam 341 to be moved along the optical axis Δ2 in the same direction as the mirror 366 without significantly changing the spatial characteristics of the collected beam. The respective movements of the mirror 366 and the collected beam 341 are shown in FIG. 3B by the two thick arrows located above the linear translation device 364 and the light sheet 340. The additional view as a top view shows the movement of the collected beam 341 along the axis Δ2 during the movement of the mirror 366. The amount of movement of the collected beam 341 caused by the movement of the mirror 366 determines the size of the light sheet 340 created by the movement of the collected beam 341 along the axis Δ2. The size of the light sheet 340 in the other direction is determined by the width of the collected beam 341. The width of the collected beam 341 itself is directly related to the size of the output beam from the laser delivery device 310. As in Figure 3A, if it is necessary to control the size of the beam at the output of the laser delivery device 310, a diaphragm can be added, which is not shown in Figure 3B but is indicated in Figure 3A by reference numeral 316.

[0106] Other embodiments of the illumination path 302 in the ASLM type principle are also possible, in particular it is also possible to use a system for controlling the focusing of the beam from the second microscope objective 375 different from the remote focusing system described above, such as the use of a deformable lens, as described, for example, in [Reference 9].

[0107] FIG. 3C shows a schematic representation of a variant of the embodiment of the illumination device of a fluorescence imaging system using light sheet illumination as shown in FIG. 3B.

[0108] As mentioned above, Figures 3A and 3B show illumination paths. When these illumination paths are implemented in a fluorescence imaging system with light sheet illumination, the microscope objective of the imaging path is generally positioned perpendicular to the microscope objective 335 (375 in Figure 3B) of the illumination device. Indeed, in most fluorescence imaging systems with light sheet illumination, the excitation light sheet is positioned in a plane perpendicular to the optical axis of the microscope objective of the imaging path, so as to make the light sheet correspond to the focal plane of the microscope objective of the imaging path, so that in the embodiment as shown in Figures 3A and 3B, the optical axes Δ1 and Δ2 are positioned perpendicular to the optical axis Δ of the microscope objective of the imaging path. In these embodiments, the maximum size of the light sheet along the axes Δ1 and Δ2 is limited by the extent, i.e. the distance, between the last optical surface of the microscope objective 335 (375 in Figure 3B) and its image plane, respectively, which in turn limits the maximum size of the object that can be imaged using such illumination paths. Figure 3C shows another embodiment. In this embodiment, the optical axis Δ2 of the microscope objective 375 of the illumination device is not perpendicular to the optical axis of the imaging lens. In this case, the creation of an excitation light sheet is possible by scanning a focused light beam 341, generated, for example, in a manner similar to that shown in FIG. 3B. This focused light beam 341 is not scanned in a direction parallel to Δ2, but in a direction that allows the definition of a light sheet 340 in a plane perpendicular to the optical axis of the imaging microscope objective. This scanning is performed by a device for scanning the focused light beam, as shown in FIG. 3B, which combines the change of the axial position of the focused light beam according to Δ2 with the change of the angle of the output beam of the microscope objective 375, for example using a galvanometric mirror (not shown in FIG. 3C). This alternative approach therefore makes it possible to create a light sheet larger than the size limit of the light sheet obtained by the illumination device described with reference to FIGS. 3A and 3B, and thus to generate images of larger objects. Details of the implementation of the illumination approach shown in FIG. 3C are described, for example, in [Reference 10].

[0109] In some embodiments, especially when a DSLM or ASLM type illumination device as described above is used, it may be possible to use a detector 220 for the fluorescence imaging path 202 of a fluorescence imaging system with light sheet illumination (see FIG. 2). This detector 220 is configured to only select light from the collected beam 341, thus filtering out the fluorescence light from other parts of the excitation beam to generate the collected beam 341. For example, a two-dimensional rolling shutter camera can be used. In such a camera, the readout of the camera pixels per row or per group of rows (or per column or per group of columns, depending on the orientation) is synchronized with the scanning of the collected beam 341.

[0110] 4A, 4B, 5A and 5B respectively illustrate embodiments of devices for spatial filtering of the fluorescence light emitted from the analysis field of view. These spatial filtering devices are part of the wavefront analyzer in this description. In these examples, the spatial filtering device comprises a filtering element arranged in a filtering plane optically conjugate with the analysis detection plane PO4 of the wavefront analyzer (see FIG. 2). This spatial filtering comprises scanning the filtering element with respect to the fluorescence image of the focused line formed in the filtering plane. This scanning is synchronized with the scanning of the focused line, so that a momentary superposition of the filtering element and the fluorescence image of the focused line is obtained. The scanning of the focused line is performed, for example, using one of the methods described with reference to FIG. 3A, FIG. 3B or FIG. 3C.

