FR3029633A1 - METHOD AND DEVICE FOR OPTICAL MEASUREMENT - Google Patents

Abstract

The invention proposes an optical measurement method and an optical measurement 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 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 length d depletion or activation wave 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 optical device for achromatic projection, 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; generating at least one optical image from the detected light; and the algorithmic analysis of the optical images to obtain location information from said at least one re-emitting source. Furthermore, the invention describes the realization of the cited achromatic projection optical apparatus, using modules based on conical diffraction or on other optical effects.

Description

FIELD OF THE INVENTION The present invention relates to a method and an optical measuring device. It finds applications in all areas of imaging, in particular but not limited to the field of microscopy, including but not limited to the fields of Biology, Medicine, Pharmacy, Semiconductors, Study of materials, metrology, control, measurement and observation and all the processes of acquisition of information from optical observations, in the macroscopic or microscopic domain.

An optical microscope is an optical instrument generally used to view, analyze or measure objects too small to be viewed by the naked eye. We will use the term biological to describe any entity of the Science of Life, 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 described technique. Microscopy is used in the field of biology, for example, to observe, study and measure biological entities (objects) and their dynamics. We will use, by extension, the term Artificial Vision to describe all applications of measurement, metrology or observation of objects or elements produced or constructed or made by a human being or a machine, for example, to observe, to study and to measure semiconductors or to characterize the materials. The usual definitions are used for optical diffraction limit, Rayleigh criterion, Airy disc and its radius and diameter. We use in the context of the invention the terms supersolution, supersoluble, superresolution imaging and supersensitive microscopy to describe the acquisition of optical data, imaging, microscopy or artificial vision, at a higher resolution at the optical diffraction limit. The usual definitions are used for fluorescence and for fluorophores. Reference is now made to the Figure. 1, which represents an illustration of the Microscopy paradigm, 100. Optical Microscopy is the illumination, by a light source, not shown, using a Microscope, 10, of a biological or nonbiological sample. 11, and the measurement as a function of time, by means of a visual observation or a detection module, 12, of the light emitted, re-emitted, scattered or reflected or 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 positions. Examples of such objects are, inter alia, a cell, a virus, a protein and a DNA fragment. In artificial industrial vision the sample may be, for example, a semiconductor element Microscopy is segmented into different modalities with different characteristics and purposes. Numerous descriptions of the different modalities, their characteristics and their advantages exist extensively in the literature and can be found, for example, on the webs of the Zeiss, Leica, Nikon or Olympus companies. The applications of Microscopy can be structured in many different ways: one of them is to distinguish the microscopic modes devolved to visualize minute point sources from those devolved to measure continuous objects. The case of tiny point sources is a priori much simpler. The object consists of a small number of luminous points; these can be described by a small number of parameters - the descriptors defined later - greatly simplifying the physical problem and the algorithmic complexity. The case of a continuous object, described by a spatial distribution - or spatio-temporal, if one takes into account the continuous dynamics, is different and is also described in this patent application. Fluorescence microscopy is one of the modalities of microscopy; in many applications it has replaced 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 the Figure. 1, describing, this time, a fluorescence microscope used either in Biology or in artificial vision to characterize, for example, materials; in fluorescence microscopy minute point sources 15-18 are fixed, for example fluorophores based on the physical phenomenon of single-photon fluorescence, at specific and predetermined positions of the objects 13 and 14; we observe the light emitted by the point sources instead of observing the light emitted by the objects, 13 and 14, themselves. The sample is illuminated by light of wavelength, or specific wavelengths, which is absorbed by the point sources, thereby inducing light emission at different wavelengths and higher. When collecting the emitted light, in fluorescence, the illumination light is separated from the emitted fluorescence, which is lower, by the use of a spectral emission filter. Fluorescence microscopy studies light emitted by small point sources, fluorophores. However, when the density of the fluorophores is high, the fluorophores are no longer analyzed individually but treated as a continuous object.

It is important to note, at 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 in the visualization of biological objects. Activity and biological information including details above the 200 nm resolution limit are systematically visualized and measured using fluorescence microscopy. This resolution limit is derived from the Rayleigh criterion, which in the best case reaches 200nm in specially designed systems. For a long time, until the emergence of the superresolution techniques described below, it was recognized that optical techniques, including fluorescence microscopy, are unable to visualize smaller details than the Rayleigh, the fluorescence and the fluorescence criteria. order of 200 nm. However, other basic biological activities also occur at scales below 200 nm in biological samples. At this level of spatial resolution, important phenomena can be observed: intracellular biological processes, cellular information transfer, protein folding and unfolding, and modifications of DNA and RNA. Thus, for example, the measurement of this intracellular information will open new avenues for understanding biological activity, and lead to progress in the knowledge and monitoring of medical research and diagnosis.

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 large field imaging microscope. Reference is now made to FIG. 2, which is a simplified representation of a confocal fluorescence microscope of the prior art 200.

A confocal fluorescence microscope, FIG. 2, is an optical instrument. Its main hardware components are shown in the figure. 2. They comprise: A light source, 20, an omnomechanical frame not shown, a filter cube, 21, a microscope objective 22, and, a detector assembly, 23, a processing unit, not shown. The light source, 20, which may be an arc lamp or a laser, creates light energy necessary for fluorescence.

The optomechanical frame, not shown, is the support for all optical elements and includes auxiliary optics and alignment capabilities. It also comprises optical elements, not shown, capable of shaping the beam, to allow its focusing of a point of minimum size, by means of the objective of the microscope. It may also comprise, in a confocal scanning fluorescence microscope, a scan mechanism, spatial or angular, not shown to modify the position of the point source with respect to the object to be measured. The scanning mechanism may alternatively: mechanically translate the object, for example by using a translation stage, optically scanning the beam on the object, for example using a set of galvanometric mirrors or acousto-optical translators, or using a any combination of these means of translation, mechanical or optical. In a confocal scanning fluorescence microscope, the information is collected point-to-point, using the scanning mechanism.

It may also comprise, in a confocal rotating disk fluorescence microscope, a rotating disc, having a plurality of microscopic holes, allowing the simultaneous projection of a plurality of points. In a confocal microscope of rotating disk fluorescence, a set of points corresponding to the microscopic holes is acquired at each instant and the rotation of the disk makes it possible to scan the entire surface of the sample for a given longitudinal position. The filter cube, 21, channels the different optical signals and avoids contamination of the fluorescence signal by the excitation light. The filter cube is decomposed into: excitation filter, 210, dichroic mirror, 211, and emission filter, 212. The filters and the dichroic mirror are chosen according to the excitation wavelength and the dichroic mirror. spectral emission characteristics of the fluorophore. The microscope objective, 22, focuses the light created by the source in the focal plane of the lens, 24, into a light distribution of reduced size, the optimum light distribution consisting of an Airy disc. The microscope objective, 22, also makes it possible to collect in return the fluorescent light emitted by the fluorophores.

For a confocal scanning fluorescence microscope, the system can be decanned, i.e. the back light can pass through the scanning mechanism to compensate for 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.

A confocal hole, 27, is theoretically placed in the image plane of the detector, 26. In most practical systems, the confocal hole, 27, is placed in an intermediate imaging plane that is not shown and reimaged on the image plane. 26. The detector assembly, 23, detects the overall fluorescent intensity in the illuminated volume, and transforms it into a digital signal. For a confocal scanning microscope, the detector assembly consists of a single element detector, such as a PMT or SPAD. For a confocal rotating disk microscope, the detector assembly consists of a matrix of detection elements, such as a CCD, an EMCCD, a CMOS or a SPAD matrix.

The assembly of components mounted from the light source to the dichroic filter is the lighting path, 201. The set of components mounted from the dichroic filter to the detector assembly is detection, 202. The basic optical process of a confocal microscope can be segmented into six steps: - Projection of light on the analysis volume - fluorescent light emission by fluorophores - Focal fluorophore imaging - Limitation in the focal plane of the light analyzed by the confocal hole - Integration of the light analyzed by a photoelectric detector 20 - Visualization of the intensity measured as a pixel value in an image Fluorescence microscopes are available from several manufacturers, for example, Nikon, Zeiss, Leica or Olympus. Fluorescence microscopes may be either standard fluorescence-adapted microscopes or specific fluorescence-optimized microscopes. 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 features with minimal modifications. Other fluorescence microscopes are dedicated instruments, customized to a specific task, such as medical or pharmaceutical diagnosis. Superresolution New optical methods, superresolution methods, are able to discriminate point sources, below the Rayleigh criterion. These methods are developed by several companies, laboratories and researchers, and some of the instruments using these methods, the superresolution microscopes, are commercially available. Several comparative analyzes of superresolution methods have recently been published in the literature, such as the articles by Schermelleh et al. [1]. An up-to-date bibliography on superresolution can be found on the website of Zeiss, and on the website of Nikon. The different existing microscopy methods and the existing microscopes, not including superresolution, allow microscopic observation in the limit of optical diffraction. This reduces their scope of use to a limited set of applications. The new superresolution techniques allow them to obtain information beyond the resolution limit. The main problem of all the existing techniques of superresolution is that the envelope of performances, expressed in terms of lateral resolution, longitudinal resolution, speed, necessary luminous intensity, photo-toxicity in the biological object, ability to measure different objects, is very limited. In addition, most existing superresolution methods and instruments can provide either good lateral resolution or good longitudinal resolution, but rarely both. In addition, all of these instruments are complex and require a high level of technical skill of the operator. In addition, these instruments can generally observe only a small portion of biological specimens, due to severe operational limitations, such as for some of them a shallow depth of field or very high intensities harmful to the cells.

Another problem with existing super resolution methods and instruments is that most of them are able to recover, in the illuminated volume, the attributes of a single fluorophore, but fail to simultaneously recognize the presence of several fluorophores and measure their attributes. A further problem with existing supersolution methods and instruments is that these existing methods and instruments are presented to users and perceived by them as a general tool, capable of replacing standard or confocal microscopes. However, existing superresolution methods and instruments lack the simplicity, robustness, ease of use and competitive prices of standard microscopes which hampers their use as general research tools or as diagnostic tools.

Another problem with existing superresolution methods and instruments is that most of these methods and instruments are constructed as stand-alone instruments designed to replace standard microscopes. Such an approach requires the replacement of existing instruments as well as the renewal of all peripheral systems and all the knowledge and know-how related to microscopy platforms and developed for many years. Another problem with most of the existing methods and instruments of fluorescence microscopy and superresolution is that these methods and instruments are designed on an image acquisition paradigm, for which the basic entity of the information is a - or multiple images, or one - or more - ROI region - Bi or three-dimensional region of interest. The algorithmic, systemic and superresolution methods described later in the context of the invention will allow, by their inherent flexibility, the development of new acquisition strategies. These dynamic and selective acquisition procedures will be defined by optimized management of the interactive and delayed acquisition and processing sequence. They will allow a more advanced optimization of the useful information, defined by criteria based on the shape, geometry and dynamics of one or more fluorescent objects, separately or with respect to each other. There is therefore still an urgent need to provide superresolution methods and instruments and algorithmic methods capable of accurately measuring the attributes of a fluorophore. It is also necessary to provide methods and instruments for detecting and quantifying the presence of several fluorophores placed in the same illuminated volume. Another problem with most existing methods and instruments of fluorescence microscopy and superresolution is that these methods and instruments are designed to study samples on microscope slides. However, the confocal microscope is now used in many medical fields as an in-vivo diagnostic instrument for internal and external examinations to the human body through optical fibers used to illuminate and visualize tissue-emitted fluorescence. to diagnose. Superresolution does not currently allow such in-vivo diagnoses. The algorithmic, systemic and superresolution methods described later in the context of the invention will allow the development of new in vivo diagnostic methods which will reduce the need for biopsies and shorten the patient's waiting time.

SUMMARY OF THE INVENTION: A first aspect of this invention relates to an optical measuring method for determining the spatial distribution or location of re-emitting sources on a sample, the sample having at least one re-emitting source of the at least one source. re-emitter re-emitting light according to the light projected on 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: using both lasers, the wavelength of one of the lasers being tuned to the excitation wavelength of said at least one re-emitting source and the length of wave of the second laser being tuned to the wavelength of depletion or activation of said at least one re-emitting source e of a polarization sub-module, for each laser, a state of controlled polarization, the projection on the sample, by means of an achromatic projection optical apparatus, for each laser a compact light distribution, propagating according to the same optical path for all the lasers, the detection of the light re-emitted by said at least one source of the sample re-emitter; Generating at least one image from the detected light. In one embodiment, the method further comprises algorithmically analyzing the images to obtain spatial distribution information or location of said at least one re-emitting source. A second aspect of the invention relates to the achromatization of the optical measuring method according to the first aspect of the invention so as to allow the use of the optical measuring method at different wavelengths of excitation and depletion, 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 STED or RELSOFT superresolution system 30 by means of an optical method of achromatic common optical path measurement and capable of different beamshaping on different beams according to their polarization. Another aspect of the invention relates to the collocation of all lasers using an optical bench preferably using an optical fiber system to collocate the beams before shaping them by the optical method described in the previous aspects.

Another aspect of the invention relates to the realization of the method according to the first aspect by means of conical diffraction. In one embodiment of the method described above, the compact light distribution of the first excitation laser is of topological family different from that of the second depletion or activation laser. In one embodiment of the method described above, said at least one 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 a conical diffraction or an assembly of uniaxial crystals.

