Classifications

• • G—PHYSICS
  • G02—OPTICS
  • G02B—OPTICAL ELEMENTS, SYSTEMS OR APPARATUS
  • G02B21/00—Microscopes
• • G—PHYSICS
  • G01—MEASURING; TESTING
  • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
  • G01N21/00—Investigating or analysing materials by the use of optical means, i.e. using sub-millimetre waves, infrared, visible or ultraviolet light
  • G01N21/62—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light
  • G01N21/63—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light optically excited
  • G01N21/64—Fluorescence; Phosphorescence
  • G01N21/645—Specially adapted constructive features of fluorimeters
  • G01N21/6456—Spatial resolved fluorescence measurements; Imaging
• • G—PHYSICS
  • G01—MEASURING; TESTING
  • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
  • G01N21/00—Investigating or analysing materials by the use of optical means, i.e. using sub-millimetre waves, infrared, visible or ultraviolet light
  • G01N21/62—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light
  • G01N21/63—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light optically excited
  • G01N21/636—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light optically excited using an arrangement of pump beam and probe beam; using the measurement of optical non-linear properties
• • G—PHYSICS
  • G01—MEASURING; TESTING
  • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
  • G01N21/00—Investigating or analysing materials by the use of optical means, i.e. using sub-millimetre waves, infrared, visible or ultraviolet light
  • G01N21/62—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light
  • G01N21/63—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light optically excited
  • G01N21/64—Fluorescence; Phosphorescence
  • G01N21/645—Specially adapted constructive features of fluorimeters
  • G01N21/6456—Spatial resolved fluorescence measurements; Imaging
  • G01N21/6458—Fluorescence microscopy
• • G—PHYSICS
  • G02—OPTICS
  • G02B—OPTICAL ELEMENTS, SYSTEMS OR APPARATUS
  • G02B21/00—Microscopes
  • G02B21/0004—Microscopes specially adapted for specific applications
  • G02B21/002—Scanning microscopes
  • G02B21/0024—Confocal scanning microscopes (CSOMs) or confocal "macroscopes"; Accessories which are not restricted to use with CSOMs, e.g. sample holders
  • G02B21/0052—Optical details of the image generation
  • G02B21/0056—Optical details of the image generation based on optical coherence, e.g. phase-contrast arrangements, interference arrangements
• • G—PHYSICS
  • G02—OPTICS
  • G02B—OPTICAL ELEMENTS, SYSTEMS OR APPARATUS
  • G02B21/00—Microscopes
  • G02B21/0004—Microscopes specially adapted for specific applications
  • G02B21/002—Scanning microscopes
  • G02B21/0024—Confocal scanning microscopes (CSOMs) or confocal "macroscopes"; Accessories which are not restricted to use with CSOMs, e.g. sample holders
  • G02B21/0052—Optical details of the image generation
  • G02B21/0072—Optical details of the image generation details concerning resolution or correction, including general design of CSOM objectives
• • G—PHYSICS
  • G02—OPTICS
  • G02B—OPTICAL ELEMENTS, SYSTEMS OR APPARATUS
  • G02B21/00—Microscopes
  • G02B21/0004—Microscopes specially adapted for specific applications
  • G02B21/002—Scanning microscopes
  • G02B21/0024—Confocal scanning microscopes (CSOMs) or confocal "macroscopes"; Accessories which are not restricted to use with CSOMs, e.g. sample holders
  • G02B21/0052—Optical details of the image generation
  • G02B21/0076—Optical details of the image generation arrangements using fluorescence or luminescence
• • G—PHYSICS
  • G02—OPTICS
  • G02B—OPTICAL ELEMENTS, SYSTEMS OR APPARATUS
  • G02B21/00—Microscopes
  • G02B21/0004—Microscopes specially adapted for specific applications
  • G02B21/002—Scanning microscopes
  • G02B21/0024—Confocal scanning microscopes (CSOMs) or confocal "macroscopes"; Accessories which are not restricted to use with CSOMs, e.g. sample holders
  • G02B21/008—Details of detection or image processing, including general computer control
• • G—PHYSICS
  • G01—MEASURING; TESTING
  • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
  • G01N21/00—Investigating or analysing materials by the use of optical means, i.e. using sub-millimetre waves, infrared, visible or ultraviolet light
  • G01N21/62—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light
  • G01N21/63—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light optically excited
  • G01N21/64—Fluorescence; Phosphorescence
  • G01N21/645—Specially adapted constructive features of fluorimeters
  • G01N2021/6484—Optical fibres

Definitions

• the present invention relates to a method and an optical measuring device. It finds applications in all fields of imaging, in particular but not limited to the field of microscopy, including but not limited to the fields of Biology, Medicine, Pharmacy, Semiconductors, Study of materials, metrology, control, measurement and observation and all information acquisition processes from optical observations, in the macroscopic or microscopic domain.
• An optical microscope is an instrument commonly used to view, analyze, or measure objects that are too small to view with the naked eye.
• Figure. 1 represents an illustration of the paradigm of Microscopy, 100.
• Optical microscopy consists of the illumination, by a light source, not shown, using a Microscope, 10, of a biological or non-biological sample, 11, and the measurement as a function of time, at using a visual observation or a detection module, 12, of the light emitted, re-emitted, scattered or reflected or transmitted by the sample.
• 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 or a DNA fragment.
• the sample may be, for example, a semiconductor element
• Microscopy is segmented into different modalities with different characteristics and purposes. Many descriptions of the different modalities, their
• Microscopy can be segmented in different ways: one of them is to distinguish the microscopic modalities devolved to the visualization of minute point sources from those devolved to the measurement of 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
• 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.
• 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.
• 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 with details above the 200-250 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 200-250 nm 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 details smaller than the Rayleigh criterion, the order of 200 - 250 nm.
• FIG. 2 is a simplified representation of a confocal fluorescence microscope of the prior art 200.