[0111] 4A and 4B show two first embodiments of a spatial filtering device with a movable slit, in which the spatial filtering of the fluorescent light emitted from the analysis field of view comprises a lateral movement of the slit, synchronized with the scanning of the collection line.

[0112] More specifically, in the example of Fig. 4A, the spatial filtering device comprises a movable slit 40 which moves in synchronism with the scanning of the focused beam. Fig. 4A therefore shows the superposition of the fluorescent image of the focused beam and the slit at each instant of time, which is shown diagrammatically by lines 411-415. The position of the slit is also shown diagrammatically at the same instant of time by lines 411-415. The arrows in Fig. 4A respectively show the movement of the fluorescent image of the focused beam (thin line) and the movement of the slit (thick line).

[0113] When the wavefront correction device (230, FIG. 2) is connected to a microscope imaging system, the slit 40 is positioned at a filtering plane that is optically conjugate with the focal plane PO1 of the imaging microscope objective.

[0114] The spatial filtering device further comprises means for laterally displacing the slit 40, i.e. in a plane perpendicular to the optical axis, said means for displacing the slit 40 being, for example, a motorized translation system, for example a stepper or a piezoelectric type motor.

[0115] The slit 40 may be a transmissive slit, which may comprise, for example, a transparent surface that defines a line of a given width, surrounded by a frame that is opaque to the fluorescent light or that reflects the fluorescent light, or may further comprise, for example, a reflective surface that defines a line of a given width.

[0116] The maximum dimension, i.e. the length, of the slit 40 can be selected so as to limit the size of the image produced by each microlens of the wavefront analyzer in the direction corresponding to the length of the slit. This selection is made so as to avoid superposition of the respective images produced by the various microlenses of the wavefront analyzer in said direction. The minimum dimension, i.e. the width, of the slit 40 can be selected so as to reach a compromise between the confocal volume defined by this width (i.e. the volume in the sample where photons are not blocked by this slit and can be detected by the wavefront analyzer), the loss of light caused by spatial filtering (directly proportional to the width of the slit) and the inability of the slit to filter aberrations of the fluorescent light measured by the wavefront analyzer for subsequent correction by the wavefront modulator, especially if the slit is too narrow. To demonstrate this compromise, the applicants have shown that the slit width can be advantageously kept within a range between a minimum and a maximum value. The minimum value is equal to the diffraction limit of the imaging microscope objective multiplied by the optical magnification between the filtering plane in which the slit 40 is located and the focal plane of the imaging microscope objective, for example advantageously equal to twice the diffraction limit multiplied by this optical magnification. The maximum value is equal to, for example, the width of the Rayleigh range corresponding to the Gaussian beam generating the fluorescent focused line multiplied by the optical magnification between the focal plane of the imaging microscope objective and the filtering plane in which the slit 40 is located.

[0117] In the example of Fig. 4B, when the wavefront correction device (230, Fig. 2) is connected to a microscope imaging system, the spatial filtering device comprises a spatial intensity modulation device 420 arranged in a filtering plane optically conjugate with the focal plane PO1 of the imaging microscope objective. The spatial intensity modulation device 420 is controlled at the same time that the focused beam is scanned to address a group of one or more rows (or columns), such that at each instant a slit is formed which is superimposed on the fluorescent image of the focused beam formed in the filtering plane.

[0118] For example, the spatial intensity modulator 420 may comprise a matrix of micromirrors or a "digital micromirror device" DMD. The DMD comprises a two-dimensional matrix of micromirrors, which are individually controlled to certain angles. Thus, a beam incident on the DMD may be reflected with an intensity pattern that is directly related to the individual positions of the micromirrors. Thus, the DMD can be controlled to direct one or more rows (or columns) of micromirrors in a direction that allows a wavefront analyzer to focus the light reflected by the group of one or more rows (or columns). The group of one or more rows (or columns) of micromirrors moves in time in the same direction as the focused beam moves, simultaneously scanning the focused beam.