In one embodiment of the method described above, the projection of light distributions of different topologies is carried out by a conical diffraction in a thin crystal. An embodiment of the method described above, comprising the coupling of light from lasers by a sub-phase. Optical module, through a single fiber or a single optical path In one embodiment of the method described above, the optical fiber comprises a photonic fiber. In one embodiment of the method described above, the optical fiber 30 comprises a fiber with a low number of modes, "few modes fiber" in English. An embodiment of the method described above, comprising controlling the lasers to jointly create a sequence of excitation distributions and a sequence of depletion or activation distributions, the two sequences being synchronized. One embodiment of the method described above, comprising controlling the lasers to jointly create a sequence of excitation distributions and a depletion or activation distribution sequence, the two sequences being synchronized, and a singular excitation pattern being concurrent with a singular distribution of Depletion An embodiment of the method described above, comprising controlling the lasers to jointly create an excitation distribution sequence and a depletion or activation distribution sequence, one of the excitation distributions being a super-oscillation. An embodiment of the method described above, comprising the control of the lasers to create simultaneously or following 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. 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 having 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 retransmitter source being able to be depleted or activated by the action of a second laser the device comprising: two lasers of different wavelengths, the length wavelength of the first laser being tuned to the excitation wavelength of said at least one retransmitter source and the wavelength of the second laser being tuned to the depletion or activation wavelength of said at least one retransmitting source, a polarization sub-module, for each laser, of a different polarization state, an achromatic projection module making it possible to create for each laser a compact light distribution propagating along the same optical path for all the lasers, a detection module able to detect light re-emitted by said at least one source of the sample; a generation module, able to generate at least one optical image, from the detected light; and an algorithmic analysis module capable of analyzing images to obtain location information of said at least one re-emitting source. In one embodiment of the method described above, the compact light distribution of the excitation laser is of topological family different from 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 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 comprises at least one conical crystal for projecting light distributions of different families of different topologies by conical diffraction or uniaxial crystal assembly. In one embodiment of the method described above, the optical fiber comprises a photonic fiber. In one embodiment of the method described above, the coupling of light from the 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 fiber Optical comprises a fiber with a limited number of modes, "few modes fiber" in English. 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 disclosed method. previously, the lasers are configured to co-create a sequence of excitation distributions and a sequence of depletion or activation distributions, the two sequences being synchronized, and a singular excitation pattern being simultaneous to a singular depletion distribution. In one embodiment of the method described above, the lasers are configured to co-create a sequence of excitation distributions and a sequence of depletion or activation distributions, one of the excitation distributions being a super-oscillation. In an embodiment of the method described above, the lasers are configured to jointly create simultaneously or in a sequence of excitation distributions and a sequence of depletion or activation distributions, the set of depletion distributions or activation being equivalent or similar to a vortex. The description of the embodiments of the invention based on confocal fluorescence microscopy can be extended mutatis mutandis, to other microscopy modalities, confocal or not, and to the artificial vision, that they observe biological objects, artificial vision or other, and that the object consists of point sources, structured objects or continuous objects. Another aspect of the invention relates to an optical measuring method for determining the spatial distribution or location of re-emitting sources on a sample, the sample having at least one re-emitting source, said at least one re-emitting source re-emitting light based on light projected onto the sample, according to a given 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 the at least one re-emitting source and the wavelength of the second laser being tuned (s) on the wavelength of depletion or activation of said at least one re-emitting source the projection on the sample, by means of n of an achromatic projection optical apparatus for the first laser and the second laser of the light distributions, the detection of light re-emitted by said at least one re-emitting source of the sample; and generating, 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 an intensity zero propagating axially in a direction of spiral movement.

In one embodiment of the methods described above, the two depletion-generating distributions come from two independent lasers at the same wavelength or at a near wavelength.

In one embodiment of the method described above, the projection on the sample is carried out by means of an achromatic projection optical apparatus, comprising 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 collocalised light distributions. In one embodiment of the method described above, the optical element comprises a matrix of microlenses. In one embodiment of the method described above, the achromatic projection optical apparatus comprises a duplicating element making it possible to duplicate the light distributions laterally for the two lasers of a matrix of compact and collocated light distributions. In one embodiment of the method described above, the duplicating element comprises a beam splitter transmission network 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 two or more laterally or axially offset beams from an incident beam. two lasers of a matrix of compact and collocated light distributions. In another embodiment of the devices described above, an additional beam derived from the depletion laser or a device comprising a second depletion laser, contains one of the light distributions with high axial dependence such as the Stokes or another distribution described in this example. invention, to reduce the axial size of the emitter volume.

In another embodiment of the devices described above, a dynamic polarization element is used before or after the biaxial crystal to correct the dynamic movement of the pupil, which in some cases may be created during the optical scanning of the microscope. confocal. This pupil movement effect being in some implementations STED technologists one of the limits of performance, without the need for an additional scanning system.

In another embodiment of the devices described above, two or more light distributions located at different wavelengths are projected using an original or modified LatSRCS module. The first light distribution makes it possible to make the scene parsimonious, that is to say isolate emitters by means of a physical effect, so as to create regions in which one can hypothesize parsimony. that is, the presence of an isolated transmitter or a small number of transmitters. The physical effects that will achieve this parsimony will be the same or will be derived effects, effects used to create parsimony for single transmitter location microscopy techniques.

In another embodiment, the microscope platform that has been described is coupled to an electron microscopy system (CLEM-Correlative Light Electron Microscopy), or any other similar system such as Transmission Electron Microscopy (TEM), or EBM (Electron Beam Microscopy), or SEM (Scanning Electron Microscopy). In another embodiment, one uses distributions with a strong axial dependence such as stokes, and so-called "half-moon shifts" to characterize the axial dependence of a point in the case of a system of Localization microscopy. In another embodiment of the invention, axial-dependent distributions such as stokes, and so-called "half-moon shifted" distributions are used to characterize the axial dependence of a point in the case of a location microscopy system in which one has been used. LatSRC module 'original or modified to make the scene parsimonious locally and measure the position of parsimonious re-emitter sources using one of the PSIT type techniques. In another embodiment of the invention, a dark tracking technique will be carried out in which the tracking of a point is achieved by the projection of a vortex or other distribution containing one or more intensity zeros. optical and cotzu: pe.nsation 3029633 15 mique .1 transmitter detected when the transmitter is found outside the zero optical intensity aforesaid. In another embodiment, a cascade of Wollaston prisms of different beam splittings is used to separate an incident beam into a large number of emerging beams. The same effect can be obtained by modifying the Wollaston prism, to create the composite Wollaston prism, by adding uniaxial crystal pieces whose index and orientation of the birefringence are chosen. It is thus possible to construct a prism of a single uniaxial crystal block which can separate an incident beam 10 into emergent beams (e.g. eight or sixteen), which are contained in a plane, and separated by equal angles. Once focused on the sample, two aligned and separated points are obtained In one embodiment of the methods described above, the method further comprises the algorithmic analysis of the images in order to obtain spatial distribution information or location of said data. Another aspect of the invention relates to an optical 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 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 device comprising: r 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 over the length of depletion or activation wave of said at least one retransmitting source an achromatic projection module for creating for the first laser and the second laser light distributions, a detection module for detecting the light re-emitted by said at least one re-emitting source sample; a generation module for generating 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 of two compact distributions one of them having a zero of intensity propagating axially following a spiral movement.

In one embodiment of the device described above, the two depletion-producing distributions are derived from two independent lasers at the same wavelength or near-wavelength.

In one embodiment of the devices described above, the achromatic projection module comprises an optical element having the function of segmenting an optical beam into a plurality of independent beams, the projection comprising for the two lasers a matrix of compact light distributions and collocalisées.

In one embodiment of the devices described above, the optical element comprises a matrix of microlenses. In one embodiment of the devices described above, the achromatic projection module comprises a duplicating element making it possible to duplicate the light distributions laterally for the two lasers of a matrix of compact and collocated light distributions. In one embodiment of the devices described above, the duplicating element has a beam splitter or uniaxial crystal transmission network. In one embodiment of the devices described above, the achromatic projection module comprises at least one additional optical element, having the function of creating, from an incident beam, two or more beams offset laterally or axially, for the two lasers. In an embodiment of the devices described above, the device 35 furthermore comprises an algorithmic analysis module able to analyze the images in order to obtain spatial distribution information or location of the said device. at least one re-reader source. In one embodiment of the devices described above, the device comprises an optical measurement method for determining the spatial distribution or the location of retransmitter 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, the method comprising: projecting on the sample, a helical topology distribution light distribution having a position of zero intensity, said intensity position 10 zero having a helicoidal 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; generating at least one image from the detected light; and direct detection or algorithmic analysis of the images to obtain spatial distribution information or location of said at least one re-transmitting source. In one embodiment of the device described in the previous embodiment, the device is based on conical diffraction for the projection step. In one embodiment of the device described in the third-party embodiment, the device includes an SLM. 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, derived from the same spatial region of an object, two-dimensional or three-dimensional, an algorithm in which the formulation of the reconstruction the reconstruction of the object and its spatial and / or temporal and / or spectral properties is considered a Bayesian inverse problem and leads the definition of a posterior distribution. A posterior law combining, thanks to Bayes' law, the probabilistic formulation of a noise model, as well as possible prior art on a light distribution created in the sample by projection; estimating the light distribution in the sample by the use of spot emitting clouds which makes it possible to favor parsimonious solutions; the estimation of the posterior average by means of a Monte Carlo Markov Chain (MCMC) algorithm, the representation of the results either in the form of an image or in the form of numerical or graphical data. BRIEF DESCRIPTION OF THE DRAWINGS The invention will now be described in connection with certain embodiments with reference to the following illustrative figures so that it can be better understood. With specific reference to the figures, it is pointed out that the illustrative indications are presented by way of example, and for purposes of illustration of the discussion of the embodiments of the invention and are presented only for the purpose of provide what can be considered the most useful and easy-to-understand description of the principles and conceptual aspects of the invention. In this regard, no attempt is made to show details of the structure of the invention in more detail than is necessary for a basic understanding of the invention, the description taken with the drawings revealing to the man of the art how the different forms of the invention can be realized in practice. In the drawings: Fig. 1 is a simplified perspective representation of a confocal fluorescence microscope of the prior art, also used as a support of the invention; Fig. 2 is a simplified pictorial representation of a fluorescence microscopy superresolution system, according to an embodiment of the present invention; Fig. 3 is a simplified schematic illustration of a setup of a conical diffraction module according to an embodiment of the present invention; Fig. 4 is a simplified pictorial representation of the two measurement paradigms according to embodiments of the invention and 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 superresolution side module, in accordance with an embodiment of the present invention; FIG. 6b is a simplified schematic illustration of another embodiment of a superresolution side module, according to an embodiment of the present invention; Fig. Fig. 7 shows light distribution tables of a conical diffraction module as a function of the polarization of the input and output polarizers for several values of the tapered diffraction parameter, po. These light distributions were calculated by simulation of the equations developed by Berry, [2]; Fig.8 is a simplified schematic illustration of one of the embodiments of dark tracking; Fig. 9 is a simplified schematic illustration of a super-resolution data algorithm method of fluorophores according to an embodiment of the present invention. Fig. 10 is a simplified schematic illustration of calculating the descriptors; Fig. 11 is a simplified schematic illustration of the control module of the SRCDP platform.

In all figures, similar reference numerals identify like parts. Definitions and Technical Supplements The usual definitions are used in the description for: phase and polarization, polarimetry, Jones vectors and matrices, Stokes parameters, and Jones and Stokes parameter measurement techniques. The usual definitions are used in the description for the TEM00 mode of a fiber and the English terms, "Photonic Crystal Fiber" - PCF -, "few modes fiber" - FMF, Vortex Fiber and "dual core Photonic Crystal Fiber". We will refer to a device for coupling several lasers at 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. Super-oscillation is a phenomenon in which a signal which is generally bandlimited may contain local segments that oscillate more rapidly than its faster 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 35 of the singular wave.

Two light distributions are collocated if their centers coincide or are separated by a small spatial value with respect to the size 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 whose definition is enlarged, to include all the optical means that transmit, refract or reflect light, optical auxiliary - optical sub-module aimed at interfacing and adjusting either the geometrical parameters or the phase and / or polarization parameters between two other submodules or optical modules -, polarizer, analyzer, delay plate, beamsplitter beamsplitter in English, polarizing and non-polarizing , beam combiner beam combine in English, polarizing and non-polarizing. In this patent application, the usual definitions are used for azimuth and radial polarizers. We will extend, implicitly or explicitly, some developments described later for azimuthal and radial polarizers, to the set of spatially variable polarizing elements. In this patent application, the usual definitions [3] are used for the various superresolution techniques; these techniques can be grouped into 20 families: - REVERSIBLE Saturable OpticaL Fluorescence Transitions (RESOLFT), regrouping the techniques: Stimulated Emission Depletion Microscopy (STED), Ground state depletion (GSD), Saturated Structured Illumination Microscopy (SSIM) and SPEM (Saturated Pattern Excitation Microscopy), Microscopy Localization, (microscopy of localization, in English) gathering the techniques 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 blinking, ground state depletion (GSD), and similar techniques (whatever the acronym used) - Structured Image Microscopy (SIM) - FRAP (fluorescence recovery after photobleaching), - TIRF (Total Internal Reflection Fluorescence Microscopy).

In this patent application, the usual definitions are used for various techniques of microscopy, standard resolution or superresolution, fluorescent or non-fluorescent, such as the terms "Computational Microscopy", "Correlative Microscopy", "Cross-platform". -microscopy ", FCS - Fluorescence Correlation 5 Spectroscopy", FCCS - "Fluorescence Cross-Correlation Spectroscopy", or PCH - Photon Counting Histogram, RICS "Raster Imaging Correlation Spectroscopy" or FRAP - "Fluorescence Recovery after Photobleaching)". In this patent application, the usual definitions are used for the Hough Transform.

We refer to a partial polarizer to describe an element or module whose absorption is different for the two linear polarizations - linear dichroism - or for both circular polarizations - circular dichroism. We refer to dynamic polarization or phase elements to describe the optical means whose polarization or phase properties vary over time in a controlled, discrete or continuous manner. These dynamic polarization or phase elements include, but are not limited to: rotating wave blades on their axis, light valves based on liquid crystal technologies, electro-optical devices, also known as Pockels cells, cells of Kerr, resonant electro-optical devices, magneto-optical devices, also known as Faraday cells, acousto- or elasto-optical 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 of the polarization dispersive sub-modules is the multimode or thick wave plate. We will refer to the "centroid algorithm" to describe the usual centroid measurement procedure and possibly width (FWHM - acronym in English for "Full width Half Maximum") of a light distribution. This algorithm has its source in Astronomy and Astrometry, and allowed the measurement of the position of the stars with a very high precision. This algorithm is used today in all optical instrumentation, including superresolution 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.