• FIG. 2 A confocal fluorescence microscope, FIG. 2, is an optical instrument. Its main hardware components are shown in the figure. 2. They include:
• a treatment 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 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 scanning mechanism, spatial or angular, not shown to modify the position of the point source with respect to the object to be measured.
• 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 microscope objective, 22, focuses the light created by the source in the focal plane of the objective, 24, into a light distribution of reduced size, the light distribution considered optimal consisting of an Airy disc.
• the microscope objective, 22, also makes it possible to collect in return the fluorescent light emitted by the fluorophores.
• the system can be decanned, ie 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 imaging plane
• the detector assembly, 23 detects the overall fluorescent intensity in the illuminated volume, and transforms it into a digital signal.
• the detector assembly consists of a single element detector, such as a PMT or SPAD.
• the detector assembly consists of a matrix of detection elements, such as a CCD, an EMCCD, a CMOS or a SPAD matrix.
• All of the components mounted from the light source to the dichroic filter are the light path, 201. All the components mounted from the dichroic filter to the detector assembly are the detection channel , 202.
• Fluorescence microscopes are available from several manufacturers, such as, for example, Nikon, Zeiss, Leica or Olympus. Fluorescence microscopes can be either standard microscopes suitable for fluorescence or specific microscopes optimized for fluorescence. Modern microscopes are versatile instruments capable of operating in many different modalities, including, but not limited to, fluorescence modalities, using the same optomechanical platform and most components. Most fluorescence microscopes are developed as an open platform, capable of performing several additional features with minimal modifications. Other fluorescence microscopes are dedicated instruments, customized to a specific task, such as medical or pharmaceutical diagnosis.
• New superresolution techniques provide information beyond the resolution limit.
• the main problem of all existing superresolution techniques is the performance envelope limit, expressed in terms of resolution, lateral and longitudinal, velocity, light intensity required and phototoxicity in the biological object, and therefore ability to measure different biological objects. This point was also emphasized by Eric Betzig, during his masterly presentation at the ceremony of reception of the Nobel Prize of Chemistry 2014.
• 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.
• a first aspect of the invention relates to an optical measuring device for determining the spatial distribution or the location of re-emitting sources on a sample, the sample comprising at least one re-emitting source, excited by light and re-emitting light according to a law. determined according to the light projected onto the sample, the device comprising
• an achromatic projection module containing a laser, whose wavelength is tuned to the excitation wavelength of said at least one re-emitting source, to create either a compact light distribution or a sequence of the compact light distributions of different topology
• a detection module for detecting the light re-emitted by said at least one re-emitting source of the sample for the or each of the different compact light distributions of topology and for each of the scanning points of the sample;
• an image acquisition module for acquiring for each scanning point, an image or a sequence of images, for the image sequence, each image corresponding to one of the compact light distributions of different topologies
• an algorithm module in which the formulation of the reconstruction of the sample and its spatial and / or temporal and / or spectral properties is considered as a Bayesian inverse problem and leads to the definition of a posterior distribution.
• a posteriori law can combine, thanks to the Bayesian law, the probabilistic formulation of a noise model, as well as possible aprioris on a distribution of light created in the sample by projection.
• the estimation of the posterior mean is carried out using a Monte Carlo Markov Chain (MCMC) algorithm.
• MCMC Monte Carlo Markov Chain
• 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, excited by light and re-emitting light according to a law. determined according to the light projected onto the sample, the device comprising an achromatic projection module, containing a laser, whose wavelength is tuned to the excitation wavelength of said at least one re-emitting source, to create either a compact light distribution or a sequence of the compact light distributions of different topology,
• a scanning module for optically scanning the sample, integrated or not in the device
• a detection module for detecting light re-emitted by said at least one re-emitting source of the sample for the or each of the different compact light distributions of topology and for each of the scanning points of the object;
• an image acquisition module for acquiring for each scanning point, an image or a sequence of images, for the image sequence, each image corresponding to one of the compact light distributions of different topologies
• the MAP algorithm furthermore contains a frequency-band limitation constraint
• the MAP algorithm uses a Nesterov accelerated numerical scheme.
• the redundancy in the frequency information is used, due to the different frequency characteristics of the different distributions projected on the sample, to compensate and greatly reduce the impact of missing points or sweep irregularities.
• the MAP algorithm is adapted to solve an inverse problem involving a sum of a small number of terms, such as a low-frequency component and a more parsimonious component.
• the MAP algorithm is adapted to impose a non-local redundancy constraint on the solution, for example by calculating weights on the images or on the different digital masks and the non-local similarity tree. being applied to the solution as a regularization.
• a mask of variable size is used in the plane of the detector to obtain images having either different axial characteristics or different or optimized stray light rejection capabilities, globally or locally.
• a computer program configured to implement the embodiments described above.
• 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, excited by light and re-emitting light according to a law. determined according to the light projected onto the sample, the method comprising
• achromatic projection by a laser, whose wavelength is tuned to the excitation wavelength of said at least one re-emitting source, to create either a compact light distribution or a sequence of different compact light distributions of topology ,
• a posteriori law can combine, thanks to the Bayesian law, the probabilistic formulation of a noise model, as well as possible aprioris on a distribution of light created in the sample by projection.
• the algorithm comprises
• the estimation of the posterior mean is carried out using a Monte Carlo Markov Chain (MCMC) algorithm.
• MCMC Monte Carlo Markov Chain
• 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, excited by light and re-emitting light according to a law. determined according to the light projected onto the sample, the method comprising achromatic projection, by a laser, whose wavelength is tuned to the excitation wavelength of said at least one re-emitting source, to create either a compact light distribution or a sequence of different compact light distributions of topology ,
• the MAP algorithm furthermore contains a frequency-band limitation constraint
• the MAP algorithm uses an accelerated numerical scheme of Nesterov type
• the redundancy in the frequency information is used, due to the different frequency characteristics of the different distributions projected on the sample, to compensate and greatly reduce the impact of missing points or sweep irregularities.
