OPTICAL MEASURING METHOD AND DEVICE

Abstract

The invention proposes an optical measuring method and an optical measuring device for determining the spatial or spatiotemporal distribution of a sample, the sample comprising at least one re-emitting source, said at least one re-emitting source re-emitting light as a function of the light projected onto the sample, according to a determined law, by a first light source comprising a first laser, and the re-emitting source being able to be depleted or activated by the action of a second light source, comprising a second laser, the method comprising: the use of the two lasers, the wavelength of one of the lasers being tuned to the excitation wavelength of said at least one re-emitting source and the wavelength of the second laser being tuned to the depletion or activation wavelength of said at least one re-emitting source, the production, using a polarization sub-module, for each laser, of a controlled polarization state,the projection onto the sample, by means of an achromatic optical projection apparatus, of at least two compact light distributions of different topological families, propagating along the same optical path, the detection of the light re-emitted by said at least one re-emitting source of the sample; the generation of at least one optical image, from the detected light; and the algorithmic analysis of the optical images to obtain location information for said at least one re-emitting source. In addition, the invention describes the production of the cited achromatic optical projection apparatus, using modules based on conical diffraction or on other optical effects.,

Description

1 METHOD AND DEVICE FOR OPTICAL MEASUREMENT The present invention relates to a method and device for optical measurement. It has applications in all fields of imaging, in particular but not limited to the field of Microscopy, including but not limited to the fields of Biology, Medicine, Pharmacy, Semiconductors, Materials Science, Metrology, control, measurement and observation and to all processes of acquiring information from optical observations, in the macroscopic or microscopic field. An optical microscope is an optical instrument generally used to view, analyze, or measure objects too small to be seen with the naked eye. We will use the term "biological" to describe any entity in the life sciences, regardless of its origin (human, animal, or plant) and the purpose of its observation (research, diagnosis, or therapy). This term includes the medical uses of the technique described. Microscopy is used in the field of biology, for example, to observe, study, and measure biological entities (objects) and their dynamics. By extension, we will use the term "artificial vision" to describe all applications of measurement, metrology, or observation of objects or elements produced or constructed or created by a human being or a machine, for example, to observe,To study and measure semiconductors or to characterize materials. The usual definitions are used for optical diffraction limit, Rayleigh criterion, Airy disk and its radius and diameter. In the context of the invention, we use the terms superresolution, super-resolved, super-resolution imaging, and super-resolution microscopy to describe the acquisition of optical data, in imaging, microscopy, or computer vision, at a resolution greater than the optical diffraction limit. The usual definitions are used for fluorescence and for fluorophores. Reference is now made to Figure 1, which illustrates the microscopy paradigm. Optical microscopy consists of illuminating a biological or non-biological sample with a light source (not shown) using a microscope and measuring it over time.using visual observation or a detection module, 12, of the light emitted, re-emitted, scattered, or reflected, or 35 transmitted by the sample. In Biology, the sample consists of one—or a plurality—of different biological object entities, 13 and 14, positioned at different locations. Examples of such objects are, among others, a cell, a virus, a protein, and a 3029633 2 fragment of DNA. In machine vision, the sample can be, for example, a semiconductor element. Microscopy is segmented into different modalities with different characteristics and purposes. Numerous descriptions of the different modalities, their 5 characteristics, and their advantages exist extensively in the literature and can be found, for example, on the websites of companies such as Zeiss, Leica, Nikon,or Olympus. Microscopy applications can be structured in many different ways: one of them is to distinguish between microscopy modalities dedicated to visualizing tiny point sources and those dedicated to measuring continuous objects. The case of tiny point sources is, a priori, much simpler. The object consists of a small number of points of light; these can be described by a small number of parameters—the descriptors defined subsequently—greatly simplifying the physical problem and the algorithmic complexity. The case of a continuous object, described by a spatial distribution—or spatiotemporal distribution, if we take into account continuous dynamics—is different and is also described in this patent application. Fluorescence microscopy is one of the microscopy modalities; it has replaced, in many applications,other microscopy techniques. A fluorescence microscope is an optical microscope used to study the properties of objects, or organic or inorganic substances, using fluorescence phenomena instead of, or in addition to, other modalities such as reflection and absorption. Reference is again made to Figure 1, which describes a fluorescence microscope used in either biology or computer vision to characterize, for example, materials. In fluorescence microscopy, tiny point sources, 15 to 18, for example, fluorophores based on the physical phenomenon of one-photon fluorescence, are fixed to specific and predetermined positions on the objects, 13 and 14. The light emitted by the point sources is observed instead of the light emitted by the objects, 13 and 14, themselves. The sample is illuminated by light of a specific wavelength, or wavelengths.which is absorbed by point sources, thus inducing the emission of light at different and higher wavelengths. During the collection of the emitted light, in fluorescence microscopy, the illumination light is separated from the weaker emitted fluorescence by the use of an emission spectral filter. Fluorescence microscopy studies the light emitted by small point sources, the fluorophores. However, when the density of fluorophores is high, the fluorophores are no longer analyzed individually but treated as a continuous object. 3029633 3 It is important to note, from this stage, that the same system allows the observation of continuous objects,and is not limited to the observation of point sources. Fluorophores have become an important tool for visualizing biological objects. Biological activity and information, including details above the 200 nm resolution limit, are routinely visualized and measured using fluorescence microscopy. This resolution limit is derived from the Rayleigh criterion, which, at best, reaches 200 nm in specially designed systems. For a long time, until the emergence of the super-resolution techniques described below, it was recognized that optical techniques, including fluorescence microscopy, are incapable of visualizing details smaller than the Rayleigh criterion, on the order of 200 nm. However, other fundamental biological activities also occur at scales smaller than 200 nm in biological samples. At this level of spatial resolution,Important phenomena can be observed: 15 biological processes at the intracellular level, cellular information transfer, protein folding and unfolding, and modifications to DNA and RNA. Thus, for example, measuring this intracellular information will open new avenues for understanding biological activity and will lead to advances in knowledge and monitoring for medical research and diagnostics. 20 The main implementations of fluorescence microscopy, described in detail in the literature, are the confocal microscope, often used in a scanning or rotating disk configuration, and the wide-field imaging microscope. Reference is now made to Figure 2, which is a simplified representation of a prior art confocal fluorescence microscope. 25 A confocal fluorescence microscope, Fig. 2,is an optical instrument. Its main hardware components are shown in Figure 2. They include: a light source, 20; an optomechanical frame (not shown); a filter cube, 21; a microscope objective, 22; a detector assembly, 23; and a processing unit (not shown). The light source, 20, which can be an arc lamp or a laser, creates the light energy necessary for fluorescence. The optomechanical frame (not shown) supports all the optical elements and includes auxiliary optics and alignment capabilities. It also includes optical elements (not shown) capable of shaping the beam to allow its focusing to a point of minimal size by means of the microscope objective. In a confocal scanning fluorescence microscope, it may also include a scanning mechanism, either spatial or angular.not shown to modify the 5th position of the point source relative to the object to be measured. The scanning mechanism can alternatively: mechanically translate the object, using for example a translation stage, optically scan the beam over the object, using for example a 10th set of galvanometric mirrors or acousto-optic translators, or use any combination of these mechanical or optical translation methods. In a scanning confocal fluorescence microscope, information is collected point by point, using the scanning mechanism. 15 It can also include, in a rotating disk confocal fluorescence microscope, a rotating disk, having a plurality of microscopic holes, allowing the simultaneous projection of a plurality of points. In a rotating disk confocal fluorescence microscope, a set of points,corresponding to the microscopic holes is acquired at each instant, and the rotation of the disk allows the entire surface of the sample to be scanned for a given longitudinal position. The filter cube, 21, channels the different optical signals and prevents contamination of the fluorescence signal by the excitation light. The filter cube is composed of: an excitation filter, 210, a dichroic mirror, 211, and an emission filter, 212. The filters and the dichroic mirror are chosen according to the excitation wavelength and the spectral emission characteristics of the fluorophore. The microscope objective, 22, focuses the light created by the source in the focal plane of the objective, 24, into a small light distribution, the optimal light distribution being an Airy disk. The microscope objective, 22,It also allows for the collection of the fluorescent light emitted by the fluorophores. 