We will use the terms: o optical image, for the spatial distribution of luminous intensity, 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 moment, in a detection plane, or digital image, to describe a matrix of numbers created by the digitization of the electronic image. To simplify the reading and understanding of the text we will also use the image term 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 the distinction between the three types of images is not necessary, we will use the generic generic term image. For images, we use the terminology used for matrix detectors, such as CCDs, EMCCDs, and CMOS. For SPADs and SPAD matrices, the result of a measurement is a time-ordered list of photon impacts detailing, for each photon, the impact time and the position of the impact. To simplify the presentation of this document, we will include this case in our definition of images. The images detailed in this document, in many cases, can be described as microimages, images of size substantially equal to a small number of diameters of the Airy disk, typically less than 5 diameters, and / or with a low number of pixels. typically 4 * 4 to 32 * 32.

In a digital image Aj, the indices m and n represent the indices of the pixels; the origin of the pixels will be chosen as the projection of the center of the analysis volume defined in a subsequent paragraph. Polarimetry and Stokes Vectors Polarimetry refers to the measurement of the polarization state of the incident light.

The state of polarization of the incident light can be described by the 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 interact with each other or not, be projected sequentially or simultaneously. In the majority of these systems and devices, the two optical paths are physically separated from each other. This physical separation creates, at the level of system engineering, a set of constraints, which, although resolvable, significantly increase the complexity of the system and its cost. We refer to common path systems in English, to refer to a set of devices in which the two differentiated beams propagate along the same physical path, with 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 It is usual in Optique to decompose the components of the field, ie its amplitude, its phase and its polarization, in orthogonal, Cartesian or polar modes. Numerous decompositions in polar orthogonal modes, such as the Gaussian, Hermite-Gaussian, and Laguerre-Gaussian modes are known to those skilled in the art.

10 We will use mainly in this document, the decomposition of the amplitude of the electric field in Hypergeometric-Gaussian HyGG modes of form: A (p, 0) oc pPHHexp (--p2 + iP, 0) (EQ. decomposition, p is the radial mode and f is the azimuth order. Singular Waves A singular wave comprises a zero intensity at its center and an azimuthal phase variation of a multiple of 2x. This research theme in Optics, initiated by the major article of J. F 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 term beam shaping to describe the transformation of a given waveform and topology into a wave of another form or topology, and in particular the transformation of a regular wave into singular and vice versa. Topology and compact light distributions A light distribution, punctual, will be deemed compact if it fulfills one of the conditions of compactness defined below, by two alternative and non-exclusive conditions: that is, more than 75% of the energy is contained in a circle of radius less than 1.75 times the radius of Airy, ie a luminous range, containing more than 65% of the energy is delimited by a line of zero intensity included in a circle of radius less than twice the radius of 'Airy, We distinguish different families of point light distributions, of different topologies: 3029633 24 Regular distributions, in their usual definition in Optics, singular distributions, also called optical vortices, topological load (azimuth order) t, in which the phase varies from 0 to 27r (, around the direction of propagation, f being an integer.

The amplitude distributions with t-order azimuthal variation, also called Laguerre-Gauss distribution, The polarization and optionally L-order azimuthal phase variation distributions also called radially Laguerre-Gausspolarized modes. Two compact light distributions, will be deemed of different topological families if they fulfill at least one, and any, of the following conditions: One is regular and the other is singular, One is punctual and the other The azimuth orders f of the amplitude of the two different light distributions are azimuthal, the azimuth orders f of the polarization or phase of the two light distributions different. Alternatively, two light distributions projected on a given volume will be deemed to have different topologies if, in a substantial part of the jointly illuminated surface, the gradients are of reversed direction.

20 Luminous Nanoemitters A nano-light transmitter is a small secondary transmitter, attached to an object; it is substantially smaller in size than a fraction of a wavelength, typically but not limited to less than one fifth of the wavelength; a light nano-transmitter absorbs the 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 light absorbed. The main 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 the light nanoemitters is determined by the two following conditions: creation of a punctual secondary light emitter, and predetermined positioning of this emitter relative to an artificial, biological or organic entity.

The physical mechanisms that can create a nano-transmitter are numerous; they include but are not limited to absorption, scattering or reflection, fluorescence, "emission-depletion", [5] for example using RESOLFT techniques, photo activation phenomena and photo depletion, fluorescence with two or more photons, the elastic diffusion or not, 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, including diffusing, absorbing and reflecting particles, attached to a biological or organic entity; the action of a scattering particle, reflective or absorbing on the electromagnetic field can indeed be described as the creation with a reverse phase, following the Babinet principle, for an absorbing particle of an auxiliary secondary field, emerging from the particle superimposed on the incident electromagnetic field.

We will refer in this patent application to the descriptors of a nanoemitter to denote the set of information describing a nanoemitter as a point source at a given time. Since the nanoemitter is considered a point source, all of the information representing it contains a limited number of parameters, namely: its position in space, its intensity, the spectral characteristics, intensity, coherence, phase and polarization of the light emitted by the fluorophore, depending on the incident light. We will refer in this patent application to the descriptors of a structured object. For example, for a uniform line, all the information representing it contains a limited number of parameters, namely: its orientation in space, its intensity, the spectral characteristics, intensity, coherence, phase and polarizing the light emitted by the object, depending on the incident light. For a continuous distribution, the object is represented, as usual in image processing, by a matrix of intensities. However, in the majority of cases, and in the description of the invention, we refer, under the description of descriptors, to a subset of the descriptors of a nanoemitter including its geometric position, intensity, and type. 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 scope all the descriptors of the light nanoemitters. To simplify the understanding of the context of the invention, the remainder of the description only refers to the simplest case, that in which the nanoemitter is a 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 light nanoemitters previously mentioned or known to those skilled in the art, regardless of the physical phenomenon below. underlying. It is striking that the nanoemitter samples the field or the incident intensity at a precise three-dimensional position, without any influence of the whole spatial distribution of the incident intensity. We will reference this remarkable property in this patent application as the sampling capacity of the light nanoemitter.

However, the embodiment of the invention described also makes it possible to measure structured objects and continuous distributions that do not have the sampling capacity of the light nanoemitter. Reference is again made to the Figure. 1; which represents a set 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 hand. Alternatively, the light emitted may consist of a continuous distribution, not shown in Figure 1, or any combination of nanoemitters, structured objects or continuous distribution. The set of nanoemitters, structured objects or continuous distribution is referred to as a set 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 reference the luminous biological object as the biological object itself, when no ambiguity can appear. The luminous biological object contains many relevant information related to the biological object, mainly spatiotemporal information, the position of the object and its orientation as a function of time, and the morphological information, for example in the case of the split of a cell in two. The measurement system according to at least one embodiment of the invention will make it possible to calculate the measured map, and perform an 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, system limitations, or measurement uncertainty. This information of the measured map can be elaborated later in different levels of abstraction. This first level of abstraction, which describes the direct results of the measurement, does not contain, a priori, any biological information but the results of a physical measurement described by nanoemitters, structured objects or continuous distributions, which could moreover, to represent any marked entity.

The second level, the level of geometric abstraction structures the nanoemitters, of structured objects or of continuous distributions in the form of 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, the information is still a physical and geometric information describing a set of objects. Geometric information uses the measured map and auxiliary information, potentially external to the system, on the relationship between light points and objects. The level of biological abstraction allows some apprehension of biological reality through a constitutive relationship between the measured objects and corresponding biological entities. It contains a set of information about the biological object, mainly the position and its dynamics, shape and morphology. Biological information uses the measured map and geometric information, and auxiliary information, potentially external to the system, on the relationship of light points and objects to biological entities. A number of conclusions about the biological functionality of the sample can be obtained at this level. The level of functional abstraction allows an apprehension of the biological reality. It consists of functional information, decorrelated from geometric information, and answering questions in terms and biological jargon, such as: "Did the virus penetrate the cell? ". An additional level of information can be defined including the process of control and instrumentation; in fact, a more advanced control and instrumentation process can be defined, making it possible to trace back to a more structured biological information, through an automation of the data acquisition process. An example of such processes is described by Steven Finkbeiner under the name of "Robotic Microscopy Systems". This description of the levels of abstraction, defined in this application, has been written, for simplicity, for Biology. It is applicable, mutatis mutandis, to all areas of vision, biological and medical, artificial and industrial.

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 light beam in the direction of the optical axis of a biaxial crystal.

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 ', 2 - L2 refraction according to the law, tan t% -2 -2 n2 - n3 Hamilton has predicted that light emerges in the form of a hollow cone of rays. Conical refraction is an important step in the history of science and has played a role in demonstrating the theory of electromagnetic waves. A renewed interest in conic diffraction took place in the last years of the twentieth century; it resulted in a complete theory by Berry et al [2], validated experimentally in 2009, [8]. Here we follow Berry's theory, terminology and definitions, including the change in denomination of the physical effect, from this point, using the more stringent term of conical diffraction. However, it is important to note that the term "conical diffraction" is also used for two other techniques not related to the technique we describe: 15 - oblique incidence diffraction is also called conic diffraction - English term "Conical diffraction mounting" refers to a diffraction grating assembly 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 creating weaker conical diffraction along a shorter optical path. However, these effects can be used within the described devices. These effects include polymers, liquid crystals, and externally induced birefringence effects. The polymers include but are not limited to: stretched polymer sheets and cascade polymerization, [10]; the liquid crystals include but are not limited to: the thermotropic biaxial nematic phase, [11]; the external effects of induced birefringence 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.

The phase in the vortex created by conical diffraction is a geometrical phase and is therefore intrinsically achromatic. The additional chromatic effects are the dispersion of the optical axis and the dependence of the various parameters present in the conical diffraction equations as a function of the wavelength. The chromatic dispersion of the optical axis creates an angle of the optical axis of the crystal, depending on the wavelength, with respect to the optical axis of the system. It is due, in most cases, to the dispersion of the refractive indices. The refractive indices depend on the wavelength, according to the Sellmeier equations. The angle of the optical axis therefore varies as a function of wavelength, it creates an angle of chromatic inclination of the optical axis in the plane created by the crystallographic x and z axes. It depends strongly on the type of crystal. In a MDT crystal, the least dispersible achromatic crystal in the visible, the direction of the optical axis varies from less than 0.1 degrees between 540 nm and 700 nm. In a KTP crystal, the more achromatic crystal in the telecommunication IR, the angle varies from 0.05 degrees, between 1.350 nm and 2.100 nm, and less than 0.02 degrees on the telecommunication window - 1450 nm to 1650 nm. On the other hand, Oo can vary greatly depending on the wavelength in some organic crystals such as DAST.

The chromatic dispersion compensation of the optical axis can be performed using geometrical optics. The chromatic dispersion of the direction of the optical axis can be compensated by using the natural dispersion of glass or other optical materials, or by using gratings or prisms. The achromatization procedure 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 available commercial optical software by defining target functions. adequate. A different concept of achromatization is based on the use of two different materials, having inverse conical diffraction effects, with high and low chromatic dispersions. The dependence of the various parameters present in the conical diffraction equations as a function of the wavelength modifies the efficiency parameters of the conical diffraction effects.

For linear conical crystals, defined later, the fundamental transfer function is identical to the unit and thus trivially independent of wavelength.

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 conical linear crystals: the fundamental wave depends on the wavelength and the vortex wave is almost independent of it. Effect, the 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 wavelength and this effect must be taken into account in the design of systems using the two waves, fundamental and vortex.

Referring now to FIG. 3, which is a simplified schematic illustration of a configuration of a conical diffraction module 300, in accordance with one embodiment of the present invention. The incident light, 30, is supposed to be collimated, although other conditions may be adapted using simple optical means.

The setup itself includes a first lens 31, a tapered 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, shown below as Uo, determines the parameters of the conical effect across the conical radius, defined below. A conical imaging plane 35 is placed at the focal plane of the first lens 31; a polarizer, or 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 projected light on the sample is, as a first approximation, neglecting the 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.

30 Let the spatial variable, R, at the conical imaging plane, and the wave vector, U, represented in cylindrical coordinates by R, OR and U, or. Let X be the wavelength of 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 standardized parameters allowing the description below of the light distribution, to be valid both in the conical imaging plane and at the focus of the microscope objective, within the limits of the theory scalar diffraction. An example of introduction of the 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, O, and the conical normalized radius , po, are given by: P = 2-U o, U = -; po = 2 -Uo. U 0 (EQ.3) R U Ro P = 2-U0, U = -; po = 2 - Uo. U 0 (EQ.4) where U0 is the numerical aperture of the system. For po <0.5, we are referring here to a thin conical crystal; for po "1, we refer here to the shape of a linear thin conical crystal, for po <0.5 and> 1, to a sinusoidal thin conical crystal and for 1 <po <6, to a medium conical crystal. The emergent wave of the thin conical crystal, E (p, OR), expressed in normalized coordinates for a circularly polarized wave, is constituted by the superposition of two waves, referenced here as the fundamental wave, EF (p), a wave regular, and the vortex wave, Ev (p, OR), a singular wave; these two waves are coherent with each other, collocalised, circular polarized and inverse chirality: (1 \ .---- i) + (p) exp /) (EQ.5) In this equation, EF ( p) is the scalar fundamental amplitude, Ft / (p) is the reduced vortex scalar amplitude; they are given by: EF (p) = 2, r of u cos (p) J '(pu); F, (p) = 2z J of u sin (p) J, (pu). (EQ 6) For a linear thin conical crystal, the fundamental wave can be approximated by an Airy spot and the vortex wave can be approximated to a linear vortex, represented by: F, (p) = 2zpo of u2 J, (pu). (EQ.7) Assuming that the action of the partial polarizer, 29, is the scaling of the vortex wave by a, the Stokes parameters can be deduced from the preceding equations, where [3 is the angle linear polarization: So = (EF (p)) 2 + (a2 Fv (p)) 2 S, = 2ocE, '(p) F, (p) sin OR; S. = latE, (p) F '(p) cos OR; (EQ.8) E (p, OR) EF (P) + E, (p, e) EF (P) s3 = (EF (p)) 2 - (ce2Fv (P)) 2 Q = OR, 3029633 32 We will use the terms of a sparse object in English, to describe a set of point light emitters, of a number less than twelve, positioned in a volume whose size, in each dimension, is smaller. at 3 wavelengths, at the emission or reflection wavelength of the emitters. The size volume smaller than 3 wavelengths, which contains the sparse object, will be referenced as a reduced size analysis volume. We will use the term continuous object to describe a set of point or continuous light emitters that do not meet the conditions previously described in the sparse object definition.