• the MAP algorithm is adapted to solve an inverse problem involving a sum of a small number of terms, such as a low-frequency component and a more parsimonious component.
• the MAP algorithm is adapted to impose a non-local redundancy constraint on the solution, for example by calculating weights on the images or on the different digital masks and the non-local similarity tree. being applied to the solution as a regularization.
• a mask of variable size is used in the plane of the detector to obtain images having either different axial characteristics or different or optimized stray light rejection capabilities, globally or locally.
• a computer program configured to implement the embodiments described above.
• an optical measurement method is used to determine 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, whose length waveform is tuned to the excitation wavelength of the re-emitting source and the re-emitting source may be depleted or activated by the action of one or more light source (s), including at least one second a laser whose wavelength is tuned to the wavelength of depletion or activation of said re-emitting source, the method comprising the two compact light distributions propagating along the same optical path for all the lasers,
• the compact light distribution of the first excitation laser being of regular topological family, ideally a Gaussian distribution or an Airy spot,
• the compact light distribution of the depletion or activation laser consisting of a superposition of a singular distribution, of the vortex type, on a first polarization, linear or circular, and a so-called distribution of black sphere or "top-hat” on the polarization orthogonal to the first polarization,
• said compact light distributions being created by a cascade of at least two conical diffraction crystals, or an assembly of uniaxial crystals, optionally separated by a chromatic polarization control element or not, dynamic or static,
• the polarization control element described is an optical element consisting of an assembly of one or two quarter-wave achromatic waves and a chromatic wave plate all being designed so that the optical element created, between the two conical crystals or between two uniaxial crystals, a rotation difference of the polarization between the excitation beam and the depletion beam close to 180 degrees and not differing by more than 30 degrees of this value
• the polarization control element is an optical element whose material has an optical activity property and the thickness of the optical element is selected so that that the natural dispersion of the optical activity of the material created between the two conical crystals or between two uniaxial crystals for a rotation difference of the polarization between the excitation beam and the depletion beam close to 180 degrees and not differing more than 30 degrees of this value
• no polarization control element is used but the two conical crystals are of different material and the natural dispersion of these two materials compensates for conical diffraction at approximately the same time. excitation wavelength and not at the wavelength of depletion.
• an optical device is used to determine 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 in a function of the light projected on the sample, according to a determined law, by a first light source comprising a first laser, the wavelength of which is tuned to the excitation wavelength of the re-emitting source and the re-emitting source capable of being depleted or activated by the action of one or more light source (s), comprising at least one second laser, the wavelength of which is tuned to the depletion or activation wavelength said re-emitting source, the method comprising the two compact light distributions propagating along the same optical path for all the lasers,
• the compact light distribution of the first excitation laser being of regular topological family, ideally a Gaussian distribution or an Airy spot,
• the compact light distribution of the depletion or activation laser consisting of a superposition of a singular distribution, of the vortex type, on a first polarization, linear or circular, and a so-called distribution of black sphere or "top-hat” on the polarization orthogonal to the first polarization,
• said compact light distributions being created by a cascade of at least two conical diffraction crystals, or an assembly of uniaxial crystals, optionally separated by a chromatic polarization control element or not, dynamic or static,
• the polarization control element described is an optical element consisting of an assembly of one or two quarter-wave achromatic waves and a chromatic wave plate all being designed so that the optical element created, between the two conical crystals or between two uniaxial crystals, a rotation difference of the polarization between the excitation beam and the depletion beam close to 180 degrees and not differing by more than 30 degrees of this value
• the polarization control element is an optical element whose material has an optical activity property and the thickness of the optical element is selected so that that the natural dispersion of the optical activity of the material created between the two conical crystals or between two uniaxial crystals for a rotation difference of the polarization between the excitation beam and the depletion beam close to 180 degrees and not differing more than 30 degrees of this value
• Another implementation of this invention describes an optical method for locally evaluating spherical aberration in each sample location, or each location of the object being imaged, using conical diffraction and its phase effects and polarization.
• the sample is illuminated with a non-uniform light distribution, which may be, for example but not limited to, a black sphere (or top hat) so that the 3D distribution has two lobes of the same intensity below and above above the focal plane. In the presence of spherical aberration in the system, these two lobes are not of the same intensity.
• By then analyzing the intensity ratio of the two images we can deduce the amount of spherical aberration of the system.
• Another implementation of this invention describes an optical device for locally evaluating spherical aberration in each sample location, or each location of the object being imaged, using conical diffraction and its phase effects and polarization.
• the sample is illuminated with a non-uniform light distribution, which may be, for example but not limited to, a black sphere (or top hat) so that the 3D distribution has two lobes of the same intensity below and above above the focal plane. In the presence of spherical aberration in the system, these two lobes are not of the same intensity.
• By then analyzing the intensity ratio of the two images we can deduce the amount of spherical aberration of the system.
• Another implementation of this invention describes an optical method for locally evaluating spherical aberration in each sample location, or each location of the object being imaged, using conical diffraction and its phase effects and polarization.
• the shaping of a light beam by conical diffraction through a cascade of crystals makes it possible to obtain an intensity distribution directly connected to the spherical aberration.
• This distribution can be obtained, for example but without limitation, between crossed linear polarizers and two biaxial crystals whose optical axes are aligned. A half-wave plate is inserted between the two crystals. Due to its characteristic shape, this distribution will be called "four-leaf clover" or "four-leaf-clover" in English.
• this distribution consists of four perfectly equal lobes and all in the same focus.
• Spherical aberration induced by an optical system breaks the symmetry of this distribution both in focus and intensity distribution in the four lobes.
• a fine measure of the spherical aberration value is possible by a lobe focus offset estimator and intensity ratios at different focus planes.
• Another implementation of this invention describes an optical device for locally evaluating spherical aberration in each sample location, or each location of the object being imaged, using conical diffraction and its phase effects and polarization.
• the shaping of a light beam by conical diffraction through a cascade of crystals makes it possible to obtain an intensity distribution directly connected to the spherical aberration.