30 For a scanning confocal fluorescence microscope, the system can be descanned, meaning that the returned light can pass through the scanning mechanism to compensate for the translation due to scanning. A detector lens, 25, creates, in the image plane of the detector 26, a magnified image of the focal plane of the objective, 24. 3029633 5 A confocal hole, 27, is theoretically located in the image plane of the detector, 26. In most practical systems, the confocal hole, 27, is located in an intermediate imaging plane not shown and reimaged onto the image plane of the detector, 26. The detector assembly, 23, detects the overall fluorescent intensity in the illuminated volume 5 and converts it into a digital signal. For a scanning confocal microscope, the detector assembly consists of a single-element detector,such as a PMT or SPAD. For a rotating disk confocal microscope, the detector assembly consists of an array of detection elements, such as a CCD, an EMCCD, a CMOS, or a SPAD array. 10 The assembly of components mounted from the light source to the dichroic filter is the illumination path, 201. The assembly of components mounted from the dichroic filter to the detector assembly is the detection path,202. The elementary optical process of a confocal microscope can be segmented into six steps: 15 - Projection of light onto the analysis volume - Emission of fluorescent light by the fluorophores - Imaging of the fluorophores on the focal plane - Limitation in the focal plane of the analyzed light by the confocal hole - Integration of the analyzed light by a photoelectric detector 20 - Visualization of the measured intensity as a pixel value in an image. Fluorescence microscopes are available from several manufacturers, such as Nikon, Zeiss, Leica, or Olympus. Fluorescence microscopes can be either standard microscopes adapted for fluorescence or 25 specific microscopes optimized for fluorescence. Modern microscopes are versatile instruments capable of operating in many different modalities, including, but not limited to, fluorescence modalities.using the same optomechanical platform and most components. Most fluorescence microscopes are developed as an open platform, capable of performing several additional functions with minimal modifications. Other fluorescence microscopes are dedicated instruments, customized for a specific task, such as medical or pharmaceutical diagnostics. Superresolution: New optical methods, superresolution methods, are capable of discriminating point sources below the Rayleigh criterion. These methods are being developed by several companies, laboratories, and researchers, and some of the instruments using these methods, superresolution microscopes, are commercially available. Several comparative analyses of superresolution methods have been published recently in the literature.such as the articles by Schermelleh et al. [1]. An updated bibliography on superresolution can be found on the Zeiss website and the Nikon website. Existing microscopy methods and microscopes, which do not incorporate superresolution, allow microscopic observation within the limits of optical diffraction. This reduces their field of use to a limited set of applications. New superresolution techniques allow us to obtain information beyond the resolution limit. The main problem with all existing superresolution techniques is that the performance envelope, expressed in terms of lateral resolution, longitudinal resolution, speed, required light intensity, phototoxicity in the biological object, and ability to measure different objects, is very limited. Furthermore,Most existing superresolution methods and instruments can provide either good lateral or good longitudinal resolution, but rarely both. Furthermore, all these instruments are complex and require a high level of operator skill. In addition, these instruments can generally only observe a small portion of biological specimens due to significant operational limitations, such as, for some, a shallow depth of field or very high intensities that are harmful to cells. Another problem with existing superresolution methods and instruments is that most of them are only able to recover the attributes of a single fluorophore within the illuminated volume.but fail to simultaneously recognize the presence of multiple fluorophores and measure their attributes. An additional problem with existing superresolution methods and instruments is that these methods and instruments are presented to users and perceived by them as a general-purpose tool, capable of replacing standard or confocal microscopes. However, existing superresolution methods and instruments lack simplicity, robustness,The ease of use and competitive prices of standard microscopes hinder their use as general research or diagnostic tools. Another problem with existing superresolution methods and instruments is that most of them are built as standalone instruments designed to replace standard microscopes. Such an approach requires replacing existing instruments as well as renewing all peripheral systems and all the knowledge and know-how related to microscopy platforms and developed over many years. Another problem with most existing fluorescence and superresolution microscopy methods and instruments is that they are designed on an image acquisition paradigm in which the basic unit of information is one or more images.or one or more two- or three-dimensional ROI (Region of Interest). The algorithmic, systemic, and superresolution methods described later in the context of the invention will, by their inherent flexibility, enable the development of new acquisition strategies. These dynamic and selective acquisition procedures will be defined by optimized management of the acquisition sequence and interactive and deferred processing. They will allow for more advanced optimization of useful information, defined by criteria based on the shape, geometry, and dynamics of one or more fluorescent objects.separately or relative to each other. Therefore, there remains an urgent need to provide super-resolution methods and instruments, as well as algorithmic methods capable of accurately measuring the attributes of a fluorophore. Furthermore, methods and instruments are needed to detect and quantify the presence of multiple fluorophores placed in the same illuminated volume. Another problem with most existing fluorescence and super-resolution microscopy methods and instruments is that they are designed to study samples on microscope slides. However,The confocal microscope is currently used in many medical fields as an in-vivo diagnostic instrument for internal and external examinations of the human body. This is achieved through optical fibers used to illuminate and visualize the fluorescence emitted by the tissues to be diagnosed. Superresolution is not currently sufficient for such in-vivo diagnostics. The algorithmic, systemic, and superresolution methods described later in the context of this invention will enable the development of new in-vivo diagnostic methods that will reduce the need for biopsies and shorten patient waiting times. 3029633 8 SUMMARY OF THE INVENTION: A first aspect of this invention relates to an optical measurement method for determining the spatial distribution or location of re-emitting sources on a sample,the sample comprising at least one re-emitting source, said at least one re-emitting source re-emitting light according to the light projected onto the sample, according to a determined law, by a first light source comprising a first laser, and the re-emitting source being able to be depleted or activated by the action of a second light source, comprising a second laser, the method comprising: the use of the two lasers, the wavelength of one of the lasers being tuned to the excitation wavelength of said at least one re-emitting source and the wavelength of the second laser being tuned to the depletion or activation wavelength of said at least one re-emitting source; the realization, using a polarization sub-module, for each laser, of a controlled polarization state; the projection onto the sample, using an achromatic projection optical device, for each laser, of a compact light distribution.propagating along the same optical path for all lasers, the detection of the light re-emitted by said at least one re-emitting source of the sample; 20 the generation of at least one image from the detected light. In one embodiment, the method further comprises the algorithmic analysis of the images to obtain spatial distribution or localization information for said at least one re-emitting source. A second aspect of the invention relates to the achromatization of the optical measurement method according to the first aspect of the invention so as to allow the use of the optical measurement method at different excitation and depletion wavelengths,so as to selectively excite the re-emitting sources according to a wavelength-selective marking. Another aspect of the invention relates to the realization of a super-resolution system of the STED or RELSOFT type using an achromatic common-path optical measurement method capable of different beamshaping on different beams depending on their polarization. Another aspect of the invention relates to the co-localization of all lasers using an optical bench preferably employing a fiber optic system to co-localize the beams before their shaping by the optical method described in the preceding aspects. Another aspect of the invention relates to the implementation of the method according to the first aspect using cone diffraction. In an embodiment of the method described above,The compact light distribution of the first excitation laser is of a different topological family than that of the second depletion or activation laser. In one embodiment of the method described above, said at least two compact light distributions of different topological families are created by interference between a regular wave and a singular wave, or between two singular waves, and a spatial differentiation between said at least two distributions is created by varying at least one of the following parameters: a) at least one of the parameters of the regular wave; b) at least one parameter of at least one singular wave; and c) a phase difference between the regular wave and the singular wave or between the two singular waves. In one embodiment of the