Referring now to FIGS. 4a to 4c, which are a simplified representation of the concept of volume confinement of the confocal microscope. The functionality of the volume confinement is to limit, in the three spatial dimensions, the observed region of the sample to a volume of the smallest possible size, the volume of analysis. The volume confinement functionality limits the volume of analysis by the combination of two effects: the confinement of light projected onto a small area, ideally the size of the Airy task, 50, and the elimination of light. defocused by the confocal hole, 28 of Figure 2. The superposition of these two effects creates a small volume, the analysis volume, 60. This volume determines the size of the elementary region detected by the system.

Consider a parsimonious or continuous object, 51, consisting of a set of nanoemitters, 53 to 59. The nanoemitters 53 to 55 positioned in the analysis volume 60, and only them, are both excited by the light source and the photons emitted by them arrive at the detector module. Nanoemitters not in the light cone, 56 and 57, are not illuminated by incident light. The light emitted by the nanoemitters 58 and 59 lying in the conjugate plane of the confocal hole 28 in FIG. 2 is blocked almost completely by the confocal hole 28 of FIG. 2. Two different Cartesian landmarks are defined in FIG. the system, Figure 4c: The reference "i": The axes referenced "i" represent a Cartesian reference centered on the center of the analysis volume, 61.

The referential "a": The axes referenced "a" represent a Cartesian reference centered, for each light nanoemitter, on the light nanoemitter considered as a discrete point, 62, when, using one embodiment of the invention which will be As described later, a vortex is projected on the sample under analysis, the center of the vortex will generally be defined as the center of the analysis volume.

At least one embodiment of the invention uses conical diffraction to realize the fundamental optical modules of the art. However, alternative implementations, replacing modules based on conical diffraction, by modules based on other optical concepts, are able to provide the same functionality. They are intrinsically part of the scope of this invention. Alternative optical concepts include but are not limited to uniaxial crystals, subwavelength gratings, structured laser modes, holographic components, and other techniques known to the art. Those skilled in the art, optical and optoelectronic concepts, techniques and devices, which may be used in embodiments of the invention are described in, for example, the book written by D. Goldstein, "Polarized light", [12]. ], the "Handbook of Confocal Microscopy", [13], the "Handbook of Optics", [14]. Optical Semaphore We use in this embodiment of the invention the term optical semaphore to describe an optical element, passive or active, capable of channeling the incident light to different channels or detectors as a function of a property of light. The simplest case is a dichroic plate that separates the light into two channels depending on the wavelength.

In this embodiment of the invention, we use the term "position dependent optical semaphore" (PDOS acronym), or optical position-dependent semaphore, to describe an optical semaphore which channels light according to the position of the transmitting point. The PDOS will be determined by a series of transfer functions, Ti (x, y, z) depending, for each channel or detector i, on the position of the transmitter (x, y, z) in a reference volume. . The order of the PDOS will be the number of channels or detectors. The PDOS will be "lossless", lossless in English, in an analysis volume, if the sum of the transfer functions, Ti (x, y, z) is equal to the unit in the analysis volume. The confocal hole, described by Minsky, [15], is considered in this embodiment of the invention as a degenerate PDOS of order 1. In most cases PDOS dependence is a complex function of lateral and longitudinal positions. . However, in embodiments of the invention we use the term "Longitudinal Position Dependent Optical Semaphore" (acronym LPDOS), or optical semaphore depending on the longitudinal position, to describe an optical semaphore which channels the light. depending on the longitudinal position of the transmitting point. The LPDOS will be determined by a series of transfer functions, Ti (z) depending, for each channel or detector i, on the longitudinal position of the transmitter (z), in a reference volume. The order of the PDOS will be the number of channels or detectors. The LPDOS will be "lossless", lossless in English, in an analysis volume, if the sum of the transfer function, Ti (z) is equal to unity in the analysis volume. The LPDOS will often be coupled to a stop, limiting the lateral field of the system. Optical fiber transmission A main use of optical fibers is the exclusive transmission of the TEM mode. However, certain configurations of optical fibers, such as FMFs or vortices Fibers, mainly but not exclusively based on so-called "Photonic Crystal" fibers. Fiber "- acronym PCF - allows the simultaneous transmission or not, more complex modes, including vortex modes, with a vorticity equal to or less than 2. It would be possible to deport optical distributions created by the optical diffraction using optical fibers, allowing a major simplification of the optical system.

The possibility of offsetting the optical distributions created by the conical refraction with the aid of optical fibers allows the application of the embodiments of the invention to many additional applications, for example but not limited to gastric or gastroenterological observation. , and to the observation of the colon and urinary tract. In addition, certain "dual-core photonic crystal fibers", in English, [16], allow interaction between two modes, one of which may be a vortex, and provide an additional physical mechanism for creating diversified transfer functions. Measurements at several wavelengths In embodiments of the invention, the object can be illuminated in monochromatic light and using, for example, a conventional laser or a monochromatic lamp. This configuration is simple because one of the main parameters of the system is fixed and well determined. However, in other embodiments of the invention the object can also be illuminated at several wavelengths, either discretely, using for example several lasers, or continuously, for example using lamp or a laser having a wider spectrum. Many existing superresolution systems measure simultaneously or sequentially at several wavelengths. Indeed, it is possible to mark similar or different elements with fluorophores having different spectral responses, to recognize and separate them. It is important to present two different cases: The use of fluorescent markers, emitting at two different wavelengths, excited by the same wavelength The use of fluorescent markers emitting at two different lengths different or similar waves, excited by two different wavelengths It should be noted that in the case of the use of fluorescent markers, emitting at two different wavelengths, excited by the same wavelength, the problem of resetting between measurements of one wavelength relative to the second, are inherently non-existent because the supersolution position information is derived from the projection of light, which is perfectly identical for different wavelengths. This allows relative calibration of the fluorophore position at two different wavelengths, with limited accuracy only by the experimental calibration system, removing the major problem of resetting between two different wavelength images. The possibility of achromatizing optical systems based on conical diffraction makes it a tool of choice for the implementation of common path optical systems for many applications, and more particularly for described embodiments of the invention. . Achromatization is also possible for optical systems based on uniaxial crystals, and for almost all alternative implementations of this invention, with, for each of them, a more or less practical complexity.

Other existing fluorescence systems use light having a broader spectral content, to reduce artifacts, and primarily speckle effects. As a corollary, the spectral properties of fluorescent proteins make it possible to measure the potential of intracellular molecular interactions using the Förster-Förster (Fluorescence) Resonance Energy Transfer (FRET) energy transfer technique. In some implementations of PSIT systems, the light from one or more incident laser beams is separated using a light splitter into two beams, the main beam that will accomplish the functionality of the PSIT system, and an additional weak beam. intensity used to measure the position of the laser beam using a camera or position detector. This device makes it possible to measure in real time the position of the laser independently of any error of wobble or other mechanical error with a very high precision. The use of nonlinear interactions in a material medium, between two beams of light, was proposed in 1994 by Hell, [5] and [5]. 17], as the basis of a super-resolution system. Many different techniques have resulted from Hell's work creating several families of techniques, such as RESOLFT and "localization microscopy". Several reviews of these techniques have been published such as that of Schermelleh et al. [1]. These non-linear interactions include but are not limited to two-photon interaction phenomena, emission-depletion, blinking, and photoactivation effects upon which the RESOLFT 10 family of technology and the family are based. of "localization microscopy" technologies. The RESOLFT family of technologies is well known and is described in several references, such as the initial article of Hell [5] and the original patent [17], or by Schermelleh et al. [1], or in recent publications, Vicidomini et al. [18], or Willig et al. [19].

In the STED technique described in the initial article of Hell [5], two beams are sequentially projected onto the object: a standard excitation beam, modeled in most cases by a light distribution described by a function of Airy, and a depletion beam - having a form of donut or vortex; the effect of depletion is to prevent the fluorescence of the fluorophores in the surface of the depletion light distribution, while not altering the emission of the fluorophores positioned in the center of the light distribution outside the depletion light distribution; this makes it possible to create an equivalent emission light distribution smaller than the initial excitation light distribution. This technique has made it possible to produce equivalent emission light distributions of very small size, while at the same time requiring very high light depletion energies. Several variants of the STED have been developed: the "CW STED", [20], the "gated STED", [21], and the "modulated STED" [22]. In CW STED the pulsed laser used in the first version of STED is replaced by a continuous laser - simpler. In the "gated STED" the emitted photons are discriminated according to their emission time 30 to reject the photons emitted from fluorophores that have not received enough time the depletion beam; the "modulated STED" uses an intensity-modulated excitation beam, in combination with synchronous detection dependent on the modulation frequency. This makes it possible to discriminate the fluorescence signal created by the excitation beam of the residual fluorescence caused by the depletion beam.

The donut - or vortex - was created, in the first version of the STED, with a spatially varying phase blade, "phase plate" in English. This implementation requires the use of two separate optical paths for the two beams, the excitation beam and the depletion beam. This optical assembly is complex, highly dependent on any mechanical drift, and creates complex optical alignments. The developed system requires high technicality and the cost of the system is important. In addition, this implementation is chromatic because the phase plate adapted to one wavelength will no longer be at another wavelength. Since the optical system is not achromatic, the use of a two-wavelength depletion STED requires an even more complex optical system. To simplify the implementation of the STED, several authors have proposed solutions for performing a STED in which the two beams, the excitation beam and the depletion beam, propagate along the same optical path: - Wildanger et al . have proposed a STED Microscope with a common optical path, insensitive to mechanical drift, based on the dispersion properties of the different optical materials, [23], - Bokhor et al. [24], proposes the use of a chromatic annular separation of the pre-aligned beams; however, this solution blocks a portion of the depletion light, - Hoeffman [25], proposes the use of a module containing the vortex-forming pre-illuminated optical elements, in the optical path of the depletion laser, to simplify the alignment, Menon et al; [26], proposes diffractive lenses that make it possible to create amplitude zeroes without phase singularities, Reuss et al. [27] introduces a beamformer placed directly in front of the objective lens. This device is based on the use of birefringent crystals, mounted as a segmented waveguide, consisting of four segments. By choosing the thickness parameter of the blade, it is possible to make a member consisting of a phase plate for depletion and a neutral blade for excitation. This technology is marketed under the name EasyDonut by Abberior [28]. However, the previously described proposed solutions are all highly chromatic and are designed for a single wavelength of excitation and depletion. However, biological applications in many cases require a system with two or more excitation wavelengths. Indeed, it is common practice to mark different biological elements with fluorescent markers differentiated by their excitation wavelength or emission. The most advanced fluorescence systems can use four to six differentiated markers. The presence of a single super-resolution channel severely limits the use of the systems. Two-wavelength STED systems are also available commercially. It is clear that for an achromatic STED microscope the use of the same optical path for two depletion beams at two different wavelengths will simplify super resolution on different fluorophores. In the prior art, for all the solutions proposed for the STED, the initial laser beams are in the form of a regular distribution and in the majority of the 5 cases of a Gaussian distribution. These initial laser beams will subsequently be transformed into an excitation beam, a regular wave, and into a depletion beam, a singular wave, by a suitable optical system, described by the various inventors. Collocating these beams upstream when they are still in Gaussian distribution form is relatively simple. Collocating these beams downstream when they have been transformed into regular and singular waves is much more complex. Collocating these beams upstream can be done commercially by laser bench systems using fiber-based techniques. It is thus relatively easy as it is also done in the set of confocal microscopes, to create a set of laser outputs at different wavelengths of the same optical fiber and therefore 15 collocated very precisely. The solution proposed in embodiments of the present invention makes it possible to rely on this collocation upstream, greatly simplifying the system and allowing the realization of a STED at several wavelengths, intrinsically. For this, it is preferable to use an optical system that combines the properties of common optical path, achromaticity and beam shaping. It is preferable to perform a different beam shaping for the different beams, ie a regular wave for the excitation beam and a singular wave for the depletion beam, and this at any wavelength in the visible spectrum or infrared. The ability to perform an achromatic beam shaping, making it possible to obtain for a polarization a regular wave and for another polarization, a singular wave, over all the visible or infrared light, different for the different polarizations, is new. The PSIT module allows the realization of such an optical system that combines the properties of common optical path, achromaticity and "beam shaping". To our knowledge, there is not in fact a system with common path, achromatic and allowing a different beam shaping for different beams, in the literature. Such a system would certainly improve the simplicity of design and use of STED.