• This distribution can be obtained, for example but without limitation, between crossed linear polarizers and two biaxial crystals whose optical axes are aligned. A half-wave plate is inserted between the two crystals. Due to its characteristic shape, this distribution will be called "four-leaf clover" or "four-leaf-clover" in English.
• this distribution consists of four perfectly equal lobes and all in the same focus.
• Spherical aberration induced by an optical system breaks the symmetry of this distribution both in focus and intensity distribution in the four lobes.
• a fine measure of the spherical aberration value is possible by a lobe focus offset estimator and intensity ratios at different focus planes.
• Another implementation of this invention describes an optical method for real-time calibration of a point tracking beam scanning system generated by a near infrared laser diode.
• An optical method is carried out making it possible to follow in real time on a camera a scanned point by a beam scanning system (galvanometric mirrors, bidirectional piezoelectric mirror or any other system).
• a beam scanning system galvanometric mirrors, bidirectional piezoelectric mirror or any other system.
• Another implementation of this invention describes an optical device for real-time calibration of a point tracking beam scanning system generated by a near infrared laser diode.
• An optical method is carried out making it possible to follow in real time on a camera a scanned point by a beam scanning system (galvanometric mirrors, bidirectional piezoelectric mirror or any other system).
• a beam scanning system galvanometric mirrors, bidirectional piezoelectric mirror or any other system.
• Another implementation of this invention describes an optical method, prism
• Modified Wollaston for duplicating a set of light distributions without changing the relationships between them, which can be used in the methods described in this invention or in the STED methods described in this invention or other standard STED methods.
• By using several Wollaston prisms in cascade it is thus possible to separate an incident beam into a large number of emerging beams.
• the same effect can be achieved by modifying the Wollaston prism, to create the composite Wollaston prism, by adding uniaxial crystal pieces whose index and orientation of the birefringence are appropriately selected. It is thus possible to construct a prism of a single block of uniaxial crystal which can separate an incident beam into 2 n emergent beams (e.g.
• Another implementation of this invention describes an optical device, modified Wollaston prism, to duplicate a set of light distributions without changing the relationships between them, which can be used in the context of the devices described in this invention or as part of the STED devices. described in this invention or other standard STED devices.
• By using several Wollaston prisms in cascade it is thus possible to separate an incident beam into a large number of emerging beams.
• the same effect can be achieved by modifying the Wollaston prism, to create the composite Wollaston prism, by adding uniaxial crystal pieces whose index and orientation of the birefringence are appropriately selected.
• Another implementation of this invention describes an optical method, and an algorithmic procedure using a Poisson noise property to generate, from a realization of a Poisson VAR of parameter I (the mean), two independent realizations of a Poisson VAR of parameter 1/2, thanks to a posteriori treatment. Thanks to a random or pseudo-random number generation process, a binomial law is simulated.
• the analysis or reconstruction of the two measurements thus generated called the Split Photon method, provides two independent results in the sense of the probabilities, and the differences between them. two results illustrate the dependence of the reconstruction algorithm on the measurement noise.
• Another implementation of this invention describes an optical device, and an algorithmic device using a Poisson noise property to generate, from a realization of a Poisson VAR of parameter I (the average), two independent realizations of a Poisson VAR of parameter 1/2, thanks to a posteriori treatment. Thanks to a random or pseudo-random number generation process, a binomial law is simulated.
• the analysis or reconstruction of the two measurements thus generated called the Split Photon method, provides two independent results in the sense of the probabilities, and the differences between them. two results illustrate the dependence of the reconstruction algorithm on the measurement noise.
• Another implementation of this invention describes an optical method, and an algorithmic method using the ICE algorithm which replaces the calculation of the expectation of posterior law performed by LSE by the iteration of the explicit calculation of the mean of the law.
• a posteriori of a pixel conditionally to its neighbors.
• Another implementation of this invention describes an optical device, and an algorithmic device using the ICE algorithm that replaces the calculation of the expectation of the posterior law performed by LSE by the iteration of the explicit calculation of the mean of the law.
• a posteriori of a pixel conditionally to its neighbors.
• Another implementation of this invention describes an optical method, and an algorithmic method for measuring the proportion of signal that belongs well to the region of interest (pinhole), for each image
• This measurement called Pinhole Ratio
• Another implementation of this invention describes an optical device, and an algorithmic device for measuring the proportion of signal that belongs well to the region of interest (pinhole), for each image
• This measurement called Pinhole Ratio
• Another implementation of this invention describes an optical method, and an algorithmic method for measuring the position of the transmitter using the features of the so-called half-moon dithered distributions, described in this invention, using an appropriate algorithmic method, by a measure of the intensity ratio between the lobes.
• This 3D location can be used either in projection, that is to say by projecting one or more half-moon distributions offset on the object and using a suitable algorithm, for example the algorithms described in this invention, or in transmission by passing the light emitted by the transmitter through an optical module creating this distribution and by analyzing the PSF back.
• Another implementation of this invention describes an optical device, and an algorithmic device for measuring the position of the transmitter using the features of the so-called half-moon shifted distributions, described in this invention, using a device algorithmic, by measuring the intensity ratio between the lobes.
• This 3D location can be used either in projection, that is to say by projecting one or more half-moon distributions offset on the object and using a suitable algorithm, for example the algorithms described in this invention, or in transmission by passing the light emitted by the transmitter through an optical module creating this distribution and by analyzing the PSF back.
• Another implementation of this invention describes an optical method, and an algorithmic method for measuring the position of the transmitter using the features of the so-called “dark helix" distributions, described in this invention, using an appropriate algorithmic method, by a measure of the angle of the axis connecting the two zeros.
• This 3D location can be used either in projection, that is to say by projecting one or more half-moon distributions offset on the object and using a suitable algorithm, for example the algorithms described in this invention, or in transmission by passing the light emitted by the transmitter through an optical module creating this distribution and by analyzing the PSF back.