[0119] Thus, by synchronizing the control of the DMD with the scanning of the focused beam, it is possible to superimpose at each instant in time a fluorescent image of the focused beam (411-415) with a group of one or more addressed rows (or columns) of each micromirror of the DMD, as shown in Figure 4B, represented by columns 421-425. In Figure 4B, the arrows respectively show how the fluorescent image of the focused beam moves over time (thin lines) and how the group of one or more addressed rows (or columns) moves over time (thick lines).

[0120] The length and width of each group of one or more rows (or columns) of micromirrors can be selected according to the same criteria as described with reference to Figure 4A. However, in the case of DMD-type spatial intensity modulators, since the size of the individual micromirrors cannot usually be customized, it may be worthwhile to fine-tune the efficiency of the spatial filtering system, such that the magnifications resulting from the various optical element groups are adjusted in the wavefront correction devices described herein.

[0121] Figures 5A and 5B show two second embodiments 51, 52 of a spatial filtering device with a filtering element, in this case with a fixed slit, indicated with reference number 510 in Figures 5A and 5B. The fixed slit 510 is arranged in a filtering plane PO6 optically conjugate with the object focal plane PO1 of the imaging microscope objective. The filtering device further comprises a set of optical components including at least one first rotatable mirror (512, 522). The set of optical components is configured to generate a fluorescence image of the focused line on a slit. Means are provided for rotating the at least one first movable mirror in synchronism with the scanning of the focused line in order to superimpose the slit and the fluorescence image of the focused line at each instant of time.

[0122] More specifically, in the example of FIG. 5A, the plane PO5 corresponds to a plane optically conjugate with the object focal plane PO1 of the imaging microscope objective. It is therefore included in the light sheet generated by the scanning of the focused light beam by the illumination device as described above. In FIG. 5A, the scanning of the fluorescent image of the focused light beam is shown in the plane PO5 as a schematic representation by the image of the focused light beam seen in a cross section perpendicular to the axis of the focused light beam. The scanning direction of the focused light beam is shown as a schematic representation by a thick arrow. The set of optical components in the example of FIG. 5A includes, for example, a plurality of groups of lenses 511 and 514 on the one hand and lenses 515 and 518 on the other hand, forming an afocal and telecentric optical system. These lens groups include, for example, achromatic lenses, such as doublet lenses. The set of optical components in the example of FIG. 5A further includes mirrors 512, 513, 516, 517. These mirrors include rotatable mirrors 512 and 517 (such as galvanometric mirrors) located in the pupil planes PP4 and PP5, respectively, in this example. These mirrors are oriented, for example, at 45° to the optical axis of the spatial filtering device and return the light by reflection at 90°. Fixed folding mirrors 513 and 516, oriented, for example, at 45° to the optical axis, allow the optical axis to be folded in one direction. This orientation makes it possible, for example, primarily to keep the optical axis in a single direction for the spatial filtering device. These folding mirrors are, for example, either treated with a metal-based treatment that provides a significant reflectivity over a very wide spectral band, or treated with a dielectric material that provides a better reflectivity than the metal-based treatment, but over a limited spectral band.

[0123] The group made up of lenses 511 and 514 allows the optical conjugation between the planes PO5 and PO6, and the group made up of lenses 515 and 518 allows the optical conjugation between the plane PO6 and the intermediate focal plane PO7. The intermediate focal plane PO7 itself is conjugated with the analysis detection plane PO4 of the detector 248 of the wavefront analyzer by the lens 242 and each microlens of the microlens matrix arranged in the pupil plane PP3 (see also FIG. 2). The pupil plane PP4 in FIG. 5A is an optically conjugated plane of the pupil plane PP1 of the imaging microscope objective 210 (see FIG. 2). When the wavefront correction device is connected to a microscope imaging system, the optical conjugation between the pupil planes PP1 and PP4 is realized, for example, by the lenses 212, 231, 241 (see FIG. 2) and 511. The pupil planes PP5 and PP4 are optically conjugated with the plane PP3. The optical conjugation between PP4 and PP5 is realized by lenses 514 and 515, which form an afocal and telecentric system. The conjugation between PP5 and PP3 is realized by lenses 518 and 242, which form an afocal and telecentric system. These two afocal and telecentric systems include, for example, achromatic doublets. Thus, the planes PO4, PO5, PO6, and PO7 are optically conjugate with each other and with the focal plane PO1 of the imaging microscope objective lens shown in FIG. 2. The pupil planes PP3, PP4, and PP5 are optically conjugate with each other and with the pupil plane PP1 of the imaging microscope objective lens 210 shown in FIG. 2. The length (maximum dimension) of the fixed slit 510, which is located at the plane PO6, is parallel to the axis of the fluorescence image of the collected light beam. When the collected light beam is scanned by the illumination path, the first movable mirror 512 performs an angle scan simultaneously with the scanning of the collected light beam. Its amplitude is adjusted to generate a fixed image of the collected beam in the vicinity of slit 510. Thus, at each instant that the collected beam is scanned, slit 510 performs in-line confocal spatial filtering of the fluorescence signal from the collected beam, so that the fluorescent light transmitted through the slit originates only from a confocal volume defined by the slit features.This allows for the reduction of the fluorescent signal from various aspects of the imaging microscope objective 210 image plane (FIG. 2) and also allows for the thickness of the transmitted collected light to be reduced.