3D STED Many systems have been proposed to extend the concept of STED to the third dimension. The solution adopted for all STED-3D is the "depletion 35 ring" proposed by the team of Stefan Hell, reference 36. The "depletion ring" allows to create a "black sphere", a distribution in which the intensity is zero at the center of the focusing field, and increases relatively rapidly with defocusing. An implementation of the "black sphere" has been described by one of the authors of this patent in reference 37. Use of helical symmetry distribution for STED 3D The solution implemented in STED or STED-3D systems uses a distribution 5 optics of zero intensity at the central point, and remains zero along the axis, along a certain distance.We propose here, the use for the STED-3D, of a distribution having a position of the preferred implementation of this distribution is the use of conical diffraction using the optical distributions 10 which we have referenced as the zero intensity, this position of zero intensity having a helical spatial variation, as a function of the axial parameter. These distributions could be realized, in particular but not exclusively, using the techniques based on the devices mentioned above, including but not exclusively the SLM (Spacial Light Modulator) and segmented mirrors that allow to create phase and / or amplitude distributions in the pupil. The use of helical topology distribution to create a zero intensity distribution having a helical movement is considered to be part of this invention. In particular, the implementation of these distributions using conical diffraction is one of the preferred implementations of this invention but the implementations of these distributions using an SLM or segmented mirror to create phase distributions or amplitude in the pupil are also considered to be one of the implementations described in this invention. A priori information and additional information Embodiments of the invention described allow the integration and the fusion of additional information external to the described platform, optical or contextual, to obtain an improvement of the accuracy of the information collected from the sample for any of the abstraction levels cited: the map, the level of geometric abstraction, the level of biological abstraction and the level of functional abstraction. More generally, the spectral diversity, the information obtained at several wavelengths, the diversity of polarization, and the information obtained by projecting different states of polarization, extends the range of information available.

The fact that the absence of energy, for example in the case of the vortex zero, is relevant information, opens additional possibilities for the acquisition of information without "cost" in photon number. This situation is of great importance for the detection of weak fluorescence phenomena, such as for example autofluorescence.

One of the methods described for certain embodiments of the invention will be referred to as dark tracking. We introduce the concept of optical integral information, the information that could be retrieved from optical or electromagnetic wave measurements, on a target, by an observer, from a given point of view. This information contains many parameters of the object, related to its position, the materials that compose it, its temperature, or its orientation. The optical integral information does not contain, on the other hand, information on regions of the object that do not have an optical path to the observer, for example an element positioned in an opaque box, or a physical information that No optical transcription. Superresolution measurements and diffraction limit It has long been considered that optics intrinsically limit the resolution of any optical system, across the diffraction limit. The appearance of superresolution techniques - in different domains and under different denominations - has shown that it is possible to exceed this diffraction limit by various means. Embodiments described in this invention, such as detecting the presence of two points of the same intensity, by projecting a vortex at the center of gravity of the light distribution created by the fundamental wave, are not limited in magnitude. resolution a priori and could ideally - with an infinite number of photons - obtain any resolution, as we will describe later for a specific case. Acronyms We will use in this patent application the acronym, SRCD, "Super Resolution 25 using Conical Diffraction" to name the platform, modules and systems specific to the implementation of this invention. We will use in this patent application the acronym, PSIT, projection method of a sequence of light intensities differing topologically, or in English: "Projected Sequence (s) of Intensities with various Topologies"; the PSIT method can also be used to project sequences of light intensities differing topologically, at two or more wavelengths, sequentially or simultaneously. We will use in this patent application the acronym, PDOS, optical semaphore with spatial dependence, in English: "Position Dependent Optical Semaphore".

The SRCDP platform, "Super Resolution using Conical Diffraction Platform", is a Microscopy platform using optical modules based on conic diffraction. We will use in this patent application the acronym, LatSRCS to name the optical module implementing the PSIT method to the implementation of this invention. We will use in this patent application the acronym, LongSRCS to name the optical module implementing the PDOS method. The SRCDP platform, described in detail later, consists mainly of two hardware modules, two new and complementary optical modules, the LatSRCS and LongSRCS optical modules, mounted on a microscope, and an algorithmic module, SRCDA, for reconstructing the supersensitive information from the sample. In addition, the SRCDP platform includes an enhanced detection module, a system control module, and computer and software support. In addition, some embodiments of the invention relate to a large number of alternative implementations of the PSIT and PDOS methods, the SRCD platform, the LatSRCS and LongSRCS optics modules, and the algorithmic SRCDA. The function of the confocal microscope is to limit, in the three spatial dimensions, the observed region of the sample to a volume of the smallest possible size, the volume of analysis.

As a corollary, in a confocal fluorescence microscope, the acquired information is a single intensity value for the entire analysis volume, designed as a single entity. More clearly, the detailed information of the position of the nanoemitters within the analysis volume is not accessible, a priori, in a confocal microscope. It was generally accepted that no additional optical information could be created that would have allowed additional discrimination within the illuminated volume. Reference is now made to Figure 4d, which is a simplified conceptual representation of the measurement paradigm according to at least one embodiment of the invention. The measurement paradigm is much more ambitious than that of the confocal fluorescence microscope, shown schematically in Figure 4a.

In Fig. 4d, an analysis volume, 60, is created in the focal plane of the microscope objective, 22; it contains a sparse object, 51, consisting of several nanoemitters, 53 to 59; the result of the system is a reconstructed parsimonious object, 63, and a list of nanoemitters and a list of their attributes, 64. However, the use of a parsimonious object in Figure 4d is for the purpose of illustration and this figure would perfectly able to represent a continuous object, mutatis mutandis.

The composite optical process comprises performing a series of optical measurement processes, controlled by the system control module, by varying the illumination sequences and / or the channel functions and / or the position of the the illumination sequence according to measured data or external information. An example of an optical process composed according to one embodiment of the invention will be detailed below.

PSIT measurement method A PSIT measurement method according to one embodiment of the invention, a sequence of light distributions of different topologies is projected on the analysis volume. The measurement method PSIT performs the following functions: Projection of a sequence, the emission sequence, of compact light distributions of different topological families on a sample; and o For each compact light distribution: - emission of light by the nanoemitters of the sample, - creation, using the microscope optics, of an optical image, 25 - acquisition of the optical image by a photoelectric detector and creating a digital image. In more detail, it should be noted that: The emission sequence consists of at least two point light distributions of different topological families. The emission sequence is projected onto a biological sample marked by nanoemiters. The emergent luminance of each nanoemitter is dependent, for each nanoemitter, on the intensity, in the incoherent case, or on the electromagnetic field in the coherent case, incidental to the spatial, three-dimensional position of the aforementioned luminous nanoemitter. discussed previously.

For each light distribution of the projected transmission sequence on the sample, an optical image is created. The set of images corresponding to all the light distributions of the transmission sequence is referenced as the sequence of images.

The PSIT method according to this embodiment makes it possible to acquire essentially lateral information, that is to say the lateral position of each of the nanoemitters. In one embodiment, the PSIT method is implemented by projecting light distributions of different topologies created by conical diffraction and modified by a variation of the input and output polarization states.

In one embodiment, the PSIT method can also be used to project sequences of light intensities that differ topologically, at two or more wavelengths, sequentially or simultaneously. PDOS Method A PDOS method according to one embodiment of the invention comprises the distribution by an "optical semaphore" of the light re-emitted by the nanoemitters or by the continuous object between at least two detectors. Ideally, the function of the optical semaphore is to separate on different detectors different regions of the analysis volume. Practically, the optical semaphore creates, for each detector, a transfer function of the light emitted by a light nano-transmitter, depending on the position in the space of the light nano-transmitter and different for the different detectors. In one embodiment, the PDOS method is implemented so as to separate on different detectors the collimated, emerging light of nanoemitters positioned at the focal plane of the objective, of the emerging non-collimated light of nanoemitters lying below or beyond the focal plane. The PDOS method makes it possible to acquire essentially longitudinal information, that is to say the longitudinal position of each of the nanoemitters. Mathematically, the method according to embodiments of the invention realizes a transfer function transforming the spatial distribution in the space of the nanoemitters into the raw information consisting of a set of images. The algorithmic performs the opposite operation: it reconstructs the spatial distribution in the space of the nanoemitters from the set of images composing the raw information. INFORMATION IN EMBODIMENTS OF THE INVENTION The intermediate result, the raw information, is obtained at the end of the detection step. The raw information consists of a set of images Aop (m, n), representing for the light distribution o, the image coming from the detection channel p.

As in a confocal microscope, the measurement process analyzes a small volume in an object of much larger size. It will therefore require the addition of additional modules, similar to those of a confocal microscope including a scanning process, a software module for integration, analysis and visualization of point data in surfaces and / or in three-dimensional objects. In mathematical terms the algorithmic solves an inverse problem or parameter estimation. The equations of the model are known and one has a priori model, parametric or not, on the configuration of nanoemitters. The most natural model is to assume a small number of nanoemitters (sparse object), but one can also use continuous models, assuming the presence of one-dimensional structures (lines, curves) or specific patterns. We can then parsimoniously use all the mathematical procedures known to those skilled in the art for the resolution of inverse problems or the estimation of parameters. We will describe later an algorithmic example specifically adapted to the measurement according to one embodiment of the invention. In addition, we present, for its emblematic value, a new solution of the problem of the discrimination of two points positioned at a short distance from each other. This problem, studied by Lord Rayleigh, is the basis of the resolution criterion in many areas of Optics.

The characteristics of the embodiments of the invention have thus been described in a rather broad manner so that the detailed description thereof can be better understood and so that the present contribution to the art can be better appreciated. . Many additional features of the invention will be described hereinafter. One embodiment of the invention is a hardware and algorithmic platform, referenced as the SRCDP platform, 500, illustrated in Figure 5. The SRCDP platform, 500, implements the method according to one embodiment of the invention, combining the two methods PSIT and PDOS described above. In one embodiment, the SRCDP platform observes, Figure 5, a biological sample, 11, integrating a set of nanoemitters. The result of the observation of the biological sample by the SRCDP platform is the acquisition of superresolution information representative of the observed sample. The SRCDP platform, 500, Figure 5, mainly comprises: In its hardware part: - A confocal200 microscope, adapted or optimized, similar to the confocal microscope described previously, and comprising all the appropriate components, as previously described 3029633 - Two new and complementary optical modules, mounted on a standard microscope. The two novel optical modules are the LatSRCS, 700, and LongSRCS, 800 optical modules, described in detail later with reference to Figures 6 and 8, respectively. The LatSRCS 700 optical module implements the illumination steps necessary for the implementation of the PSIT method according to one embodiment of the invention. The LongSRCS optical module, 800, implements the steps of distribution of the emerging light intensity into a plurality of images of the PDOS method according to one embodiment of the invention and the algorithm module SRCDA, 600, capable of from the images created by the SRCDP platform to reconstruct the superresolution information of the biological sample. Other auxiliary elements, such as the computer 66 and the software 67, necessary for the realization of the platform. LatSRCS Optical Module Implementing the PSIT Method We describe, with reference to FIG. 6a, an optical module according to FIG. embodiment of the invention, the LatSRCS optical module, 700, and its specific function in microscopy. The optical module, LatSRCS, 700 according to this embodiment, is an optical module, projecting on a plurality of nanoemitters of a sample, a sequence of compact light distributions of different topology. Each nano-transmitter fluoresces with a sequence of luminous fluorescent intensities depending on the intensity incident on the nanoemitter and characterizing the lateral position of the nanoemitter. In most embodiments, compact light distributions of different topologies are created by interference, with varying amplitudes and phases between a regular wave and a singular wave. In the preferred embodiment, the regular and singular waves are created by a thin conical crystal. The LatSRCS optical module, 700, is positioned in the illumination path of the confocal microscope 200; it projects a sequence of compact light distributions of different topologies on the sample 11 using the objective of the confocal microscope 200. In one embodiment using conical diffraction, the incident intensity at a specific position on the sample 11 will be proportional, for each light distribution, to a specific combination of Stokes parameters. The LatSRCS optics module, 700, utilizes an inherent feature, previously described, specific to the nanoemitter, which samples the incident light intensity at its precise position (from the nanoemitter) and re-emits a fluorescent light dependent on the incident light. It is remarkable that the measured information is directly related to the position of the nano-transmitter within the compact light distribution. This information is fixed by the functionality of the nanoemitter, its property of absorbing and reemitting light, which break the optical chain. This information is carried by the fluorescent light, in the form of an emergent light distribution, recoverable by a detection assembly 65. If the incident light varies over time as a function of a sequence of compact light distributions of different topologies, the The intensity of the fluorescent light re-emitted varies in the same proportions. The sequence of the re-emitted fluorescent light will be proportional to the sequence of the compact light distributions of different topologies. From this information, it is possible to recover the position of the nanoemitter, as explained below. The PSIT method according to embodiments of the invention refers to the projection of a sequence of compact light distributions of different topologies in a microscope, the interaction with the parsimonious object or the continuous object, the collection of the light re-emitted by the objective of the microscope, 22, the detection of light, fluorescent or not, by the improved detection assembly 65, and the analysis of the information by an appropriate algorithm. In some embodiments, the enhanced detection set, 65, consists of a single detector, and only retrieves the overall intensity as a function of time, while in other embodiments, the entire Enhanced detection consists of a small area of pixels and also recovers the spatial distribution of fluorescent light. The set of information retrieved, consisting of a plurality of images, designated as the superresolution side images. In one embodiment, the contribution of a nano-transmitter positioned in the illuminated volume to a specific superresolution side image is proportional to a combination of the Stokes parameters of the incident light at the position of the nanoemitter. The superresolution side images, the information created by the compact light distributions of different topologies is new, and not present in the prior art. This new information makes it possible to refine the position of the nanoemitters or the spatial distribution of the continuous object, to quantify the number of nanoemiters present in the illuminated volume and to differentiate several nanoemitters present in the same volume. Reference is now made to Figure 6a, which is a simplified schematic illustration of a LatSRCS optics module, 700, in accordance with one embodiment of the present invention.