• Another implementation of this invention describes an optical device, and an algorithmic device for measuring the position of the transmitter using the features of the so-called “dark helix" distributions, described in this invention, using an appropriate algorithmic device, for example a measure of the angle of the axis connecting the two zeros.
• This 3D location can be used either in projection, that is to say by projecting one or more half-moon distributions offset on the object and using a suitable algorithm, for example the algorithms described in this invention, or in transmission by passing the light emitted by the transmitter through an optical module creating this distribution and by analyzing the PSF back.
• Fig. 1 is a simplified 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 microscopy system of
• Fig. 3 is a simplified schematic illustration of a conical diffraction module according to an embodiment of the present invention.
• Fig. 4a to 4f are simplified pictorial representations of the measurement and volume confinement 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. Figures 7a to 7c show 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]; Figure 7d completes these distribution tables by showing the variation of so-called half-moon distributions offset as a function of the axial position (z axis). Figure 7e presents a new distribution, "dark helix" including two zeros, whose axis connecting these two zeros turns according to the propagation.
• Fig.8 is a simplified schematic illustration of one of the embodiments of dark tracking
• Fig. 9 is a simplified schematic illustration of a fluorophore data superresolution algorithm method, in accordance with an embodiment of the present invention.
• Fig. 10 is a simplified schematic illustration of the calculation of the descriptors
• Fig. 11 is a simplified schematic illustration of the control module of the SRCDP platform.
• Fig. 12 presents the light distributions obtained "Four-leaf-clover" distribution at different spherical aberration values.
• Microscopy is used in the field of biology, for example, to observe, study and measure biological entities (objects) and their dynamics.
• 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, study and measure semiconductors or to characterize materials.
• phase and polarization polarimetry
• Jones vectors and matrices Jones vectors and matrices
• Stokes parameters Jones and Stokes parameter measurement techniques.
• optical diffraction limit optical diffraction limit
• Rayleigh criterion Rayleigh criterion
• Airy disc radius and diameter.
• supersolution, supersoluble, superresolution imaging and supersensitive microscopy to describe the acquisition of optical data, in imaging, microscopy or artificial vision, at a resolution greater than the optical diffraction limit.
• fluorescence and for fluorophores are used for fluorescence and for fluorophores.
• superoscillations are that of Yakir Aaronov and Sir Michael Berry.
• a super-oscillation is a phenomenon in which a signal that is generally band-limited may contain local segments that oscillate more rapidly than its faster Fourier components.
• the center or centroid of a light distribution is the center of gravity of the intensity.
• the diameter of a light distribution is the diameter of the first intensity zero, for regular and singular waves, without taking into account the central zero of the singular wave. Two light distributions are co-located if their centers coincide or are separated by a small spatial value relative to the size of the light distribution.
• 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 geometric parameters or the phase and / or polarization parameters between two other submodules or optical modules -, polarizer, analyzer, delay plate, beamsplitter beam splitter in English, polarizing and non-polarizing polarizing, "beam combine" beam combiner in English, polarizing and non-polarizing.
• REVERSIBLE Saturated OpticaL Fluorescence Transitions grouping the techniques: Stimulated Emission Depletion Microscopy (STED), Ground State Depletion (GSD), Saturated Structured Illumination Microscopy (SSIM) and SPEM (Saturated Pattern Excitation Microscopy), Microscopy Location, (Microscopy of Localization, in English) grouping Photo-Activated Localization Microscopy (PALM), FPALM (3D Localization in Fluorescence Photoactivation Localization Microscopy), Stochastic Optical Reconstruction Microscopy (STORM), dSTORM (direct STORM), SPDM (Spectral Precision Distance Microscopy) ,
• a partial polarizer to describe an element or a module whose absorption is different for the two linear polarizations - linear dichroism - or for the two circular polarizations - circular dichroism.
• 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, for example using PLZT material components, resonant electro-optical devices, magneto-optical devices, also known as Faraday cells, acousto- or elasto-optical devices or any combination thereof.
• polarization or phase dispersive elements we refer to polarization or phase dispersive elements to describe elements whose polarization state depends on the wavelength.
• the simplest of the polarization dispersive submodules is the multimode or thick waveguide,
• centroid algorithm we will refer to the "centroid algorithm” to describe the usual procedure for measuring the centroid and possibly the width (FWHM) of a light distribution.
• Optoelectronics photoelectric detector, CCD, EMCCD, CMOS, SPAD Single Photon Avalanche Diode and SPAD Matrix.
• an electronic image for describing 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,
• 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.
• 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 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.
• the beams can interact with each other or not, be projected
• beam shaping to describe the transformation of a waveform of given form and topology into a wave of another form or topology, and in particular the transformation of a regular wave into singular and vice versa.
• 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:
• Singular distributions or waves also called optical vortices, of topological charge (azimuthal order) 1, in which the phase varies from 0 to 2 ⁇ ⁇ , around the direction of propagation, where £ is an integer.
• Amplitude distributions with azimuthal variation of order i also called Laguerre-Gauss distribution
• the polarization and optionally phase-shift distributions with C-order azimuthal variation are also radially polarized Laguerre-Gauss modes.
• the azimuth orders i of the polarization or phase of the two light distributions differs.
• 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.
• a luminous nano-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 physical mechanisms that can create a nano-transmitter are numerous; they include but are not limited to absorption, diffusion or reflection, fluorescence, "emission-depletion", [5] for example using RESOLFT techniques, photo activation phenomena and photo depletion, fluorescence with two or more photons, elastic diffusion or not, Raman scattering, or other physical mechanisms known to those skilled in the art.
• nanoemitters including scattering, absorbing and reflective 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.
• a nanoemitter denotes the set of information describing a nanoemitter as a point source at a given time. Since the nano-transmitter is considered as a point source, all 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.
• the object is represented, as usual in image processing, by a matrix of intensities.
• the rest 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.
• 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, whatever the underlying physical phenomenon .
• 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.