method described above,The projection of light distributions of different topological families is carried out by conical diffraction or an assembly of uniaxial crystals. In one embodiment of the process described above, the projection of light distributions of different topologies is carried out by conical diffraction in a thin crystal. An embodiment of the process described above, comprising the coupling of light from lasers by an optical sub-module, through a single fiber or a single optical path. In one embodiment of the process described above, the optical fiber comprises a photonic fiber. In one embodiment of the process described above, the optical fiber comprises a few-mode fiber. An embodiment of the process described above,comprising laser control to jointly create a sequence of excitation distributions and a sequence of depletion or activation distributions, the two sequences being synchronized. An embodiment of the method described above, comprising controlling 35 lasers to jointly create a sequence of excitation distributions and a sequence of depletion or activation distributions, the two sequences being synchronized, and a singular excitation distribution being simultaneous with a singular depletion distribution. An embodiment of the method described above, comprising controlling 5 lasers to jointly create a sequence of excitation distributions and a sequence of depletion or activation distributions, one of the excitation distributions being a superoscillation. An embodiment of the method described above,comprising laser control to create simultaneously or sequentially excitation distributions and a sequence of depletion or activation distributions, the set of depletion or activation distributions being equivalent or similar to a vortex. Another aspect of this invention relates to a measuring device for determining the spatial distribution or location of re-emitting sources on a sample, the sample comprising at least one re-emitting source, said at least one re-emitting source re-emitting light as a function of the light projected onto the sample by a first laser, according to a determined law, and the re-emitting source being able to be depleted or activated by the action of a second laser, the device comprising: two lasers of different wavelengths,the wavelength of the first laser being tuned to the excitation wavelength of said at least one re-emitting source 20 and the wavelength of the second laser being tuned to the depletion or activation wavelength of said at least one re-emitting source; a polarization sub-module, for each laser, of a different polarization state; an achromatic projection module enabling the creation, for each laser, of a compact light distribution 25, propagating along the same optical path for all lasers; a detection module capable of detecting light re-emitted by said at least one re-emitting source of the sample; a generation module, capable of generating at least one optical image, from the detected light 30; and an algorithmic analysis module capable of analyzing images to obtain location information for said at least one re-emitting source. In an embodiment of the method described above,The compact light distribution of the excitation laser is of a different topological family than that of the depletion or activation laser. In one embodiment of the method described above, the projection module is capable of creating said at least two compact light distributions of different topological families by interference between a regular wave and a singular wave, or between two singular waves, and a spatial differentiation between said at least two distributions is created by varying at least one of the following parameters: a) at least one of the parameters of the regular wave; b) at least one parameter of at least one singular wave; and c) a phase difference between the regular wave and the singular wave, or between the two singular waves. In one embodiment of the method described above,The projection module includes at least one conical crystal for projecting light distributions of different topological families by conical diffraction or a 15 uniaxial crystal assembly. In one embodiment of the method described above, the optical fiber includes a photonic fiber. In one embodiment of the method described above, the coupling of light from lasers is accomplished by an optical sub-module, through a single fiber or a single optical channel. In one embodiment of the method described above, the optical fiber includes a few-mode fiber. In one embodiment of the method described above, said at least one conical crystal, defined later, is a thin crystal. In one embodiment of the method described above,The lasers are configured to jointly create a sequence of excitation distributions and a sequence of depletion or activation distributions, the two sequences being synchronized. In one embodiment of the process described above, the lasers are configured to jointly create a sequence of excitation distributions and a sequence of depletion or activation distributions, the two sequences being synchronized, and a singular excitation distribution being simultaneous with a singular depletion distribution. In one embodiment of the process described above, the lasers are configured to jointly create a sequence of excitation distributions and a sequence of depletion or activation distributions, one of the excitation distributions being a superoscillation. In one embodiment of the process described above,The lasers are configured to create jointly, simultaneously, or sequentially, a sequence of excitation distributions and a sequence of depletion or activation distributions, the set of depletion or activation distributions being equivalent or similar to a vortex. The description of embodiments of the invention, based on confocal fluorescence microscopy, can be extended, mutatis mutandis, to other microscopy modalities, confocal or otherwise, and to computer vision, whether they observe biological, computer vision, or other objects, and whether the object consists of point sources, structured objects, or continuous objects. Another aspect of the invention relates to an optical measurement method for determining the spatial distribution or localization of re-emitting sources on a sample.the sample comprising at least one re-emitting source, said at least one re-emitting source re-emitting light according to the light projected onto the sample, according to a determined law, by a first light source comprising a first laser, and said at least one re-emitting source being able to be depleted or activated by the action of a second light source, comprising a second laser, the method comprising: the use of two lasers, the wavelength of the first laser being tuned to the excitation wavelength of said at least one re-emitting source and the wavelength of the second laser being tuned to the depletion or activation wavelength of said at least one re-emitting source, the projection onto the sample, by means of an achromatic projection optical apparatus for the first laser and the second laser, of the light distributions,the detection of the light re-emitted by said at least one re-emitting source of the sample; and 30 the generation of at least one image from the detected light. In one embodiment of the method described above, the projection comprises, for the first laser, a compact light distribution, and for the second laser, two compact distributions, one of them having a zero intensity, propagating axially in a spiral motion. 3029633 13 In one embodiment of the methods described above, the two distributions creating a depletion originate from two independent lasers at the same or a close wavelength. 5 In one embodiment of the method described above, the projection onto the sample is carried out by means of an achromatic projection optical device, comprising an optical element having the functionality of segmenting an optical beam into a plurality of independent beams,The projection comprises, for both lasers, a matrix of compact and collocalized light distributions. In one embodiment of the method described above, the optical element comprises a microlens array. In one embodiment of the method described above, the achromatic projection optical apparatus comprises a duplication element for laterally duplicating the light distributions for both lasers from a matrix of compact and collocalized light distributions. In one embodiment of the method described above, the duplication element comprises a beam splitter transmission grating or a uniaxial crystal. In one embodiment of the method described above, the achromatic projection optical apparatus includes at least one additional optical element having the functionality of creating, from an incident beam, two or more beams offset laterally or axially.for the two lasers of a matrix of compact and collocalized light distributions. In another embodiment of the devices described above, an additional beam 30 derived from the depletion laser, or a device comprising a second depletion laser, contains one of the light distributions with strong axial dependence, such as the Stokes distributions or another distribution described in this invention, to reduce the axial size of the emitting volume. 35 In another embodiment of the devices described above, a dynamic polarizing element is used before or after the biaxial crystal to correct the 3029633 14 dynamic pupil movement, which in some cases can be created during the optical scanning of the confocal microscope. This pupil movement effect being, in some implementations by STED technologists, one of the performance limitations,without requiring an additional scanning system. 5 In another embodiment of the devices described above, two or more localized light distributions at different wavelengths are projected using an original or modified LatSRCS module. The first light distribution makes the scene parsimonious, that is, emitters are isolated using a physical effect, so as to create regions in which the hypothesis of parsimony can be made, i.e., the presence of an isolated emitter or a small number of emitters. The physical effects that make this parsimony possible will be the same as, or derived from, effects used to create parsimony for single-emitter localization microscopy techniques. 