FIG. 6a shows an optical module, LatSRCS, 700, it includes all the components of the conical diffraction module, of FIG. 3, which are implemented in the same way as in the conical diffraction module 300. The optics of the scanning confocal microscope light source are assumed to be achromatic and in infinite conjugation, although other conditions may be adapted using auxiliary optics. The incident light entering from the light source is parallel, 30. The optical module 700 itself comprises a first lens, 31, an achromatic 32, or a subset achromatically realizing the functionality of a conical crystal as explained above, and a second lens 33; a partial polarizer, 29, described above, may also be added. The first two lenses, 31 and 33, are preferably configured as a 1: 1 Kepler telescope; the conical imaging plane, 35, is placed in the common focal plane of the lenses 31 and 33. The numerical aperture of the first lens, 31, determines the parameters of the conical diffraction effect across the conical standardized radius, defined below. The second objective, 33, 15 restores the parallelism of the light, to inject it under the microscope. It further comprises a polarization control submodule 71, including, for example, a rotating quarter wave plate, a pair of liquid crystal light valves or a Pockels cell, 72, and an analyzer 73. The information of the Stokes parameters can be transformed into sequential information, through a sequence of spatially differentiated light distributions, and carrying sequential information, as described above. Referring to Figure 7a, this figure shows the light distributions, created through a 0.388 standard conic tapered crystal, calculated by a scalar approximation, for different input and output bias states, including input or at the output is a circular or linear polarizer or a radial or azimuth polarizer. These light distributions were calculated in an intermediate imaging plane and not at the focal point of the lens to separate conical refraction from vector effects. The polarization states - input and output - are characterized by their angle for linear polarizations and by their chirality for circular polarizations.

Referring to FIG. 7b, this figure shows the light distributions, created through a conically standardized tapered con crystal of 0.818, calculated by a scalar approximation, for different input and output bias states, including as input or at the output is a circular or linear polarizer or a radial or azimuth polarizer. These light distributions were calculated using Diffract software from MMresearch. These light distributions were calculated in an intermediate imaging plane and not at the focal point of the lens to separate conical refraction from vector effects. The input and output polarization states are characterized by their angle for linear polarizations and their chirality for circular polarizations. We present in these tables a large number of different transfer functions, including cases including input or output of circular, linear, azimuth or radial polarizers. This description must be supplemented by additionally including circular, linear, azimuth or radial polarizers described in the figures, the cases of elliptic, dichroic or partially dichroic polarizers, and spatially varying polarizers. Moreover, as illustrated in FIGS. 7a and 7b, these transfer functions vary greatly according to the standardized conical parameter po. In addition, the introduction of two conical crystals, or a conical crystal and a uniaxial or biaxial crystal (in which light propagates in a different direction of propagation from that of conical diffraction) in cascade allows a even larger number of transfer functions as illustrated for two tapered crystals in Figure 7c. In summary, in this patent application, reference will be made to the term "tapered diffraction transfer function", the set of transfer functions that can be obtained by means of a small (<6) number of crystals in cascade and polarization elements, static or dynamic, uniform or spatially varying We will note mainly the following light distributions: - The fundamental: Figure 7a00 and 7a11, obtained between parallel circular polarizers, which is a distribution close to the distribution of Airy - The vortex: Figure 7a01 and 7a10 obtained between circular polarizers, crossed - The distribution which we called the distribution in "crescent moon" or Stokes, the sub-figures 7a0,2_5, 7a1,2-5, 7a2-5, 0 and 7a2-5,1, and Figure 7c describing the axial variation of the "crescent moon" or Stokes distributions. These distributions are obtained between a circular polarizer and a variable linear polarizer; this distribution is antisymmetric and the axis rotates with the axis of the linear polarizer, - The distribution which we have called the distribution in "half-moons", sub-figures 7a42, 7a35, 7a24 and 7a53 are obtained between two crossed polarizers; this distribution is symmetrical. The distribution which we have called the distribution in "offset half-moons", figure 7d, representing the distribution in "offset half-moons" at different axial positions, is obtained between two elliptical polarizers for certain ellipticity values. The more complex light distributions, FIG. 7b, for a standardized conical parameter crystal, po, greater than 0.5; the production of additional light distributions using two or more conical crystals in cascade, (not shown) ) with or without static or dynamic polarization elements between the crystals The different light distributions are produced by the modification of the input or output polarization. The different light distributions follow the same optical path and the optical system creating these distributions is a common path optical system, as we have defined it previously. There are a number of polarization elements having a different polarization at different wavelengths. The use of one of these elements makes it possible to create two compact waves, either regular or singular at two wavelengths, or a regular wave at one wavelength and a singular wave at another wavelength. Such a device allows a much simpler implementation of emission-depletion concepts limited in some cases by tolerances or vibrations of the optical system. Redundancy and Random Phase Variations The elementary light distributions described in Figure 7 can be obtained in several different ways. In addition, some of them can be obtained as a linear combination of other elementary light distributions; for example the vortex can be obtained by any sum of two orthogonal "half-moon" light distributions.

This redundancy makes it possible to average part of the random phase errors present inexorably in many processes for measuring biological objects. New light distributions can also be obtained as mathematical combinations of elementary light distributions. The "pseudo-vortex" light distribution, calculated from arithmetic combinations of the four "crescent moon" distributions, has the particularity of having a very strong curvature at the origin. The PSIT Method was initially designed to allow lateral super-resolution; however, the PSIT method can also be used to obtain the longitudinal position of a nanoemitter. Indeed, certain elementary light distributions are relatively insensitive - within reasonable limits - to a variation of the longitudinal position of the nanoemitter, while others are very sensitive. A sequence of compact light distributions, some of them independent and some of them dependent on the longitudinal position, would make it possible to go back to the longitudinal position of the nanoemitters.

In addition, for light distributions strongly dependent on the longitudinal position of the nanoemitter, a series of elementary light distributions shifted slightly longitudinally relative to each other can be projected onto the sample, allowing a set of images. containing longitudinal information. In addition, some more complex elementary light distributions, consisting of more complex superimpositions of waves, exist with a strong longitudinal dependence; for example, the "dark three-dimensional spots" described by Zhang, [29], create a black spot surrounded in three dimensions by a luminous sphere. These "dark three-dimensional spots" consist of a superposition of LaguerreGauss functions, which can be realized inside a laser cavity or using a hologram or a phase plate, as suggested by Zhang or using conical or uniaxial crystals as suggested by the inventor. Conical diffraction can be used to change and shape the PSF so that it has lateral and axial variations. After the crystal, the incident beam is transformed into two Bessel beams: a Bo part (fundamental mode) and a B1 part (vortex mode). All homogeneously polarized incident beams can decompose on the orthogonal basis composed of the right and left circular circular polarizations. The outgoing beam is the sum of the contributions of the two components, one giving rise to a part Bo and the other to a part B1. Different combinations of Bo and B1 can be achieved by choosing the input and output polarizations, which allows PSFs of different shapes to be obtained. We are particularly interested in the case where the input polarization and the output polarization are elliptic with an orientation of the major axes of each ellipse having an angle of 90 ° between them. Among the distributions generated under these conditions, some have significant variation along the Z axis, and these variations can be exploited to measure the position of a transmitter with high axial accuracy. We separate these distributions into two groups, distributions that have a single lobe and those with two lobes. The distributions with a single lobe present a rotation effect along the Z axis. Thus, the Stokes distributions, made from a linear polarization (ellipticity = 0 °) and a circular polarization (ellipticity = 45 °) present variations. which appear in the table below. By using an appropriate algorithm, the orientation of the distribution 30 can be detected and the position of the transmitter deduced with high axial accuracy. (Figure 7d). The distributions with two lobes present an offset effect of the two lobes along the Z axis. Thus, the so-called offset half-moon distributions, made from two elliptical polarizations oriented at 90 ° but with the same ellipticity, exhibit variations that appear in the table below. By using an appropriate algorithm, the position of the transmitter can be deduced with high axial accuracy. (Figure 7d) All these variants are deemed to be part of the invention. The inventor has nevertheless chosen, in certain implementations, to separate into two disjoint optical modules but the lateral measurements of the longitudinal measurements to reduce the complexity of each of the complementary modules. Vector Effects The theory developed so far describes the light distribution in the imaging plane of the microscope, 35. The distribution of the light projected onto the sample is, according to the theory of geometrical imaging, a reduced image. of the distribution of light in the image plane. However, as is abundantly described in the literature, for a high numerical aperture objective, the theory of geometric imaging is not accurate and the vector effects must be taken into account. These effects consist essentially of the presence of a longitudinally polarized component. Referring back to FIG. 6a, to mitigate the vector effects, it may be advantageous to maintain the fixed final analyzer and to add an additional fixed or variable element, the output bias adaptation sub-module, 74, to control the output bias. We have found that output polarization with circular symmetry substantially reduces vector effects. Such polarization can be circular, radial or azimuthal. For circular polarization, the output polarization adaptation sub-module 74 is simply a four-wave delay plate. In this case, the longitudinal bias elements have vortex symmetry and blend smoothly into the system with only a small change in the shape of the Stokes parameters, even for microscope objectives with very high numerical aperture. Alternatively, the output bias matching sub-module, 74, may be variable and adapt to the topology and symmetry of each of the compact light distributions. It is recalled that a Wollaston prism can be used to separate an incident beam into two emerging beams separated by an angle. Using multiple prisms in cascade, or can thus separate an incident beam into a large number of emerging beams. The same effect can be obtained by modifying the Wollaston prism, to create the composite Wollaston prism, by adding uniaxial crystal pieces whose index and orientation of the birefringence are chosen. It is thus possible to construct a prism of a single uniaxial crystal block which can separate an incident beam into emerging beams (e.g. eight or sixteen) which are contained in a plane, and separated by equal angles. Once focused on the sample, r points are aligned and also separated. If the incident beam passed through a LatSRC module these rpoints are not Airy's tasks, but the distributions created by the module and are all identical.

3029633 52 The advantage of using this beamsplitter is to sweep the sample faster because you have 2 'light spots instead of one. PDOS Method and Lateral Measurements The PDOS method was originally designed to allow longitudinal superresolution; however, the PDOS method can also be used to obtain the lateral position of a nanoemitter. Indeed, the elementary light distributions are also sensitive to a variation of the lateral position of the nanoemitter. For a flat sample, in the case where light projection is not feasible, the PDOS method can replace the PSIT method to make superresolved measurements.

All these variants are deemed to be part of the invention. The inventor has nevertheless chosen, in one of the implementations, to separate into two disjoint but complementary optical modules the lateral measurements of the longitudinal measurements to reduce the complexity of each of the complementary modules. Detection Module In confocal scanning microscopy the detector is a detector consisting of a single element such as a PMT or a SPAD. The acquisition time of the detector is determined by the scanning mechanism. An improved detection module, 65, can be implemented using small-sized, low-pixel detectors. Such a module would not have been possible ten or twenty years ago, because of the lack of adequate technologies. Today, high-speed, low-pixel, small-size detectors with low noise characteristics are available on a number of technologies. SPAD matrices with a low pixel count, such as 32 * 32, have been recently demonstrated with acquisition rates up to 1 MHz. The improved detector module 65, 25 can also be implemented using CCD, EMCCD or CMOS sensors. CCD, CMOS or EMCCD sensors with a small number of pixels exist or can be specifically designed. In addition, CCD, CMOS EMCCD detectors can be used by using region of interest, sub-fenestration or "binning" functions, "crop" or "fast kinetics" modes, available for certain detectors.

Spatiotemporal information referenced herein is the position and time of the impact of each fluorescent photon. In real systems, spatio-temporal information is corrupted by detector noise, creating erroneous photons, and inefficient detection, which creates photons that are not detected, reducing performance. In the SPAD matrices, for each photon, the pixel that has detected it and the impact time are received, i.e. the complete spatio-temporal information is available. For CCD, CMOS or EMCCD detectors, the acquisition of several frames is necessary to approximate the spatio-temporal information. In several implementations we will refer to separate detectors; in very many cases the detectors may be either physically separated or consisting of different zones on the same detector, or a combination of the two previous cases. Algorithmic SRCDA The reconstruction algorithm that we have detailed applies not only in the case of a given field analyzed using the PSIT and P methods but also in the case of a larger field analyzed. by scanning. In the case of a scan, the direct model is enriched by the fact that a recovery between the different positions of the projected signals makes it possible to take into account, at a given point, more measurements. The taking into account of offset projected signals does not however bring any additional complexity, since these off-set signals only add to the list of projected signals. Multi-image system including the E-LSE reconstruction algorithm We will refer under the name of multi-image system, all optical and optoelectronic systems, in which a set of different and differentiated images from the same region two-dimensional or three-dimensional object spatial - are recorded and analyzed through an appropriate algorithm to analyze the spatial - and / or spectral - distribution of the emitting spatial region. This differentiation may be due to the spatially different illumination projection, as previously described; it can also be due to a variation of the spectral content of the illumination; it can also be due to a natural movement or imposed from outside objects. Finally, it may be due to a stochastic variation in the content of the spatial region of the object, two-dimensional or three-dimensional, through a natural or imposed stochastic effect, such as the systems used in super-resolution based on stochastic detection, including the PALM and STORM processes and their innumerable 30 variants, each with a different acronym. Other means of differentiating images of the same emitting spatial region are known to those skilled in the art and are considered an integral part of this invention. We will refer under the name of multi-image system including the E-LSE reconstruction algorithm, a multi-image system using the E-LSE algorithm described below. E-LSE reconstruction algorithm The proposed algorithm allows from the images recorded by the camera (or the cameras) following the excitation of the sample by all the selected illuminations, to reconstruct a high resolution image, two-dimensional or three-dimensional, of the sample. This algorithm is based on the combination of several principles: 1) the formulation of the reconstruction as a Bayesian inverse problem that leads to the definition of a posterior distribution. This posterior law 10 combines, thanks to Bayes' law, the probabilistic formulation of the noise model (Poisson noise inherent to the quantum nature of photon emission, which may be superimposed on the modeling of other sources of noise, in particular the reading noise of the camera), as well as possible aprioris (positivity, regularity, etc.) on the distribution of light in the sample; 2) the estimation of the light distribution in the sample by the mean (mathematical expectation) of the posterior distribution. This approach, considered by Besag in 1984 [30], has recently been implemented numerically in the case of image denoising with a total variation type apriori [31,32]; 3) the use of spot emitter clouds which makes it possible to favor parsimonious solutions (samples with a limited number of emitters, or whose emitters are concentrated on small structures such as curves, or surfaces in the case three-dimensional (4) the estimation of the posterior mean using a Monte-Carlo Markov Chain (MCMC) algorithm [33,34], as in the references [31,32] mentioned above. According to one embodiment of the algorithm, the principle 3 is not used and the average of the posterior distribution is calculated on all the possible images as in [31,32].