• the sampling capacity of the light nanoemitter We will reference this remarkable property in this patent application as the sampling capacity of the light nanoemitter.
• 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.
• the Figure. 1 represents a set of nanoemitters or structured objects, positioned on a given biological object, 15 and 16 on the one hand and 17 and 18 on the other hand.
• 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.
• 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 morphological information, for example in the case of division of a cell in two.
• the measurement system will make it possible to calculate the measured map, and to evaluate 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, by structured objects or by continuous distributions, which could moreover, to represent any marked entity.
• the second level the level of geometric abstraction structures nanoemitters, structured objects or continuous distributions as geometric objects. It consists of a description of luminous objects and their dynamic characteristics, such as their position or orientation, or their morphology.
• 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 a certain apprehension of the biological reality thanks to a constitutive relation between the measured objects and biological entities
• Bio information contains a set of information about the biological object, mainly the position and its dynamics, shape and morphology.
• Biological information uses the map measured 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 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 "Robotic Microscopy Systems”.
• Conical diffraction describes the propagation of a light beam in the direction of the optical axis of a biaxial crystal.
• biaxial crystals Historically, conical diffraction has been observed in biaxial crystals.
• a conical crystal to describe an inorganic or organic biaxial crystal exhibiting the conical diffraction phenomenon.
• biaxial crystals include, Aragonite, KTP, KTA, KBiW, LBO, KNbO3, MDT, YCOB, BIBO, DAST, POM, NPP, LAP, LiInS2 and LiInSe2.
• effects exist, creating either inherently weak 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, [1 1]; the external effects of induced birefringence
• 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 equations of the conical diffraction 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 according to the wavelength, it creates an angle
• the angle ⁇ can vary greatly depending on the wavelength in some organic crystals such as DAST.
• Compensation of the chromatic dispersion 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 geometrical optics. This procedure can be designed and optimized using one of the available commercial optical software by defining appropriate target functions.
• 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 fundamental transfer function is identical to the unit and thus trivially independent of the wavelength.
• the vortex transfer function depends on the wavelength and can be represented by a chromatic factor equal to ⁇ ( ⁇ ).
• the fundamental wave depends on the wavelength and the vortex wave is almost independent of it. Indeed, the simulations show that the shape of the vortex wave is only slightly modified by a variation of the parameter, ⁇ from 0.5 to 0.75.
• the shape of the fundamental wave depends on the wavelength and this effect must be taken into account in the design of systems using the two waves, fundamental and vortex.
• FIG. 3 is a simplified schematic illustration of a configuration of a conical diffraction module 300, in accordance with an 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 fixture 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 Kepler telescope 1: 1.
• the numerical representation 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 the microscope objective.
• R spatial variable
• U wave vector
• ⁇ 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, R0; the conical radius depends on the material and the thickness of the crystal.
• the normalized radial position, p, the normalized wave vector, u, represented in cylindrical coordinates by p, 9R and u, ⁇ , and the conical normalized radius, po, are given by:
• Uo being the numerical aperture of the system.
• Low interaction volume diffraction effects have the following properties:
• linear Raman Nath regime previously referenced, [37]
• linear thin conical crystal in which a simple surface approximation of the effect can be used, po ⁇ 1 and> 0.5, sinusoidal Raman Nath regime , previously referenced, [37], "sinusoidal thin conical crystal”, in which a surface approximation of the effect can be used, po ⁇ 3 and> 1, intermediate regime, referenced previously, [37], "medium conical crystal” , in which complex effects, coupling the effects of Raman Nath regimes and the Hamilton-Lloyd regime described later,
• the emergent wave of the thin conical crystal, E (p, 9R), 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, 6R), a singular wave; these two waves are coherent with each other, co-localized, circular polarized and inverse chirality:
• EF (p) is the scalar fundamental amplitude
• Fv (p) is the reduced vortex scalar amplitude
• the emerging wave of the conical crystal for a circularly polarized wave, is constituted by the superposition of two waves, the fundamental wave, a regular wave, and the vortex wave, a singular wave.
• Homogeneous polarization can decompose on the orthogonal basis composed of circular right and left circular polarizations.
• the incident beam is therefore the coherent superposition of two beams, one polarized in right circular polarization and the second in left circular polarization.
• the beam emerging is the coherent superposition of four beams; two fundamental waves, the first created by the polarized beam in right circular polarization and the second by the polarized beam in left circular polarization and two vortex waves created by the polarized beams in right and left circular polarization.
• both fundamental waves have the same spatial distribution and can interfere by keeping their topology, both vortices are opposite chirality and create complex distributions.
• Different combinations of these waves can be achieved by choosing the polarizations at the input and output, which allows to obtain PSF of different shapes.
• parsimonious object in English, to describe either a set of parsimonious or sparse transmitters, spot light emitters, or a set of parsimonious objects, parsimony being defined in the references [ 41-43] and not limited to point objects but including, for example, filaments.
• the limit chosen is a number less than twelve, positioned in a volume whose size, in each of the dimensions, is less than 3 wavelengths, at the emission or reflection wavelength. issuers. The size smaller than 3 wavelengths, which contains the parsimonious object, will be referenced as a small size analysis volume.
• FIGS. 4a to 4c are a simplified representation of the concept of volume confinement of the confocal microscope.
• volume confinement 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.
• Volume containment 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 defocused light 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.
• the reference “i” The axes referenced “i” represent a Cartesian coordinate system centered on the center of the analysis volume, 61.
• the referential "a” represent a Cartesian coordinate system centered, for each light nanoemitter, on the light nanoemitter considered as a discrete point, 62,
• 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.
• alternative implementations replacing modules based on conical diffraction, by modules based on other optical concepts, are able to provide the same functionalities. 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.
• optical and optoelectronic concepts, techniques and devices that can 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 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.
• PDOS Position Depends Optical Semaphore
• 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 in English, in an analysis volume, if the sum of the transfer functions, Ti (x, y, z) is equal to unity in the analysis volume.