[0124] Similarly, simultaneously with the scanning of the condenser line by the illumination path, the second movable mirror 517 performs an angular scan simultaneously with the scanning of the condenser line and the scanning of the first movable mirror 512. This allows the reconstruction of a fluorescence image generated by the time succession of fluorescence images of the condenser line generated by the scanning of the condenser line. This reconstruction is possible in an intermediate focal plane PO7 conjugate to the analytical detection plane, based on the time succession of multiple fluorescence images of multiple condensers located in the vicinity of the slit 510. This reconstructed fluorescence image is then imaged on a detector 248 located in a plane PO4 of the wavefront device described herein, using the optical element 242 and a matrix of microlenses located in a plane PP3. The detector 248 thus allows the detection of an image composed of multiple fluorescence images of an object arranged in a similar manner to the arrangement of the microlenses in the plane PP3. This image allows the estimation of the wavefront therefrom by calculation of the relative shift between the images, using the methods described above, such as the cross-correlation calculation method.

[0125] The dimensions of the slit 510 can be selected similarly to the dimensions of the slit described with reference to FIG. 4A.

[0126] In the example of Figure 5B, optical elements 521, 523, 526, and 527 are equivalent to optical elements 511, 514, 515, and 518, respectively, of Figure 5A, and have substantially similar features. The various conjugates of surfaces identified with the same reference numbers are similar to the conjugates shown in Figure 5A. In this embodiment, a single rotatable mirror 522 is used, e.g., a galvanometric mirror of the same type as mirrors 512 and 517 of Figure 5A. Folding mirrors 524 and 525, of the same type as mirrors 513 and 516 of Figure 5A, are positioned to return light back towards movable mirror 522.

[0127] Thus, when the illumination path scans the focused line, the rotatable mirror 522 performs an angular scan simultaneously with the scanning of the focused line. Its amplitude is adjusted so as to generate a fixed image of the focused line located in the vicinity of the slit 510, in particular by reflection on the first folding mirror 524. The second folding mirror 525 is directed to return the light spatially filtered by the slit 510 towards the rotatable mirror 522. The second scanning of this mirror therefore makes it possible to reconstruct a fluorescence image generated by the temporal succession of a plurality of fluorescence images of the focused line generated by the scanning of the focused line. This reconstruction is made possible in an intermediate focal plane PO7 conjugate to the analytical detection plane, based on the temporal succession of the fluorescence lines located in the vicinity of the slit 510. The selection criteria for the characteristics of the slit 510, in particular its width, are similar to those described with reference to FIG. 4A or 5A.

[0128] Spatial filtering device 52 is an advantageous variation of spatial filtering device 51 because it allows for the use of a single rotatable mirror. This allows the opto-mechanical implementation of spatial filtering device 52 to be more compact than the opto-mechanical implementation of spatial filtering device 51, reducing implementation costs while minimizing potential performance degradation of spatial filtering device 51 due to alignment and synchronization failures.

[0129] 6 is a schematic diagram showing a synchronization diagram of each main module of the microscope imaging system described in the present specification according to an example shown in FIG. 2 to FIG. 5B are shown as an example, but are not limited to this.

[0130] As shown in Fig. 6, signal 61 corresponds to a clock signal and is configured to trigger the imaging and analysis detectors of the scanning system of the focused line of the illumination path and the imaging and analysis detectors of the scanning system of the spatial filtering device as described herein (220 and 248, respectively, Fig. 2). The trigger signal in this example comprises two square wave logic voltage signals. The first square wave signal starts with its rising edge, for example, at time t1. The second square wave signal starts with its rising edge, for example, at time t2. t2 is, for example, offset in time with respect to t1.