The algorithm uses as input the measurements made on the sample, but also data inherent to the system that are obtained after a so-called calibration step. In this step, measurements are performed on a reference sample in order to accurately measure the illumination functions and the optical point-of-return function of the optical system.

In the standard embodiment, where only the Poisson noise is modeled, the density of the posterior probability law is written in the form E (x, λ) p (s'1) = 40 where Z is a normalization constant which does not intervene in the algorithm, and 3029633} The emitter cloud is here represented by the vector x = (xi, x2, ... 4 (n discrete positions in the field of the image high resolution to be reconstructed) and the vector À = (À ,, A2, ... / 1,) which encodes the intensities of the emitters at the points xbx2, ... xn. Each quantity of type ui (x, y) is determined during the calibration step: it represents the intensity emitted at the pixel y of the camera by an emitter situated at the pixel x of the high resolution image, in response to an illumination of index i (i therefore codes here at both the position of the illumination signal, but also its shape.) The positive real B corresponds to the intensity of the continuous background, which generally results both from the ntillon (diffuse fluorescence for example) but also the sensor. Finally, the quantities mi (y) correspond simply to the measurements (images recorded by the camera): mi (y) is the intensity measured at the pixel y of the index image i, that is to say the image recorded after index illumination i).

The proposed algorithm consists in changing the emitters represented by the vectors x and λ in accordance with the law given by the density p (x, λ). The algorithm is iterative: at each iteration, one of the transmitters is perturbed (in position or in intensity) and this disturbance is accepted or not according to the principle of the Metropolis-Hastings algorithm [3]. The reconstructed image is obtained by averaging, with equal weight for each iteration, the emitters thus constructed. If x 'and λ respectively correspond to the position and intensity of the transmitters at the iteration j of the algorithm, then the image I reconstructed after N iterations is given by several improvements can be made to this algorithm: introduction of a burn-in step (the first iterations are not used in the reconstruction), the optimization of the initialization of the emitters, the optimization of the disturbances carried out at each iteration (proposition law), use of a post-filtering step (for example a slight Gaussian blur), etc. The results of the algorithm can be transmitted to the user either in the form of an image or in the form of digital or graphical data.

The same reconstruction algorithm may be used in a second version including a set of additional parameters describing global spatial region parameters of the object either known a priori or determined a posteriori. This algorithm, in its two versions, can be used in all 5 multi-image systems, in which a set of different and differentiated images, from the same object spatial region, two-dimensional or three-dimensional - are recorded and analyzed. . Algorithm of the Compound Optical Process The optical process composed in at least one embodiment of the invention is a logical complement to the SRCDA algorithm. Indeed, the reconstruction obtained by the algorithm SRCDA can lead to the conclusion that an additional image would improve the performance of the measurement. The SRCDP microscopy platform makes it possible to acquire one or more complementary images chosen from a set of light distributions of the PSIT or PDOS methods.

An example is explained later. Measuring the position of a point by the PSIT method The PSIT method can be used as a technique for measuring the position of a nano-transmitter with high precision. Let us consider a nanoemitter positioned at the x, y position in Cartesian coordinates and p, 0 in polar coordinates. An illumination sequence consisting of a fundamental wave, and a pair of distributions called "half-moons", aligned along orthogonal axes is projected on the nanoemitter. The pretreatment procedure creates two images: A "top hat" image consisting of the sum of the three images in the sequence, A vortex image consisting of the sum of the two half-moon images. A first descriptor is the Cartesian position calculated using the centroid algorithm on the "top-hat" image. Referring to FIG. 10, the radial position p can be measured univocally by measuring a parameter, pa, equal to the normalized tangent arc by a factor x, of the intensity ratio between the normalized intensity emitted by the nano-transmitter illuminated by the vortex wave, IV, and the normalized intensity emitted by the nano-transmitter illuminated by the fundamental wave, IF. In fact: the normalized intensity emitted by the nano-transmitter illuminated by the fundamental wave varies from 1 to the center for the fundamental wave at 0 to the Airy ray, the normalized intensity emitted by the nano-transmitter illuminated by the The vortex wave varies from 0 in the center to at most 1 of the vortex to reach 0 for a value slightly greater than the radius of Airy. The tangent arc of the report is a monotonous function. The azimuth position can be measured by measuring the intensity ratio between the total intensity emitted by the nanoemitter illuminated by the first half-moon distribution, IH, and the total intensity emitted by the nanoemitter illuminated by the second half distribution. moon, Ive. The ratio between these two intensities is a square tangent geometric law: IVE = tank 16, E = tan - 0 111 (EQ.9) (EQ.9) Both measurements are redundant. This redundancy is a measure of qualifying the object observed as a single point and of separating this case from other objects potentially present on the sample. A direct application of the use of the PSIT method according to one embodiment of the invention for measuring the position of a nanoemitter with high precision is the integration of this measurement technique in a new technique of local stochastic optical reconstruction. . One of the limits of the applicability of stochastic techniques is the measurement process, which requires a large number of images and therefore a long measurement time and a high phototoxicity. The use of the PSIT technique according to at least one embodiment of the invention, which makes it possible to measure the position of a light emitter, at a resolution much greater than the Airy disk, at rates that can reach the micro or nanoseconds allows the extension of stochastic techniques to many new applications. The images resulting from the use of the PSIT method can also be processed using the generalized Hough method, allowing to recognize structured objects, line, circle or others, in an image. Conic diffraction could make it possible to apply a whole family of super resolution techniques or super localization, the localization of a single molecule. Referring now to Figure 8, which schematically illustrates an original technique we have named "DarkTracking". Referring to FIG. 8, 80 represents the initialization phase, i.e., the detection of the transmitter using a confocal scanner or any other known optical method. 81 represents the positioning phase of the vortex beam on the transmitter; Represents the case where the emitter moves with respect to the center of the vortex and is excited by a fraction of the beam creating a fluorescence which can be detected; 83 represents the repositioning of the emitter at the center of the vortex beam such that no fluorescence is created and detected (recorded position Xi, Yi) and 84 corresponds to a feedback loop in which an independent location system repositions the element followed at a corrected position. The light distributions used by this technique are vortices generated by conical diffraction. However, this technique can use other vortices or other distributions generated by conical diffraction. We assume that the molecule of interest has been labeled by one or more fluorophores that can be excited at X. The position of the molecule is first detected by a conventional confocal image at X. The position of the scanner is then adjusted to stimulate the sample so that the center of the vortex coincides exactly with the position of the transmitter. The fluorescence signal is detected by a very sensitive camera (eg EMCCD or sCMOS), or PMT, allowing a detection of the low amplitude signal of the transmitter thanks to a significant quantum efficiency. The localization process is based on the absence of a fluorescent signal when the emitter is exactly in the center of the vortex. The fact that the intensity gradient is large near the center of the vortex allows a precise location of the transmitter. If the transmitter moves slightly, the intensity it absorbs will no longer be zero, and it will emit a fluorescence signal whose position and intensity are deduced from the image. A feedback loop then makes it possible to refocus the vortex on the transmitter and to memorize the position of the transmitter. The feedback loop can be executed at the speed of execution of the camera (up to 1kHz) or the detector (several MHz), which makes it possible to track a molecule in real time and over a period of time. consequent time. Since we are always trying to minimize the fluorescence signal by minimizing the transmitter exciting signal, the localization can be conducted over a long period of time, bleaching being less likely to occur with such small doses of light. The location accuracy is highly dependent on the signal-to-noise ratio of the image, therefore the background noise (camera noise and autofluorescence signal) must be taken into account. Suppose now that the sample is labeled with two different fluorophores that can be excited at two different wavelengths, Xi and X2. We propagate two beams together with different topologies that depend on their wavelength. The first (X1) has a classical Gaussian form and makes it possible to obtain a global confocal image of the sample. The second (X2) is a vortex beam used for DarkTracking. By controlling the galvo mirror that scans the sample, the dark-tracking can be performed each time a line for the camera image is scanned, so that the transmitter's position is tracked to a frequency much higher than the frame rate. The main advantages of this technique, compared to other single molecule tracking technique, are the use of a single scanning system and the power sent to the monitored molecules is extremely low. In practice, the use, alignment and shaping of these two beams is not a trivial task if conventional beam shaping techniques, such as computer-generated holograms, are used. Spatial modulators or spiral wave plates, mainly because of their inherent chromaticity. Tapered diffraction can be used to simplify mounting. By using the adapted optics, a crystal can be adapted to generate a vortex at one wavelength, and a Gaussian beam at another wavelength along the same optical path. This makes it possible to use a simple optical path starting from a fiber that solves many practical problems. Dark-tracking technology can be easily used to investigate many biological issues in which conventional particle-tracking techniques are penalized by bleaching of particles. Dark tracking has been described above in the case of the use of a vortex. However, many variations, using one or more distributions, for example but not limited to half-moons or Stokes vectors, are any distributions having an intensity zero and a gradient - if possible the highest possible - depending of one of the spatial dimensions may allow the realization of Dark Tracking and are considered part of this invention. The capabilities of conic diffraction to create many different light distributions make it a tool of choice for the realization of Dark Tracking.

25 Recognizing and Measuring Two Points: A New Resolution Criterion Consider now two nanoemitters of the same intensity positioned symmetrically around the center at positions p, 0 and p, -0 in polar coordinates. We will use the system described in the previous paragraphs. The three descriptors will give the following results: - The centroid will measure the center of gravity of the light distribution, which will be the origin, - The descriptor p, will measure the common radial value of the two nanoemitters, - the descriptor 0, which in the case of the half-moons contains a degeneracy between 0 and -0, will measure the value 0.

As mentioned earlier, if the value of the descriptor p is not zero, we know that the case studied is not a point but two or more. In addition, descriptors p and 0 allow us to measure the characteristics of two points at a resolution much greater than that defined by the Rayleigh criterion. Moreover, using a compound process, it is possible to separate this case from the vast majority of cases of 3 points or more. An additional light distribution may be projected on the sample, a half moon 5 inclined at an angle O; the hypothesis of the presence of two points will be confirmed or invalidated according to the results of this image. Indeed, the measured energy will be zero only for two points, for a line or for a series of points aligned in the direction of the angle O. This measurement is not limited a priori. Of course, a practical resolution limit exists first, related to the signal quality, various fluctuations and imperfections. If we neglect the practical limits, the resolution limit is linked to the number of photons detected. STED Modalities Referring now to Figure 6b, which is a simplified schematic illustration of a modified LatSRCS optical module, achromatic or otherwise, 700, in accordance with one embodiment of the present invention. This module differs from the implementation of a LatSRCS optical module, 700, previously described and shown in FIG. 6a, by the addition of several elements before the module: lasers, 79a, 79b and 79c, optionally at lengths of different waves, optical fibers, 78a, 78b and 78c, from the lasers 79a, 79b and 79c, optionally, polarization control sub-modules, 77a, 77b and 77c, for statically or dynamically modifying the polarization of the lasers 79a, 79b and 79c, the polarization control sub-modules can be positioned before or after the fiber; However, in certain implementations, for example using PM fibers, polarization maintaining in English, the polarization control sub-modules 77a, 77b and 77c may not be necessary and the polarization of the light coming from the fiber is determined. by the relative position of the laser and the fiber, a "laser combine" (76a), combining two or more laser sources optionally through optical fibers, 78a, 78b and 78c, into a common output fiber, 76b, an optical element, 75, for transforming the light from the fiber into collimated light that can be used by the rest of the LatSRCS module, 700, similarly to that described above. Numerous variations of this device, known to those skilled in the art, can be added to this scheme and are claimed in this invention: for example, the two optical elements 31 and 75 can be integrated into a single element, or even removed if the output parameters of the common fiber are adequate. Similarly, the common fiber, 76b, may not be necessary for some implementations. In addition, the introduction, through a common optical fiber, or directly into light propagating in space, of two or more wavelengths having different polarizations, makes it possible to create a method and / or a device capable of performing the RESOLFT or STED techniques in all their different modalities. In this method and / or device, the excitation and depletion waves propagate along a common path and the LatSRCS module, 700, can be totally achromatic, or not in a simplified version, as previously described. In the simplest variant of this method, the excitation and depletion waves propagate along two orthogonal linear polarizations and a quarter-wave plate - optionally achromatic - is positioned at the input of the system 15 to transform these polarizations into orthogonal circular polarizations to obtain, for one a fundamental wave and for the other a vortex. Reference is now made to Figure 6b again. The polarization control sub-modules, 77a, 77b and 77c, may be actuated to create, simultaneously, sequences of light distributions, identical or different, for the excitation path 20 and the depletion path. In addition, the intensity of the lasers can be modulated, creating a complex timing diagram of the intensity of each laser, the excitation light distribution sequence and the sequence of light depletion distributions. In the preferred implementation, using a confocal microscope and an original or modified, achromatic or non-achromatic LatSRCS module, one projects sequentially: In the first place, an Airy distribution or a fundamental excitation simultaneously with a depletion vortex. The resulting emission image will be called the positive image. In the second step, an excitation vortex simultaneously with a depletion vortex. The resulting emission image will be called the negative image.

In a variant of the first locus, only an Airy distribution or a fundamental excitation is projected. The difference image, consisting of the weighted subtraction of these two images. This difference image, if we choose the right parameters, will have a size - in terms of PSF - finer than a conventional STED, while requiring only a relatively low depletion intensity. Indeed, the purpose of vortex depletion will no longer be to reduce the size of an Airy spot, thus requiring significant energy, as in conventional STED or RESOLFT, but to reduce the excess energy present in the excitation vortex with respect to the Airy distribution or the fundamental one. In addition, the depletion of the Airy or of the fundamental will couple with the subtraction of the negative image, created by the excitation vortex, which is equivalent to the mathematical subtraction of the two illuminations. Reducing the size of the resulting PSF will be the combination of these two effects.