• the confocal hole, described by Minsky, [15] could be considered in this embodiment of the invention as a degenerate PDOS of order 1.
• LPDOS dependence is a complex function of lateral and longitudinal positions.
• LPDOS Longitudinal Position Depends Optical Semaphore
• optical semaphore dependent on the longitudinal position - to describe an optical semaphore which channels the light in function of the longitudinal position of the emitter 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.
• 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 often be coupled to a stop, limiting the lateral field of the system.
• optical fibers A main use of optical fibers is the exclusive transmission of the TEMoo mode.
• certain configurations of optical fibers such as FMF or Vortex Fibers, mainly but not exclusively based on fibers called "Photonic Crystal Fiber” (acronym PCF) and fibers called “vortex fiber” allow the transmission simultaneous or not, more complex modes, including vortex modes, with a vorticity equal to or less than 2. It would therefore be possible to deport the optical distributions created by conical diffraction using optical fibers, allowing simplification major part of the optical system.
• optical fibers allow the application of the embodiments of the invention to many additional applications, for example but not limited to gastric or gastroenterological observation, and observation of the colon and urinary tract.
• a broad-spectrum laser - called a white laser - may be used.
• This configuration is simple because one of the main parameters of the system is fixed and well determined.
• the object can also be illuminated at several wavelengths, either discretely, using for example several lasers, or continuously, for example using a lamp or a laser having a wider spectrum.
• Many existing superresolution systems measure simultaneously or sequentially at multiple 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:
• 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.
• FRET Förster-Förster energy transfer
• 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.
• Ge 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.
• Embodiments of the disclosed invention allow the integration and fusion of additional information external to the described platform, optical or contextual, to obtain an improvement in the accuracy of the information collected from the sample for one.
• 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.
• 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 towards the observer, for example an element positioned in an opaque box, or a physical information that does not No optical transcription.
• Embodiments described in this invention are not limited in resolution. a priori and could ideally - with an infinite number of photons - obtain any resolution, as we will describe later for a specific case. Use of non-linear interactions in the superresolution and family of RESOLFT and STED techniques
• non-linear interactions include but are not limited to two-photon interaction phenomena, emission-depletion, blinking, and photoactivation effects upon which the RESOLFT family of technology and the family of "localization microscopy" technologies.
• CW STED the pulsed laser used in the first version of STED is replaced by a continuous laser - simpler.
• the emitted photons are discriminated according to their emission time to separate 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.
• Another variant proposed by Gould et al. [45], uses an SLM to create, either separately or jointly, the lateral and / or axial depletion distributions.
• phase plate 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 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.
• this implementation is chromatic, because the phase plate adapted to a wavelength will not 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.
• Reuss et al. introduces a beamformer placed directly in front of the objective lens.
• This device is based on the use of birefringent crystals, mounted in the form of a segmented wave plate, 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.
• the initial laser beams are in the form of a regular distribution and in most cases 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. Co-locating these beams upstream when they are still in Gaussian distribution form is relatively simple. Co-localizing these beams downstream when they have been transformed into a regular and singular wave is much more complex. Co-localizing these beams upstream can be done commercially by laser bench systems using fiber optic techniques. It is therefore relatively easy, as it is also done in all confocal microscopes, to create a set of laser outputs at different wavelengths of the same optical fiber and therefore co-located very precisely.
• the solution proposed in embodiments of the present invention allows to base on this co-location upstream, greatly simplifying the system and allowing the realization of a STED at several wavelengths, intrinsically.
• 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 in fact no common system, achromatic and allowing a different beam shaping for different beams, in the literature. Such a system certainly improves the simplicity of design and use of STED.
• PSIT method can also be used to project sequences of light intensities that differ topologically, at two or more wavelengths, sequentially or simultaneously.
• the SRCDP platform "Super Resolution using Conical Diffraction Platform" is a Microscopy platform using optical modules based on conic diffraction.
• the SRCDP platform 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, "Super Resolution using Conical Diffraction Algorithm ", to rebuild
• the SRCDP platform includes an enhanced detection module, a system control module, and computer and software support.
• certain 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 optical modules and the algorithmic SRCDA.
• 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 polarization states, including input and output.
• / 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.
• this figure shows the light distributions, created through a conically standardized tapered crystal of 0.818, calculated by a scalar approximation, for different input and output polarization states, including as input or at the output is a circular or linear polarizer or a radial or azimuth polarizer.
• input and output polarization states 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 by their chirality for circular polarizations.
• tapeered diffraction transfer function the set of transfer functions that can be obtained using a small ( ⁇ 6) number of crystals in cascade, and polarization elements, static or dynamic, uniform or spatially varying
• the different light distributions are produced by modifying 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.
• the elementary light distributions described in Figure 7 can be obtained in several different ways. Moreover, some of them can be obtained as an incoherent superimposition of other elementary light distributions; for example the vortex can be obtained by any sum of two orthogonal "half-moon" light distributions.
• 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 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.
• the output bias adaptation sub-module, 74 to control the output bias.
• the output polarization has a circular symmetry substantially reduces the vector effects and makes it possible to adapt to the direction of the fluorescent dipole.
• Such polarization can be circular, radial, azimuthal or position-dependent.
• the output polarization adaptation sub-module 74 is simply a four-wave delay plate.
• the longitudinal polarization elements have a vortex symmetry and blend harmoniously into the system with only a small change in the shape of Stokes parameters, even for microscope objectives with very high numerical aperture.
• the output polarization adaptation sub-module, 74 may be variable and / or controllable and adapt to the topology and symmetry of each of the compact light distributions.
• a Wollaston prism can be used to separate an incident beam into two emerging beams separated by an angle. By using several prisms in cascade, it is thus possible 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 are appropriately selected. It is thus possible to construct a prism of a single block of uniaxial crystal which can separate an incident beam into 2 n emergent beams (e.g. eight or sixteen) which are contained in a plane, and separated by equal angles.
• the modified Wollaston prism can be coupled with the methods described in this invention to duplicate all light distributions without changing the relationships between them.