15 In another embodiment,The microscope platform described is coupled to a CLEM (Correlative Light Electron Microscopy) system, or any other similar system such as TEM (Transmission Electron Microscopy), EBM (Electron Beam Microscopy), or SEM (Scanning Electron Microscopy). In another embodiment, distributions with strong axial dependence, such as Stokes distributions and so-called "shifted half-moon" distributions, are used to characterize the axial dependence of a point in the case of a localization microscopy system. In another embodiment of the invention, distributions with axial dependence, such as Stokes distributions, are used.and the so-called "offset half-moon" distributions to characterize the axial dependence of a point in the case of a localization microscopy system in which an original or modified LatSRC module has been used to make the scene locally sparse and to measure the position of sparse re-emitting sources using one of the PSIT-type techniques. In another embodiment of the invention, a dark tracking technique is implemented in which the tracking of a point is performed by the projection of a vortex or other distribution containing one or more optical intensity zeros and the emission detected when the emitter is outside the aforementioned optical intensity zero. In another embodiment,A cascade of Wollaston prisms with five different beam splittings is used to separate an incident beam into a large number of emergent beams. The same effect can be achieved by modifying the Wollaston prism to create a compound Wollaston prism by adding pieces of uniaxial crystal whose refractive index and birefringence orientation are chosen. A prism can thus be constructed from a single block of uniaxial crystal that can split an incident beam into emergent beams (for example, eight or sixteen), which lie in a plane and are separated by equal angles. Once focused on the sample, two aligned and equally separated points are obtained. In one embodiment of the processes described above,The method further comprises the algorithmic analysis of images to obtain spatial distribution or localization information for said at least one re-emitting source. Another aspect of the invention relates to an optical measuring device for determining the spatial distribution or localization of re-emitting sources on a sample, the sample comprising at least one re-emitting source, said at least one re-emitting source re-emitting light according to the light projected onto the sample, following a determined law, by a first light source comprising a first laser, and said at least one re-emitting source being able to be depleted or activated by the action of a second light source, comprising a second laser, the device comprising: a first laser and a second laser,the wavelength of the first laser being tuned to the excitation wavelength of said at least one re-emitting source and the wavelength of the second laser being tuned to the depletion or activation wavelength of said at least one re-emitting source; an achromatic projection module to create light distributions for the first and second lasers; a detection module to detect the light re-emitted by said at least one re-emitting source of the sample; a generation module to generate at least one image from the detected light. In one embodiment of the device described above, the projection module is capable of creating a compact light distribution for the first laser.and for the second laser, two compact distributions, one of them having a zero intensity, propagating axially in a spiral motion. 5 In one embodiment of the device described above, the two distributions creating a depletion originate from two independent lasers at the same or a close wavelength. 10 In one embodiment of the devices described above, the achromatic projection module includes an optical element having the function of segmenting an optical beam into a plurality of independent beams, the projection comprising, for both lasers, a matrix of compact and collocalized light distributions. 15 In one embodiment of the devices described above, the optical element comprises a microlens matrix. In one embodiment of the devices described above, the achromatic projection module,includes a duplication element for laterally duplicating the light distributions for the two lasers of a compact and collocalized light distribution matrix. In one embodiment of the devices described above, the duplication element comprises a beam splitter transmission grating or a uniaxial crystal. In one embodiment of the devices described above, the achromatic projection module includes at least one additional optical element having the functionality of creating, from an incident beam, two or more beams offset laterally or axially, for the two lasers of a compact and collocalized light distribution matrix. In one embodiment of the devices described above,Device 35 further includes an algorithmic analysis module capable of analyzing images to obtain spatial distribution or location information for at least one re-emitting source. In one embodiment of the devices described above, the device includes an optical measurement method for determining the spatial distribution or location of re-emitting sources on a sample, the sample comprising at least one re-emitting source, said at least one re-emitting source re-emitting light as a function of the light projected onto the sample, the method comprising: the projection onto the sample of a light distribution with a helical topology having a zero intensity position, said zero intensity position having a helical spatial variation as a function of the axial parameter; the detection of the light re-emitted by said at least one re-emitting source of the sample; the generation,of at least one image, from the detected light; and the direct detection or algorithmic analysis of the images to obtain spatial distribution information or the location of said at least one re-emitting source. In one embodiment of the device described in the preceding embodiment, the device is based on conical diffraction for the projection step. In another embodiment of the device described in the third-to-last embodiment, the device includes an SLM or a segmented mirror for the projection step. Another aspect of the invention relates to an optical and / or optoelectronic system, comprising an image acquisition module for acquiring a set of different and differentiated images, originating from the same spatial region of a two-dimensional or three-dimensional object.30 an algorithm in which the formulation of the reconstruction of the object and its spatial and / or temporal and / or spectral properties is considered as an inverse Bayesian problem and leads to the definition of a posteriori distribution. 3029633 18 a posteriori law combining, thanks to Bayes' law, the probabilistic formulation of a noise model, as well as possible priors on a light distribution created in the sample by projection; the estimation of the light distribution in the sample by the use of clouds of point emitters which allows for parsimonious solutions; the estimation of the a posteriori mean by means of a Markov Chain Monte Carlo (MCMC) type algorithm,The representation of the results, either in the form of an image or in the form of numerical or graphical data. 10 BRIEF DESCRIPTION OF THE DRAWINGS The invention will now be described in relation to certain embodiments with reference to the following illustrative figures so that it may be better understood. With specific reference to the figures, it is emphasized that the indications shown 15 are presented by way of example and for the purpose of illustrating the discussion of embodiments of the invention and are presented only for the purpose of providing what may be considered the most useful and easily understood description of the principles and conceptual aspects of the invention. In this regard, no attempt is made to show structural details of the invention in greater detail than is necessary for a basic understanding of the invention.The description, taken with the drawings, shows to those skilled in the art how the various forms of the invention can be implemented in practice. In the drawings: Fig. 1 is a simplified perspective representation of a prior art confocal fluorescence microscope 25, also used as a support for the invention; Fig. 2 is a simplified pictorial representation of a super-resolution fluorescence microscopy system, according to one embodiment of the present invention; Fig. 3 is a simplified schematic illustration of a setup for a conical diffraction module 30,in accordance with one embodiment of the present invention; Fig. 4 is a simplified pictorial representation of the two measurement paradigms according to embodiments of the invention and of confocal microscopy; Fig. 5 is a simplified pictorial representation of a particular embodiment of the SRCDP microscopy platform; Fig. 6a is a simplified schematic illustration of a super-resolution lateral module, in accordance with one embodiment of the present invention; Fig. 6b is a simplified schematic illustration of another embodiment of a super-resolution lateral module, in accordance with one embodiment of the present invention; Fig. 7 presents tables of light distributions of a conical diffraction module as a function of the polarization of the input and output polarizers for several values ​​of the conical diffraction parameter,These light distributions were calculated by simulating the equations developed by Berry, [2]; Fig. 8 is a simplified schematic illustration of one embodiment of dark tracking; Fig. 9 is a simplified schematic illustration of a 10-superresolution algorithm method for fluorophore data, according to an embodiment of the present invention. Fig. 10 is a simplified schematic illustration of the descriptor calculation; Fig. 11 is a simplified schematic illustration of the SRCDP platform control module. 15 In all figures, similar reference numerals identify similar parts. Definitions and technical additions The usual definitions are used in the description for: phase and polarization, polarimetry, Jones vectors and matrices,Stokes parameters and measurement techniques 20 of Jones and Stokes parameters. The usual definitions are used in the description for the TEM00 mode of a fiber and the English terms, "Photonic Crystal Fiber" - PCF -, "few-mode fiber" - FMF, vortex Fiber and "dual-core Photonic Crystal Fiber". We will refer to a device allowing the coupling of several lasers at 25 different wavelengths or at the same wavelength, with the same polarization or with different polarizations, in one or more optical fibers,using the term laser bench. The definition of superoscillations is that of Yakir Aaronov and Sir Michael Berry. A superoscillation is a phenomenon in which a signal that is globally band-limited can contain local segments that oscillate faster than its fastest Fourier components.