In addition, this preferred embodiment may in some cases overcome the need to trigger excitation as necessary in the "Gated STED". Indeed, the photons arrived before the complete application of the depletion, can be taken into account by an adequate choice of parameters, without requiring the addition of a complex and binding system. Finally, the need to modulate the STED, "ModSted" can also be avoided because the emission photons emitted by the depletion vortex do not differ substantially from those emitted by the excitation vortex and can also be compensated. In a second embodiment, using a confocal microscope and an original or modified LatSRCS module, an excitation beam sequence, a depletion beam sequence, are simultaneously projected, the two beam sequences being able to differ in their polarization, creating light distributions of different topologies. This device makes it possible to produce a sequence of light distributions of smaller sizes than would have been obtained without the depletion beam. The algorithmic SRCDA, will be used in this implementation to determine the spatial distribution or the position of point emitters.

In another embodiment, using a confocal microscope and an original or modified LatSRCS module, an excitation beam, in the form of an Airy and a depletion beam in the form of a vortex, are simultaneously projected. beams differing in their polarization. This device makes it possible, without dynamic elements, to produce a totally achromatic STED device.

In another embodiment, using a confocal microscope, an original or modified LatSRCS module, and a LongSRCS module, an excitation beam sequence, a depletion beam sequence, the two beam sequences can be simultaneously projected. differ by their polarization, creating light distributions of different topologies. The LatSRCS module makes it possible to carry out a sequence of light distributions of smaller sizes than would have been obtained without the depletion beam. The LongSRCS module implements the PDOS method so as to separate on different detectors the collimated light, emerging from nanoemiters positioned in the focal plane of the objective, of the emerging non-collimated light of nanoemitters lying below or beyond the focal plane. The PDOS method, in this implementation, makes it possible to acquire essentially longitudinal information, that is to say the longitudinal position of each of the nanoemitters, complementary to the lateral information obtained using the original or modified LatSRCS module. .

The algorithmic SRCDA, will be used in this implementation to determine the spatial distribution or the position of point emitters. In another embodiment using one of the previously described implementations and using a biaxial crystal to create the beam shaping, a dynamic polarization element is used before or after the biaxial crystal to correct the dynamic movement of the pupil, which in some cases can be created during optical scanning of the confocal microscope. This pupil movement effect being in some implementations STED technologists one of the limits of performance, without the need for an additional scanning system. [35] In another embodiment using a confocal microscope, two or more light distributions located at different wavelengths are projected using an original or modified LatSRCS module. The first light distribution makes it possible to make the scene parsimonious, that is to say to isolate emitters by means of a physical effect, which dilutes the density of the emitters capable of emitting fluorescence, so to create regions in which the assumption of parsimony is valid, that is to say the presence of an isolated transmitter or a small number of transmitters. The physical effects that will achieve this parsimony will be the same or will be derived effects, effects used to create parsimony for single transmitter location microscopy techniques. Such techniques include, for example, PALM STORM, DSTORM, FPALM and the like. The second light distribution at another wavelength will create a fluorescence whose intensity will be variable over time. This second light distribution will use one of the PSIT techniques, either a discrete distribution sequence or a sequence of continuous distributions. In the case of discrete distributions, the light will be detected either by a matrix detector or by a single detector. In the case of continuous distribution, although it is also possible to use a matrix detector, the most likely implementation will be the use of a single detector; in this case the lateral position information xy and potentially the longitudinal distribution information z can be obtained by intensity ratios. One of the most interesting cases is the case of harmonic distributions in which the electro-optical cells are actuated by a sinusoidal voltage. In this case, the xy position can be retrieved using a measurement of the temporal harmonics of the signal measured by the detector, which may be a single detector, which indirectly contain the lateral position information.

Numerous other implementations of this general method will be clear to those skilled in the art, for example: 3029633 64 Some of the distributions created by the LatSRCS module, 700, are super oscillations, In FIG. 7b, the cases under FIGS. and 7b11 are superoscillations. It consists of a naturally small spot, surrounded by a wide and intense ring. By projecting a depletion ring on this super-oscillation, it will be possible to prevent, at a low energy cost, the fluorophores contained in the ring from emitting and the size of the spot will be the size of the central spot of the super-oscillation. successively or simultaneously two or more depletion light distributions may advantageously replace the depletion vortex in all RELSOFT or STED modes. One of the possible effects is the improvement of the shape of the light distribution of depletion taking into account the vector effects. It is also possible to create a number larger than 2 of distributions called "half-moons", to explore different states of polarization.

Control Module Referring to Figure 11 and Figure 5, in a preferred embodiment of this invention, the various control elements integrated in the SRCDP platform 500 will be described: Control Module, 1100, at using the systemic control procedure, 1101, 20 control and modify the optical parameters of the platform SRCDP, 500, the electronic parameters of the enhanced detection set, 65, and the mathematical parameters of the algorithmic procedures SRCDA, 900, for optimize emerging information, according to criteria defined by the system or by the user. The control is carried out by varying the control systems 1102, 1103 and 1104, the various elements of the platform, 600, 800 and 900. The control system, 1100, will also use, if available external information, 1105 , relayed by the computer support. Note: 1105 is not present in Figure 11. It is to be understood that the invention is not limited in its application to the details set forth in the description contained herein or illustrated in the drawings. The invention is capable of other embodiments and of being practiced and carried out in various ways. Those skilled in the art will readily appreciate that various modifications and changes may be applied to the embodiments of the invention as described. The present embodiments of the invention can be integrated on a confocal fluorescence microscope. The superresolution system according to embodiments of the invention consists of a new measurement mode, in addition to or in replacement of the existing modes of microscopy. However, the superresolution system according to embodiments of the invention can just as easily be integrated on the other microscopy platforms. These microscopy platforms, described for example, include, but are not limited to: wide field microscopes, wide field microscopes, darkfield microscopes, dark field microscopes, polarization microscopes, phase, differential interference interference microscopes, stereoscopic microscopes, Raman microscopes, dedicated spot microscopes, such as "live cell imaging", "cell sorting", "cell motility" or any other microscopy instrument optical. In another embodiment, the microscope platform that has been described is coupled to an electron microscopy system (CLEM-Correlative Light Electron Microscopy), or any other similar system such as Transmission Electron Microscopy (TEM), or EBM (Electron Beam Microscopy), or SEM (Scanning Electron Microscopy) In another embodiment of the invention, the microscope platform is a complete SRCDP platform, and includes a LongSRCS module, implementing the PDOS method and using algorithmic SRCDA. In another embodiment of the invention, the microscope platform is a partial SRCDP platform, and uses the algorithm SRCDA. In another embodiment of the invention, the microscope platform is a partial SRCDP platform, and uses the control module. In another embodiment of the invention, the microscope platform further includes a LongSRCS module, implementing the PDOS method. As for a more in-depth discussion of how to use and exploit the invention, it should come out of the description above. Therefore, no discussion of the form of use and operation will be described. In this regard, before explaining at least one embodiment of the invention in detail, it is understood that the invention is not limited in its application to the details of the construction and the regime of the components set forth in the present invention. following description or illustrated in the drawing. The invention is capable of other embodiments and can be practiced and performed in a variety of ways. In addition, it is understood that the phraseology and terminology used in this document are for the purpose of the description and should not be construed as limiting. The references cited here teach many principles that are applicable to the present invention. Therefore, all of the contents of these publications are hereby incorporated by reference, where appropriate for teaching additional or replacement details, features and / or technical information. The advantageous use of optical fibers is the transmission of the fundamental mode, the TEM00 mode and only it. However, certain configurations of optical fibers, mainly but not exclusively based on fibers called "Photonic Crystal 10 Fiber" (in English) allows the simultaneous transmission or not, more complex modes, including vortex modes. It would therefore be possible to deport the optical distributions created by the conical refraction using optical fibers, allowing a major simplification of the optical system. In addition, some dual-core photonic crystal fibers (bers), [16], allow an interaction between two modes, one of which may be a vortex, and provide an additional physical mechanism for creating functions. Many super-resolution techniques are based on the measurement of point sources, smaller than a fraction of a wavelength, and the superresolution techniques according to described embodiments allow the measurement of point sources, but also of structured objects, for example and mainly line segments, circles or even continuous objects.In Biology, this extension will allow the measurement of important biological entities, such as filaments, neurons and some microtubules. the descriptions of the embodiments, to simplify the understanding of the invention, show the applications in Microscopy, more specifically in Biology, and even more specifically in Fluorescence Biology, the applications can be extended to the general applications of Microscopy and to the whole field of Vision, including the artificial Vision. Embodiments of the invention may be applied, by choosing a different optical system, to many medical applications, for example but not limited to ophthalmological observation. This field of application corresponds to the measurement of biological or medical objects of micron resolution, the resolution being between 1 and 10 pin. In addition, embodiments of the invention may be applied, as explained later, through an optical fiber. This allows many additional applications, for example but not limited to gastric and gastroenterological observation, and observation of the colon and urinary tract.

It is understood that the invention is not limited in its application to the details set forth in the description contained herein or illustrated in the drawings. The invention is capable of other embodiments and to be practiced and performed in a variety of ways. Those skilled in the art will readily understand that various modifications and changes may be applied to the embodiments of the invention as previously described without departing from its scope as defined in and by the appended claims. References 1. L. Schermelleh, R. Heintzniann, and H. Leonhardt, "A guide to super-resolution fluorescence microscopy," The Journal of Biology 190, 165-175 (2010). 2. M. V. Berry, "Conical Diffraction Asymptotics: Fine Structure of Poggendorff Rings and Axial Spike," Journal Of Optics A-Pure And Applied Optics 6, 289-300 (2004). 4. J. F. Nye and M. V. Berry, "Dislocations in Wave Trains," Proceedings of the Royal Society of London. 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Claims (2)

REVENDICATIONS1. An optical measuring method for determining the spatial distribution or the location of retransmitter sources on a sample, the sample comprising at least one retransmitting 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 retransmitter 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 wavelength or activating said at least one retransmitting source, with the aid of a polarization sub-module, for each laser, a polarization state co controlled, the projection on the sample, by means of an achromatic projection optical apparatus, for each laser of a compact light distribution, propagating according to the same optical path for all the lasers, the detection of the light re-emitted by said at least one re-emitting source of the sample; generating at least one image from the detected light; and algorithmically analyzing the images to obtain spatial distribution information or location of said at least one re-transmitter source. A method according to the preceding claim wherein the compact light distribution of the first excitation laser is of different topological family from that of the second depletion or activation laser. A method according to claim 2, wherein said at least two distributions compact luminos 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. 4. A method according to claim 2 or 3 wherein the projection of light distributions of different topological families is effected by conical diffraction or uniaxial crystal assembly. 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 one any one of the preceding claims, comprising controlling the lasers to jointly create a sequence of excitation distributions and a sequence of depletion or activation distributions, the two sequences being synchronized. 7. A measurement device for determining the spatial distribution or locating retransmitting 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 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: wavelengths, the wavelength of the first laser being tuned to the excitation wavelength of the at least one re-emitting source and the wavelength of the second laser being tuned to the wavelength for depleting or activating said at least one retransmitting source a polarization sub-module, for producing for each laser, a different polarization state, - an achromatic projection module making it possible to create for each laser a compact light distribution , propagating along the same optical path 25 for all the lasers, - a detection module able to detect 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 an algorithmic analysis module capable of analyzing images in order to obtain location information for said at least one re-emitting source. 8. A device according to the preceding claim wherein the compact light distribution of the excitation laser is of topological family different from that of the depletion or activation laser. 9. A device according to claim 7 or 8 comprising a fiber photonics. An optical measuring method for determining the spatial distribution or location of retransmitter sources on a sample, the sample having at least one re-emitting source, said at least one re-emitting source re-emitting light in dependence on the light projected onto the sample sample, according to a given 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: use of two lasers, the wavelength of the first laser being tuned to the excitation wavelength of the at least one re-emitting source and the wavelength of the second laser being tuned over the length of wave of depletion or activation of said at least one re-emitting source projection on the sample, by means of a projection optical apparatus ac hromatic for the first laser and the second laser light distributions, detecting the light re-emitted by said at least one re-emitting source of the sample; and generating, at least one image, from the detected light. 11. A method according to claim 10, wherein the projection comprises for the first laser a compact light distribution, and for the second laser of two compact distributions one of them having an intensity zero propagating axially according to a spiral movement. 12. An optical measuring method according to claim 11 wherein the two depletion distributions are derived from two independent lasers at the same wavelength or wavelength. An optical measuring method according to claim 11, wherein the projection on the sample is carried out by means of an achromatic optical projection apparatus comprising an optical element having the function of segmenting an optical beam into a plurality of beams. independent, the projection comprising for the two lasers a matrix of compact and collocalised light distributions, 14. An optical measuring method according to any one of claims 13 to 13 furthermore the algorithmic analysis of the images to obtain information spatial distribution or location of said at least one re-emitting source. 15. An optical measuring device for determining the spatial distribution or the location of retransmitter sources on a sample, the sample comprising at least one retransmitting source, said at least one retransmitting source retransmitting light as a function of the light projected onto the sample. sample, according to a determined law, by a first light source comprising a first laser, and said at least one retransmitting 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 the at least one re-emitting source and the wavelength of the second laser being tuned to the wavelength of depletion or activation of said at least one re-emitting source an achromatic projection module for creating for the first laser and the second laser of the light distributions, a detection module for detecting 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. 16. A device according to claim 15, in which the projection module is able to create a compact light distribution for the first laser, and for the second laser two compact distributions, one of them having an intensity zero. propagating axially in a spiral motion. 17. A device according to claim 16 wherein the two depletion-generating distributions are derived from two independent lasers at the same wavelength or near-wavelength. 30