[0131] In this example, signal 62 corresponds to a signal for controlling the scanning of the focused beam of the illumination path. In the example of Fig. 6, this signal is a lamp voltage, which corresponds for example to the signal sent to the galvanometric rotatable mirror 320 of Fig. 3A, or also to the signal sent to the piezoelectric motor-driven translation system 364 of Fig. 3B, allowing the movement of the mirror 366 to scan the focused beam. The voltage received by this type of movable device actually allows the direct control of the rotation angle or translation of said movable device. Thus, to generate a light sheet corresponding to an imaging field, the focused beam is scanned at times t1 and t2 corresponding to low and high levels of said lamp voltage. i is scanned between

[0132] Signal 63 represents diagrammatically a period during which an image of the target object is acquired, for example using imaging detector 220 in the embodiment of FIG.

[0133] Signal 64 corresponds to an example of a signal for controlling the scanning of a spatial filtering device as described herein. For example, signal 64 is a ramp voltage, which corresponds to a signal sent, for example, to a galvanometric type rotatable mirror (512 and 517 in FIG. 5A and 522 in FIG. 5B). The voltage received by this type of mirror allows direct control of the rotation angle of said mirror. The analysis field of view is therefore determined by the time instants t2 and t3 corresponding to low and high levels of said ramp voltage of signal 64. a4B , or a system for controlling an array of DMD-type spatial intensity modulators, or any other controllable system for implementing a scanning of a spatial filtering device as described herein.

[0134] Signal 65 represents diagrammatically a period during which acquisition of the analysis field of view of the wavefront analysis device according to the invention is performed, for example using analysis detector 248 (FIG. 2). Voltage signals 61, 62 and 64 are generated, for example, using an electronic synchronization system (such as an input / output electronic card) providing both a logic output generating a signal of the type shown on line 61 and two analog outputs generating signals of the type shown on lines 62 and 64, these various outputs being precisely controllable over time relative to one another by said electronic synchronization system.

[0135] The electronic synchronization system may form one unit of the processing unit 250.

[0136] Thus, the first slot for triggering signal 61 at time t1 allows for synchronous triggering of the emission of signals 62 and 63. The scanning of the collection line of the illumination path controlled by signal 62 and the acquisition by the imaging detector (220, FIG. 2) are performed for the same period t i This ensures that the entire imaging field is covered by scanning the illumination path, by suitable adjustment of the amplitude and speed of the scanning of the focused light beam of the illumination path.

[0137] This means that one can benefit from the rolling shutter readout mode that is typically present in the fluorescence imaging cameras of e.g. light sheet fluorescence imaging devices, which allows the pixel columns of the camera to be integrated in a sequentially synchronous manner with the scanning of the collected light beam, i.e. one can enjoy the advantages of confocal detection on the camera.

[0138] The second slot for triggering signal 61 at instant t2 allows for synchronous triggering of signals 64 and 65. The scanning of one or more rotatable mirrors of the spatial filtering system controlled by signal 64 and the acquisition of the analysis field of view by the analysis detector (248, FIG. 2) of the wavefront analyzer controlled by signal 65 occur during the same period t a t2, whereby by appropriate adjustment of the scanning amplitude and speed of one or more rotatable mirrors of the spatial filtering system, the entire analysis field of view benefits from the effect of the spatial filtering system.

[0139] In the device according to the invention, the size of the analysis field defined by the wavefront analyzer is usually significantly smaller than the size of the imaging field defined by the imaging camera. Therefore, it is preferred to delay the start of the acquisition of the image of the analysis field and the scanning of the scanning device of the spatial filtering system from the start of the acquisition of the image of the imaging field. The adjustment of this delay t2 to t1 is performed on an electronic synchronization system, taking into account the speed and amplitude characteristics for the scanning of the illumination path so that the moment t2 corresponds to the moment when the scanned focused beam is located along the first edge of the analysis field.

[0140] All signals shown in FIG. 6 correspond to acquisition sequences in the microscopy imaging system described herein.

[0141] Clearly, this sequence can be repeated over time to acquire successive images and measurements of the wavefront, and other acquisition sequences are possible depending on the characteristics of each module of the microscope imaging system.