[26] The center or centroid of a light distribution is the center of gravity of the intensity. The diameter of a light distribution is the diameter of the first intensity zero, for regular and singular waves, without taking into account the central zero of the singular wave. Two light distributions are collocalized if their centers coincide or are separated by a small spatial value compared to the dimension of the light distribution. In this patent application, we will use the emission wavelength as the basic metric of the system. In this patent application,The usual definitions are used for the following optical components: lens, the definition of which is broadened to include all optical means that transmit, refract, or reflect light; auxiliary optics—optical sub-modules designed to interface and adjust either the geometric parameters or the phase and / or polarization parameters between two other sub-modules or optical modules; polarizer; analyzer; retarder; beam splitter (polarizing and non-polarizing); beam combiner (polarizing and non-polarizing). In this patent application, the usual definitions are used for azimuthal and radial polarizers. We will extend, implicitly or explicitly, certain developments described later for azimuthal and radial polarizers to all spatially variable polarizing elements. In this patent application,The usual definitions, [3], are used for the different superresolution techniques; These techniques can be grouped into 20 families: - Reversible Saturable Optical Fluorescence Transitions (RESOLFT), including techniques such as Stimulated Emission Depletion microscopy (STED), Ground state depletion (GSD), Saturated Structured Illumination Microscopy (SSIM), and SPEM (Saturated Pattern Excitation Microscopy); - Localization Microscopy, including techniques such as Photoactivated Localization Microscopy (PALM), FPALM (3D Localization in Fluorescence Photoactivation Localization Microscopy), Stochastic Optical Reconstruction Microscopy (STORM), dSTORM (direct STORM), SPDM (Spectral Precision Distance Microscopy), "stochastic 30 blinking", Ground state depletion (GSD), and similar techniques (regardless of the acronym used); - Structured Image Microscopy (SIM); - FRAP (fluorescence recovery after photobleaching).- TIRF (Total Internal Reflection Fluorescence Microscopy). 35 3029633 21 In this patent application, common definitions are used for various microscopy techniques, standard or super-resolution, fluorescent or non-fluorescent, such as the English terms "Computational Microscopy," "Correlative Microscopy," "Cross-platform microscopy," FCS (Fluorescence Correlation Spectroscopy), FCCS (Fluorescence Cross-Correlation Spectroscopy), PCH (Photon Counting Histogram), RICS (Raster Imaging Correlation Spectroscopy), or FRAP (Fluorescence Recovery after Photobleaching analysis). In this patent application,The usual definitions are used for the Hough Transform. 10 We refer to a partial polarizer to describe an element or module whose absorption differs for both linear polarizations—linear dichroism—or for both circular polarizations—circular dichroism. We refer to dynamic polarization or phase elements to describe optical means whose polarization or phase properties vary 15 as a function of time in a controlled manner, either discretely or continuously. These dynamic polarization or phase elements include, but are not limited to: wave plates rotating on their axis, light valves based on liquid crystal technologies, electro-optical devices, also known as Pockels cells, Kerr cells, resonant electro-optical devices, magneto-optical devices, also known as Faraday cells, 20acousto- or elasto-optic devices or any combination thereof. We refer to polarization or phase dispersive elements to describe elements whose polarization state depends on the wavelength. The simplest polarization dispersive submodule is the multimode or thick waveplate. We will refer to the "centroid algorithm" to describe the usual procedure for measuring the centroid and possibly the full width half maximum (FWHM) of a light distribution. This algorithm originated in astronomy and astrometry and enabled the measurement of star positions with very high precision. This algorithm is used today in all optical instrumentation, including super-resolution biology. In this document, the usual definitions are used for the following optoelectronic components: photoelectric detector, CCD, EMCCD, CMOS,SPAD Single Photon Avalanche Diode and SPAD matrix. 35 We will use the terms: o optical image, for the spatial distribution of light intensity, 3029633 22 o electronic image, to describe the spatial distribution of charges for a CCD, current for a CMOS, or events for a SPAD, created by the optical image at a given instant in a detection plane, o digital image, to describe a matrix of numbers created by the digitization of the electronic image. To simplify reading and understanding the text, we will also use the term image for the output of a single-pixel detector such as a PMT or SPAD, considering it as an image consisting of a single pixel. When no ambiguity exists, or when the distinction between the three types of images is not necessary, we will use the simplified generic term image. For images, we use the terminology used for matrix detectors, such as CCDs,EMCCDs and CMOS. For SPADs and SPAD arrays, the result of a measurement is a time-ordered list of photon impacts, detailing, for each photon, the impact time and position. To simplify the presentation of this document, we will include this case in our definition of images. The images detailed in this document can, in many cases, be described as microimages, images with a size approximately equal to a small number of Airy disk diameters, typically less than 5 diameters, and / or a low number of pixels, typically 4x4 to 32x32. In a digital image Aj,The indices m and n represent the pixel indices; the pixel origin will be chosen as the projection of the center of the analysis volume defined in a later paragraph. Polarimetry and Stokes Vectors Polarimetry refers to the measurement of the polarization state of incident light. The polarization state of incident light can be described by Stokes parameters, a set of values ​​introduced by George Gabriel Stokes in 1852 and used in optics. Copropagation of Two Optical Beams Many optical systems and devices use two—or more—beams with different properties. The beams may or may not interact with each other, and may be projected sequentially or simultaneously. In most of these systems and devices, the two optical paths are physically separated from each other. This physical separation creates, at the system engineering level, a set of constraints which, although solvable,significantly increase the system's complexity and cost. We refer to common-path systems to designate a set of devices in which the two differentiated beams propagate along the same physical path, with only minor variations. Electric field in polar coordinates and angular modes E(p, 0) = A(p, 0) Uexp [iq)(p, 0)]u(p, 0) (EQ-1) 5 In optics, it is common to decompose the field components—its amplitude, phase, and polarization—into orthogonal, Cartesian, or polar modes. Many decompositions into polar orthogonal modes, such as Gaussian, Hermite-Gaussian, and Laguerre-Gaussian modes, are known to those skilled in the art. 10 In this document, we will primarily use the decomposition of the electric field amplitude into Hypergeometric-Gaussian modes HyGG of the form: A(p, 0) oc pPHHexp (--p2 + iP, 0) (EQ. 2) In this decomposition,p is the radial mode and f the azimuthal order. Singular Waves 15 A singular wave has zero intensity at its center and an azimuthal phase variation of a multiple of 2x. This research topic in Optics, initiated by the major article by JF Nye and M. Berry in 1974, [4], is now known as "singular optics." Examples of regular and singular waves are presented below. We use the English term beam shaping to describe the transformation of a wave of a given shape and topology into a wave of another shape or topology, and in particular the transformation of a regular wave into a singular wave and vice versa. Compact Topology and Light Distributions A point-like light distribution will be considered compact if it fulfills one of the compactness conditions defined below by two alternative and non-exclusive conditions: either,More than 75% of the energy is contained within a circle with a radius less than 1.75 times the Airy radius, meaning a luminous domain containing more than 65% of the energy is delimited by a line of zero intensity contained within a circle with a radius less than twice the Airy radius. We distinguish different families of point light distributions with different topologies: Regular distributions, in their usual definition in optics; Singular distributions, also called optical vortices, with topological charge (azimuthal order) t, in which the phase varies from 0 to 2πr around the direction of propagation, where f is an integer; Amplitude distributions with azimuthal variation of order t, also called Laguerre-Gauss distributions; Polarization and optionally phase distributions with azimuthal variation of order t. Also called radially polarized Laguerre-Gauss modes. Two compact light distributions,will be deemed to belong to 10 different topological families if they satisfy at least one, and any one, of the following conditions: One is regular and the other is singular, One is point-like and the other is annular, The azimuthal orders f of the amplitude of the two light distributions differ, 15 The azimuthal orders f of the polarization or phase of the two light distributions differ. Alternatively, two light distributions projected onto a given volume will be deemed to belong to different topologies if, in a substantial part of the jointly illuminated surface, the gradients are in opposite directions. 