[0142] Although described with respect to several embodiments, the wavefront analyzer and the microscopy imaging system and method using the wavefront analyzer include various alternative embodiments, modifications and improvements that will be apparent to those skilled in the art, and it is to be understood that these various alternative embodiments, modifications and improvements form part of the scope of the invention as defined by the appended claims. References Reference 1: MJ Booth, et al., "Adaptive optics for fluorescence microscopy", excerpted from the following literature: A Cornea, et al., "Fluorescence Microscopy: Super-resolution and other Novel Techniques", Academic Press, (USA), 2014 Reference 2: N. Ji, "Adaptive optical fluorescence microscopy", Nature Methods 14, (English), 2017, p.374-380 Reference 3: US Patent No. 855730 B2 Reference 4: U.S. Patent Application Publication No. 2015 / 0362713 Reference 5: K. Lawrence, et al., "Scene-based Shack-Hartmann wavefront sensor for light-sheet microscopy", Adaptive optics and Wavefront Control for Biological Systems IV, International Society for Optical Engineering, 2018, Abstract 10502 Reference 6: International Publication No. 2020 / 157265 Reference 7: OEOlarte et al., “Light-Sheet microscopy: a tutorial”, Advances in optics and photonics, (USA), 2018, Volume 10, No. 1, p. 122-125 Reference 8: Dean, et al., "Deconvolution-free subcellular imaging with axially swept light sheet microscopy", Biophysical Journal, (USA), 2015, No. 108, p. 2807-2815 Reference 9: Hedde et al., “Selective plane Illumination Microscopy with a Light Sheet of uniform thickness formed by an electrically tunable lens)”, Microscopy Research and Technique, (USA), 2016 Reference 10: Migliori, et al., "Light-Sheet theta microscopy for rapid high-resolution imaging of large biological samples," BMC Biology 16, (English), 2018, No. 57

Claims

1. A method for fluorescence microscopy imaging of a three-dimensional fluorescent object (10) by a fluorescence microscopy imaging system (200) using light sheet illumination, comprising: The system is arranged at a pupil plane (PP １ ) and an optical axis (Δ), The method comprises: Illuminating the object with a light sheet through an illumination path (201) equipped with an illumination device (205), The irradiating step comprises: generating a focused beam of light; scanning the focused light beam in an illumination plane substantially perpendicular to the optical axis of the imaging microscope objective to generate a light sheet (100); Including, The focal plane (PO １ ) is included in the optical sheet, The light sheet causes the emission of fluorescent light by the object. Steps and The imaging microscope objective (210) and the imaging detection surface (PO ３ ) through an imaging path (202) including an imaging detector (220) having a focal plane (PO) of the imaging microscope objective. １ generating at least one first fluorescent image of an optical section of the object superimposed on the first fluorescent image, the at least one first fluorescent image is generated in a spectral imaging band; Steps and Analysing the wavefront of the fluorescent light emitted by the object through an analysis path (203) comprising the imaging microscope objective and a wavefront analyzer (240), The wavefront analyzer includes: An analytical detection plane (PO) conjugate with the focal plane of the imaging microscope objective lens. ４ a two-dimensional detector (248) having a The pupil plane (PP １ ) and the conjugate analytical surface (PP ３ a two-dimensional arrangement (243) of a plurality of microlenses (244) arranged on the Equipped with Analyzing the wavefront includes: The focal plane (PO １ each microlens generating a fluorescence image of a given analysis field of view of the object located at said analysis detection plane, The fluorescence image is generated in a spectral analysis band. Sub-steps and The fluorescent light emitted by the analysis field of view is detected by the analysis detection plane (PO ４ a sub-step of spatial filtering by a spatial filtering device (245) having a filtering element arranged in a filtering plane optically conjugate with the said spatial filtering step including scanning said filtering element relative to the fluorescence image of the focused light beam formed on said filtering surface in synchronization with said scanning of said focused light beam to obtain a superposition at each instant of time of said filtering element and said fluorescence image of the focused light beam. Sub-steps and processing the plurality of fluorescence images produced by the plurality of microlenses by a processing unit (250) to determine a two-dimensional map of characteristic parameters of the wavefront in the analysis plane; Contains analyzing the wavefront; Based on the two-dimensional map of characteristic parameters of the wavefront, the optical slice of the object and the imaging detection plane (PP １ At least a part of the optical defect between the pupil plane (PP １ ) by a wavefront modulation device (232) having a correction surface conjugate with the wavefront modulation device (232); Includes Fluorescence microscopy imaging methods.