20 Light Nanoemitters A light nanoemitter is a small secondary emitter, attached to an object; it is substantially smaller than a fraction of a wavelength,Typically, but not limited to a size less than one-fifth of the wavelength, a light nanoemitter absorbs incident energy and re-emits light at the same wavelength as the incident light or at different wavelengths. The light emitted by the nanoemitter may be coherent, partially coherent, or incoherent with the absorbed light. The principal examples of light nanoemitters are fluorophores and nanoparticles, but they also include a large number of other elements. The definition, in the context of the invention, of light nanoemitters is determined by the following two conditions: creation of a point-like secondary light emitter, and predetermined positioning of this emitter relative to an artificial, biological, or organic entity. The physical mechanisms that can create a nanoemitter are numerous; they include, but are not limited to, absorption.scattering or reflection, fluorescence, “emission-depletion”, [5], for example using RESOLFT techniques, photoactivation and photodepletion phenomena, fluorescence with two or more photons, elastic or inelastic scattering, Raman scattering, or other physical mechanisms known to those skilled in the art. We will use the term light emission to describe the emission of electromagnetic waves by the light nanoemitter, whether the light is coherent, incoherent, or partially coherent. We will extend our definition of nanoemitters to include scattering, absorbing, and reflecting particles attached to a biological or organic entity; the action of a scattering, reflecting, or absorbing particle on the electromagnetic field can indeed be described as the creation, with an inverse phase, following Babinet's principle, for an absorbing particle, of an auxiliary secondary field.emerging from the particle, superimposed on the incident electromagnetic field. 15 In this patent application, we will refer to the descriptors of a nanoemitter to denote the set of information describing a nanoemitter as a point source at a given instant. Since the nanoemitter is considered a point source, the set of information representing it contains a limited number of parameters, namely: its position in space, its intensity, and the spectral, intensity, coherence, phase, and polarization characteristics of the light emitted by the fluorophore, as a function of the incident light. In this patent application, we will refer to the descriptors of a structured object. For example, for a uniform line, the set of information representing it contains a limited number of parameters, namely: its orientation in space, its intensity, and the spectral, intensity, and coherence characteristics.phase and polarization of the light emitted by the object, as a function of the incident light. For a continuous distribution, the object is represented, as is customary in image processing, by an intensity matrix. However, in most cases, and in the description of the invention, we refer, under the designation of descriptors, to a subset of the descriptors of a nanoemitter comprising its geometric position, its intensity, and the type of fluorophore, when several populations of light nanoemitters, differentiated for example by their emission spectrum, are present in the same sample. This simplification used in the description does not alter the scope of the invention, which will include in its field of application all the descriptors of light nanoemitters. To simplify the understanding of the context of the invention, the remainder of the description refers only to the simplest case,the one in which the nanoemitter is a 3029633 26 fluorophore and the physical interaction is one-photon fluorescence. However, this description should be understood as a simplified illustration of a general description of the methods and concepts applicable to all the light nanoemitters mentioned previously or known to those skilled in the art, regardless of the underlying physical phenomenon. It is noteworthy that the nanoemitter samples the incident field or intensity at a precise three-dimensional position, without influence from the overall spatial distribution of the incident intensity. We will refer to this remarkable property in this patent application as the sampling capability of the light nanoemitter. 10 However,The described embodiment of the invention also allows for the measurement of structured objects and continuous distributions that do not possess the sampling capacity of the light nanoemitter. Reference is again made to Figure 1, which represents an array of nanoemitters or structured objects positioned on a given biological object, 15 and 16 on the one hand, and 17, 15, and 18 on the other. Alternatively, the emitted light may consist of a continuous distribution, not shown in Figure 1, or any combination of nanoemitters, structured objects, or continuous distributions. The array of nanoemitters, structured objects, or continuous distributions is referred to as an array of "luminous biological objects"; they represent a map of the biological object, in the sense defined by Alfred Korzybski in general semantics. However, it is common practice to simplify the description,to refer to the luminous biological object as the biological object itself, when no ambiguity can arise. The luminous biological object contains a great deal of relevant information related to the biological object, primarily spatiotemporal information, the object's position and orientation as a function of time, and morphological information, for example, in the case of a cell splitting in two. The measurement system according to at least one embodiment of the invention will allow the calculation of the measured map and the evaluation of the descriptors of any combination of nanoemitters, structured objects, or an evaluation of the spatial distribution of continuous distributions. This measured map differs from the original map due to noise, measurement conditions,limits of the system or measurement uncertainty. This information from the measured map can be further developed at different levels of abstraction. This first level of abstraction, which describes the direct results of the measurement, does not, a priori, contain any biological information but rather the results of a physical measurement described by nanoemitters, structured objects, or continuous distributions, which could, moreover, represent any labeled entity. The second level, the level of geometric abstraction, structures the nanoemitters, structured objects, or continuous distributions as geometric objects. It consists of a description of luminous objects and their dynamic characteristics, such as their position or orientation, or their morphology. At this level,Information is still physical and geometric information describing a set of objects. Geometric information uses the measured map and auxiliary information, potentially external to the system, about the relationship between points of light and objects. The biological level of abstraction allows a certain apprehension of biological reality through a constitutive relationship between measured objects and corresponding biological entities. It contains a set of information about the biological object, primarily its position and dynamics, its shape and morphology. Biological information uses the measured map and geometric information, and auxiliary information, potentially external to the system.on the relationship of the 15 points of light and objects to biological entities. A number of conclusions about the biological functionality of the sample can be obtained at this level. The functional level of abstraction allows for an understanding of biological reality. It consists of functional information, uncorrelated with geometric information, and answers questions in biological terms and jargon, such as: "Did the virus penetrate the cell?". An additional level of information can be defined that includes the control and instrumentation process; indeed, a more advanced control and instrumentation process can be defined, allowing access to more structured biological information through automation of the data acquisition process. An example of such a process is described by Steven Finkbeiner under the name "Robotic Microscopy systems". This description of the levels of abstraction,defined in this application, was written, for simplicity, for Biology. It is applicable, mutatis mutandis, to all areas of Vision, biological and medical, artificial and industrial. 30 Conical Diffraction Conical diffraction or refraction is an optical phenomenon predicted by Hamilton, [6] in 1832, and experimentally confirmed two months later by Lloyd, [7]. Conical diffraction describes the propagation of a beam of light in the direction of the optical axis of a biaxial crystal. 3029633 28 Indeed, in a biaxial crystal, the optical axis is positioned in the plane created by the crystallographic axes x and z; the angle with respect to the z-axis is 00, depending on the three indices of refraction according to the law,Hamilton predicted that light emerges in the form of a hollow cone of rays. Conical refraction is an important milestone in the history of science and played a role in the demonstration of the electromagnetic wave theory. A renewed interest in conical diffraction occurred in the latter years of the 20th century; it resulted in a comprehensive theory by Berry et al.[2], experimentally validated in 2009,[8]. Here, we follow Berry's theory, terminology, and definitions, including the change in nomenclature of the physical effect, from this point onward, using the more rigorous term conical diffraction. However, it is important to note that the term "conical diffraction" is also used for two other techniques unrelated to the one we are describing: oblique incidence diffraction is also called conical diffraction. The English term,"Conical diffraction mounting" refers to a diffraction grating mounting in which the grating is mounted on a curved surface. Conical diffraction has attracted considerable theoretical and experimental interest, but "no practical application seems to have been found" [9]. Historically, conical diffraction has been observed in biaxial crystals. We refer to a conical crystal to describe an inorganic or organic biaxial crystal exhibiting the conical diffraction phenomenon. Some non-limiting examples of biaxial crystals include aragonite, KTP, KTA, KBiW, LBO, KNbO3, MDT, YCOB, BIBO, DAST, POM, NPP, LAP, LiInS2, and LiInSe2. Other effects exist, creating intrinsically weaker conical diffraction or by creating weaker conical diffraction along a shorter optical path. However, these effects can be used within the devices described. These effects include polymers,liquid crystals and externally induced birefringence effects. Polymers include, but are not limited to: stretched polymer sheets and cascade polymerization,

[10] ; liquid crystals include, but are not limited to: the thermotropic biaxial nematic phase,

[11] ; external induced birefringence effects include, but are not limited to: the application of an electric field creating an electro-optical effect, on a non-centrosymmetric cubic crystal, and the photoelastic modulator. 3029633 29 The phase in the vortex created by cone diffraction is a geometric phase and is therefore intrinsically achromatic. Additional chromatic effects are optical axis dispersion and the wavelength dependence of the various parameters present in the cone diffraction equations. Chromatic dispersion of the optical axis creates an angle of the crystal's optical axis.dependent on the wavelength, relative to the optical axis of the system, it is due, in most cases, to the dispersion of refractive indices. Refractive indices depend on wavelength, according to Sellmeier's equations. The angle of the optical axis therefore varies with wavelength, creating a chromatic inclination angle of the optical axis in the plane formed by the crystallographic x and z axes. It is highly dependent on the type of crystal. In a DTM crystal, the least dispersive achromatic crystal in the visible spectrum, the direction of the optical axis varies by less than 0.1 degrees between 540 nm and 700 nm. In a KTP crystal, the most achromatic crystal in the telecommunications IR, the angle varies by 0.05 degrees between 1,350 nm and 2,100 nm, and by less than 0.02 degrees over the telecommunications window (1450 nm to 1650 nm). On the other hand,Oo can vary greatly depending on the wavelength in certain organic crystals such as DAST. 20 The compensation of chromatic dispersion of the optical axis can be performed using geometric optics. Chromatic dispersion in the direction of the optical axis can be compensated using the natural dispersion of glass or other optical materials, or by using gratings or prisms. The achromatization procedure 25 does not differ, in this case, from the standard procedure for correcting any chromatic aberration in geometric optics. This procedure can be designed and optimized using one of the commercially available optical software programs by defining suitable target functions. A different achromatization concept is based on the use of two 30 different materials, having inverse conic diffraction effects.at high and low chromatic dispersions. The dependence of the various parameters present in the conical diffraction equations on wavelength modifies the efficiency parameters of the conical diffraction effects. 35 For linear conical crystals, defined later, the fundamental transfer function is identical to unity and thus trivially independent of wavelength. 3029633 30 On the other hand, the vortex transfer function depends on the wavelength and can be represented by a chromatic factor equal to i(X). For sinusoidal conical crystals, defined later, the behavior is different from that of linear conical crystals: the fundamental wave depends on the wavelength and the vortex wave is almost independent of it. Indeed, simulations show that the shape of the vortex wave is only slightly modified by a variation of the parameter, Oo, from 0.5 to 0.75. On the other hand,The shape of the fundamental wave depends on the wavelength, and this effect must be taken into account in the design of systems using both the fundamental and vortex waves. Reference is now made to Figure 3, which is a simplified schematic illustration of a conical diffraction module configuration, 300, according to an embodiment of the present invention. The incident light, 30, is assumed to be collimated, although other conditions can be adapted using simple optical means. The setup itself comprises a first lens, 31, a conical crystal, 32, and an optional lens, 33. The first two lenses, 31 and 33, are preferably configured as a 1:1 Kepler telescope. The numerical aperture of the first lens, 31, in the image space, represented below by Uo, determines the parameters of the conical effect through the conical ray, defined below. A conical imaging plane, 20 35,is placed at the focal plane of the first lens 31; a polarizer, or a partial polarizer, 29, described above, may also be added. However, in some optical systems where the incident light is already polarized, this element is not necessary. A focusing lens, 36, determines the final size of the light spot. It may be an external microscope objective, or may be fused with the second lens 33, as implemented in another embodiment of this invention. The distribution of the light projected onto the sample is, to a first approximation, neglecting vector effects, a reduced image of the distribution of light in the image plane. The influence of vector effects will be discussed below. The scale ratio or magnification is determined by a microscope objective. Let the spatial variable, R, be in the conical imaging plane, and the wave vector, U,represented in cylindrical coordinates by R, OR and U, Ou. Let X be the wavelength of the light. The behavior of the electric field emerging from the conical crystal 32 is entirely characterized by a single parameter, the conical radius, Ro; the conical radius depends on the material and the thickness of the crystal. We introduce normalized parameters allowing the description below of the light distribution to be valid both at the conical imaging plane and at the focus of the microscope objective, within the limits of the scalar theory of diffraction. An example of the introduction of normalized parameters is described in reference [2]. The normalized radial position, p, the normalized wave vector, u, represented in cylindrical coordinates by p, OR and u, Ou, and the normalized conical radius, po, are given by: P = 2 - U₀, U = -; po = 2 - U₀. U0 (EQ. 3) RU Ro P = 2 - U0, U = - ; po = 2 - U0. U0 (EQ. 4) U0 being the numerical aperture of the system. For po < 0.5,we are referring here to a thin conical crystal; for po < 1, we are referring here to the form of a linear thin conical crystal, for po < 0.5 and > 1, to a sinusoidal thin conical crystal and for 1,