2. 2. The method of claim 1, further comprising: The filtering element comprises a movable slit (40, 425); the spatial filtering of the fluorescent light emitted from the analysis field of view includes a lateral movement of the slit synchronized with the scanning of the collection beam. Fluorescence microscopy imaging methods.

3. 2. The method of claim 1, further comprising: The filtering element comprises a fixed slit (510); The spatial filtering device further comprises a set of optical components including at least a first rotatable mirror (512, 522); the set of optics is configured to generate a fluorescence image of the collected light beam onto the slit; The spatial filtering of the fluorescent light emitted from the analysis field of view includes: rotating the at least one first movable mirror in synchronism with the scanning of the focused beam to superimpose the slit and the fluorescent image of the focused beam at each instant of time; Including, Fluorescence microscopy imaging methods.

4. The fluorescence microscope imaging method according to any one of claims 1 to 3, The device for illuminating the illumination path comprises: a laser emission device (310) for emitting a light beam; Light irradiation axis (Δ １ A light irradiation system having Equipped with the illumination system is configured to generate from the light beam a focused light beam (341) parallel to the illumination axis; the light sheet (340) is generated by scanning the focused light beam in a direction perpendicular to the optical axis; Fluorescence microscopy imaging methods.

5. The fluorescence microscope imaging method according to any one of claims 1 to 3, The device for illuminating the illumination path comprises: a laser emission device (310) for emitting a light beam; Light irradiation axis (Δ ２ a light projection system configured to generate a focused light beam (341) from the light beam perpendicular to the light projection axis; Equipped with the light sheet is generated by scanning the focused light beam; Fluorescence microscopy imaging methods.

6. A method for fluorescence microscopy imaging according to any one of claims 1 to 5, comprising: determining the two-dimensional map includes determining a positional variation of the plurality of fluorescent images formed by the plurality of microlenses; The positional variation of one of the fluorescent images formed by one of the microlenses is measured relative to a reference position of a reference image; the positional change is determined by a calculation between the image and the reference image; The operation is selected from a cross-correlation operation, a phase correlation operation, and a sum of squared differences operation. Fluorescence microscopy imaging methods.

7. A wavefront correction device (230) adapted to be connected to a fluorescence microscopy imaging system with light sheet illumination for performing the method according to any one of claims 1 to 6, comprising: The fluorescence microscope imaging system includes: Pupil plane (PP １ ) and an imaging microscope objective (210) having a pupil (211) and an optical axis (Δ) at an imaging detection plane (PO ３ an imaging path including an imaging detector having a an illumination path with an illumination device (205) configured to scan a focused light beam in an illumination plane substantially perpendicular to the optical axis to generate a light sheet (100); Equipped with The wavefront correction device comprises a wavefront analysis device (240); The wavefront analyzer (240) comprises: Analysis detection surface (PO ４ a two-dimensional detector (248) having a Analysis side (PP ３ a two-dimensional arrangement (243) of a plurality of microlenses (244) arranged on the Each of the microlenses is located at the focal plane (PO １ ) at the analysis detection plane when the wavefront correction device is connected to the microscope imaging system; The fluorescence image is generated in a spectral analysis band. A two-dimensional arrangement (243) of a plurality of microlenses (244); A spatial filtering device (245), comprising: the spatial filtering device is configured to spatially filter the fluorescent light emitted by the analysis field of view when the wavefront correction device is connected to the microscope imaging system; The spatial filtering device comprises: The analysis detection surface (PO ４ ) and a filtering surface (PO ５ , P.O. ６ ) a filtering element located at a scanning means configured to scan the filtering element relative to the fluorescence image of the focused light beam formed on the filtering surface in synchronization with the scanning of the focused light beam so as to obtain a superposition at each instant of time of the filtering element and the fluorescence image of the focused light beam, A spatial filtering device (245); a processing unit (250) configured to determine a two-dimensional map of characteristic parameters of the wavefront at the analysis plane based on all of the images formed by the plurality of microlenses; Equipped with The wavefront correction device comprises: Correction surface (PP ２ A wavefront modulation device (232) comprising: When the wavefront correction device is coupled to the microscope imaging system, the wavefront correction device corrects the optical slice of the object and the imaging detection plane (PO) based on the two-dimensional map of characteristic parameters of the wavefront. ３ ) configured to correct at least a portion of the optical imperfections between the A wavefront modulator (232); The wavefront correction device is connected to the microscope imaging system, and the pupil plane (PP １ ) and the correction surface (PP ２ ) and the analysis surface (PP ３ a first optical relay system (231, 241, 242) configured to be optically conjugate with the first optical relay system (231, 241, 242); The wavefront correction device is adapted to correct the focal plane (PO) of the imaging microscope objective when connected to the microscope imaging system. １ ) and the analysis detection surface (PO ４ ) and the imaging detection surface (PO ３ ) and the filtering surface (PO ５ , P.O. ６ a second optical relay system (231, 236, 241, 242) configured to be optically conjugate with the first optical relay system (231, 236, 241, 242); The wavefront correction device (230) further comprises:

8. 8. A wavefront correction device (230) according to claim 7, comprising: The filtering element of the confocal filtering device comprises a movable slit (40, 425); the scanning means is configured to cause a lateral movement of the slit synchronized with the scanning of the focused beam. A wavefront correction device (230).

9. 9. A wavefront correction device (230) according to claim 8, comprising: the movable slit is formed by a movable opto-mechanical element (40) configured to transmit or reflect fluorescent light; A wavefront correction device (230).

10. 9. A wavefront correction device (230) according to claim 8, comprising: the movable slit is formed by addressing a group of one or more rows (or columns) of a spatial intensity modulator (420); A wavefront correction device (230).

11. 8. A wavefront correction device according to claim 7, The filtering element comprises a fixed slit (510); The spatial filtering device further comprises a set of optical components including at least a first rotatable mirror (512, 522); the set of optics is configured to generate a fluorescence image of the collected light beam onto the slit; the scanning means is configured to cause a rotation of at least one first movable mirror in synchronism with the scanning of the focused light beam in order to superimpose at each instant of time the slit and the fluorescent image of the focused light beam. Wavefront correction device.

12. The wavefront correction device according to any one of claims 8 to 11, The width of the slit (40, 425, 510) is a minimum value equal to twice the diffraction limit of the imaging microscope objective multiplied by the optical magnification between the filtering plane where the slit is located and the focal plane of the imaging microscope objective; a maximum value equal to the width of the Rayleigh range corresponding to the Gaussian beam generating the fluorescent light beam multiplied by the optical magnification between the focal plane of the imaging microscope objective and the filtering plane where the slit is located; Between Wavefront correction device.

13. A fluorescence microscopy imaging system (200) for a three-dimensional fluorescent object (10) using light sheet illumination, comprising: an imaging path (202) configured to generate at least one first image of an optical slice of the object in a spectral imaging band, The imaging pathway includes: Pupil plane (PP １ an imaging microscope objective (210) having a pupil at Imaging detection surface (PO ３ an imaging detector (220) having a Equipped with The optical section is located at the focal plane (PO) of the imaging microscope objective. １ ) is superimposed on an imaging path (202); An illumination path of the object, the illumination path comprises an illumination device (205) configured to scan a focused light beam in an illumination plane substantially perpendicular to an optical axis of the imaging microscope objective to generate a light sheet (100); The focal plane (PO １ ) is included in the optical sheet, The light sheet is configured to produce an emission of fluorescent light. The irradiation path; An analysis and correction path (203), the imaging microscope objective lens; A wavefront correction device (230) according to any one of claims 7 to 12, Equipped with The wavefront correction device (230) is adapted to correct the optical slice of the object and the imaging detection plane (PP) based on the two-dimensional map of characteristic parameters of the wavefront. １ ) configured to correct at least a portion of the optical imperfections between the An analysis and correction path (203); A fluorescence microscope imaging system (200) comprising:

14. 14. A fluorescence microscopy imaging system (200) according to claim 13, comprising: The device for illuminating the illumination path comprises: a laser emission device (310) for emitting a light beam; Light irradiation axis (Δ １ A light irradiation system having Equipped with the illumination system is configured to generate from the light beam a focused light beam (341) parallel to the illumination axis; the light sheet (340) is generated by scanning the focused light beam in a direction perpendicular to the light irradiation axis; A fluorescence microscopy imaging system (200).

15. 14. A fluorescence microscopy imaging system (200) according to claim 13, comprising: The device for illuminating the illumination path comprises: a laser emission device (310) for emitting a light beam; an illumination system configured to generate from the collimated light beam a focused light beam (341) having an illumination axis and perpendicular to the illumination axis; Equipped with the light sheet is generated by scanning the focused light beam; A fluorescence microscopy imaging system (200).

Images