Claims

CLAIMS 1. An optical measurement method for determining the spatial distribution or location of re-emitting sources on a sample, the sample comprising at least one re-emitting source, said at least one re-emitting source re-emitting light as a function of the light projected onto the sample, according to a determined law, by a first light source comprising a first laser, and the re-emitting source being able to be depleted or activated by the action of a second light source, comprising a second laser, the method comprising: the use of the two lasers, the wavelength of one of the lasers being tuned to the excitation wavelength of said at least one re-emitting source and the wavelength of the second laser being tuned to the depletion or activation wavelength of said at least one re-emitting source; the realization, using a polarization sub-module, for each laser, of a controlled polarization state,the projection onto the sample, by means of an achromatic projection optical device, for each laser, of a compact light distribution propagating along the same optical path for all lasers; the detection of the light re-emitted by said at least one re-emitting source of the sample; the generation of at least one image from the detected light; and the algorithmic analysis of the images to obtain spatial distribution information or the localization of said at least one re-emitting source. 2 A method according to the preceding claim wherein the compact light distribution of the first excitation laser is of a different topological family than that of the second depletion or activation laser. 3 A method according to claim 2, wherein said at least two compact light distributions of different topological families are created by interference between a regular wave and a singular wave.or between two singular waves, and a spatial differentiation between said at least two distributions is created by varying at least one of the following parameters: a) at least one of the parameters of the regular wave; b) at least one parameter of at least one singular wave; and c) a phase difference between the regular wave and the singular wave or between the two singular waves. 3029633 71 4. A method according to claim 2 or 3 wherein the projection of light distributions of different topological families is carried out by conical diffraction or an assembly of uniaxial crystals.

5. A method according to any one of claims 1 to 4, comprising coupling the light from the lasers by an optical sub-module, through a single fiber or a single optical path.

6. A method according to any one of the preceding claims,comprising laser control to jointly create a sequence of excitation distributions and a sequence of depletion or activation distributions, the two sequences being 10 synchronized 7. A measuring device for determining the spatial distribution or location of re-emitting sources on a sample, the sample comprising at least one re-emitting source, said at least one re-emitting source re-emitting light as a function of the light projected onto the sample by a first laser, according to a determined law, and the re-emitting source being able to be depleted or activated by the action of a second laser, the device comprising: two lasers of wavelengths,the wavelength of the first laser being tuned to the excitation wavelength of said at least one re-emitting source and the wavelength of the second laser being tuned to the depletion or activation wavelength of said at least one re-emitting source; a polarization sub-module, to achieve for each laser, a different polarization state; - an achromatic projection module enabling the creation for each laser of a compact light distribution, propagating along the same optical path for all lasers; - a detection module capable of detecting light re-emitted by said at least one re-emitting source of the sample; - a generation module, capable of generating at least one optical image.from the detected light; and 30 - an algorithmic analysis module capable of analyzing images to obtain location information for said at least one re-emitting source.

8. A device according to the preceding claim in which the compact light distribution of the excitation laser is of a different topological family than that of the depletion or activation laser.

9. A device according to claim 7 or 8 comprising a photonic fiber. 10 An optical measurement method for determining the spatial distribution or location of re-emitting sources on a sample, the sample comprising at least one re-emitting source, said at least one re-emitting source re-emitting light as a function of the light projected onto the sample, according to a determined law, by a first light source comprising a first laser.and said at least one re-emitting source that can be depleted or activated by the action of a second light source, comprising a second laser, the method comprising: the use of two lasers, the wavelength of the first laser being tuned to the excitation wavelength of said at least one re-emitting source and the wavelength of the second laser being tuned to the depletion or activation wavelength of said at least one re-emitting source; the projection onto the sample, by means of an achromatic projection optical apparatus for the first and second lasers, of the light distributions; the detection of the light re-emitted by said at least one re-emitting source from the sample; and the generation of at least one image from the detected light. A method according to claim 10, wherein the projection comprises, for the first laser, a compact light distribution.and for the second laser, two compact distributions, one of them having a zero intensity, propagating axially in a spiral motion.

12. An optical measurement method according to claim 11, wherein the two distributions creating a depletion are produced by two independent lasers at the same or a close wavelength.

13. An optical measurement method according to claim 11, wherein the projection onto the sample is carried out by means of an achromatic optical projection device comprising an optical element having the functionality of segmenting an optical beam into a plurality of independent beams, the projection comprising, for both lasers, a matrix of compact and collocalized light distributions.

14. An optical measurement method according to any one of claims 11 3029633 73 35 to 13, further including algorithmic image analysis for obtaining spatial distribution or location information for said at least one re-emitting source.

15. An optical measurement device for determining the spatial distribution or location of re-emitting sources on a sample, the sample comprising at least one re-emitting source, said at least one re-emitting source re-emitting light as a function of the light projected onto the sample, according to a determined law, by a first light source comprising a first laser, and said at least one re-emitting source capable of being depleted or activated by the action of a second light source, comprising a second laser, the device comprising: a first laser and a second laser.the wavelength of the first laser being tuned to the excitation wavelength of said at least one re-emitting source and the wavelength of the second laser being tuned to the depletion or activation wavelength of said at least one re-emitting source; an achromatic projection module for creating light distributions for the first and second lasers; a detection module for detecting the light re-emitted by said at least one re-emitting source of the sample; a generation module for generating at least one image from the detected light. A device according to claim 15, wherein the projection module is capable of creating a compact light distribution for the first laser.and for the second laser, two compact distributions, one of them having a zero intensity, propagating axially in a spiral motion. 25 17. A device according to claim 16, wherein the two distributions creating a depletion originate from two independent lasers at the same or a close wavelength. 30,