• the modified Wollaston prism can be coupled to the STED methods described in this invention or other standard STED methods to duplicate all light distributions without changing the relationships between them.
• 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.
• 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.
• Figure 4d is a simplified conceptual representation of a measurement paradigm according to at least one embodiment of the invention.
• the paradigm of measurement is much more ambitious than that of the confocal fluorescence microscope, shown schematically in Figure 4a.
• an analysis volume, 60 is created at the focal plane of the microscope objective, 22; it contains a parsimonious 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.
• FIG 4e is a simplified conceptual representation of another measurement paradigm, based on STED concepts, according to at least one embodiment of the invention.
• This paradigm of measurement limits in two (STED 2D) or in the three spatial dimensions (STED 3D), the observed region of the sample to a volume of the smallest possible size, the analysis volume.
• an analysis volume, 60 is created in the focal plane of the microscope objective, it contains a parsimonious object, 51, comprising several nanoemitters, 53 to 59; one or more depletion waves, 2000, reduce by a depletion effect, the volume of analysis to a smaller volume represented by 2001.
• the superposition of the set of effects creates a small volume, the analysis volume, 60. This volume determines the size of the elementary region detected by the system.
• an analysis volume, 60 is created in the focal plane of the microscope objective, it contains a parsimonious object, 51, comprising several nanoemitters, 53 to 59; one or more depletion waves, 2000, reduce by a depletion effect the analysis volume to a smaller volume represented by 2001.
• this volume contains a parsimonious object, 51, consisting of several
• 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 PSIT measurement method performs the following functions:
• the transmission sequence consists of at least two point light distributions of different topological families
• the emission sequence is projected on a biological sample marked by nanoemiters.
• the emergent light emission of each nanoemitter is dependent, for each nanoemitter, on the intensity, in the incoherent case, or on the electromagnetic field in the coherent case, incidents at the spatial position, three-dimensional luminous nanoemitter aforesaid sampling property of the light nanoemitter previously discussed.
• the PS1T method according to this embodiment makes it possible to acquire information
• 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.
• the PSIT method can also be used to project sequences of light intensities that differ topologically, at two or more wavelengths, sequentially or simultaneously.
• the PSIT Method was initially designed to allow lateral super-resolution
• the PSIT method can also be used to obtain the longitudinal position of a nanoemitter. Indeed, some 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.
• the PDOS method 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. It has been described by one of the inventors, [37].
• the function of the optical semaphore is to separate on different detectors different regions of the analysis volume.
• 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.
• the PDOS method is implemented so as to separate on different detectors the collimated, emerging light of nanoemiters 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.
• 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.
• the modified PDOS method an optical method for shaping the emission beam, for the axial and / or lateral location of nanoemitters.
• This method implements a single-channel PDOS method, in which the variation of one of the parameters describing the created distribution is used to measure the axial or lateral parameters.
• the parameters used can be either an angle parameter for a helical axial variation distribution, or the ratio between the lobes of the distribution - for a two-lobe distribution (or more) with axial variation.
• This method has a certain similarity with the original PDOS method, described by one of the inventors in [37], but differs in that the variation of the topology of the created distribution is used to measure the axial or lateral parameters and not by an intensity ratio between two detectors, as described in the original version of the PDOS method.
• This method like the original method, has applications as a complementary method of the PSIT method, but also for the axial location of nanoemitters, for example for the localization modalities, such as, for example, the PALM, STORM or GSDIM modalities or the like. .
• the PDOS method was initially designed to allow longitudinal superresolution; however, the PDOS method can also be used to obtain the lateral position of a nanoemitter. In fact, 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.
• the intermediate result is obtained at the end of the detection step.
• the raw information consists of a set of images A op (m, n), representing for the light distribution o, the image resulting from the detection channel p.
• 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.
• 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 (parsimonious object), but one can also use continuous models, assuming the presence of one-dimensional structures (lines, curves) or specific patterns. We can then use all the procedures
• One embodiment of the invention is a hardware and algorithmic platform referenced as the SRCDP platform 500, illustrated in FIG. 5.
• the SRCDP platform, 500 implements the method according to one embodiment of the invention, either by combining the two methods PSIT and PDOS, original or modified, described above, either using the STED techniques or by combining the techniques. STED with PSIT or PDOS methods, original or modified.
• the SRCDP platform observes, Figure 5, a biological sample, 1 1, integrating a set of nanoemitters.
• the result of the observation of the biological sample through the SRCDP platform is the acquisition of information from
• the SRCDP platform, 500, Figure 5 mainly includes:
• a confocal microscope 200 adapted or optimized, similar to the confocal microscope, described previously, and comprising all the appropriate components, as previously described.
• 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 LatSRCS 700 optical module implements the illumination steps necessary for the implementation of a STED or RELSOFT technique, according to another embodiment of the invention.
• the original or modified LongSRCS optical module, 800 implements the steps of distributing the emerging light intensity into a plurality of images of the PDOS method or implements the variation of the emerging PSF, as a function of lateral or axial parameters, according to one embodiment of the invention and,
• 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 feasible 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 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.
• the spatio-temporal information referenced here is the position and the time of the impact of each fluorescent photon.
• spatio-temporal information is corrupted by detector noise, creating erroneous photons, and inefficient detection, which creates photons that are not detected, reducing performance.
• SPAD matrices for each photon, the pixel that has detected it and the impact time are received, ie the complete spatio-temporal information is available.
• CCD, CMOS or EMCCD detectors the acquisition of several frames is necessary to approximate the spatio-temporal information.
• detectors in 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.
• the Control Module, 1,100 using the systemic control procedure, 1101, controls and modifies the optical parameters of the SRCDP platform, 500, the electronic parameters of the enhanced detection set, 65, and the parameters mathematical algorithmic procedures SRCDA, 900, to optimize emerging information, according to criteria defined by the system or by the user.
• the control is performed by varying the control systems 1 102, 1 103 and 1 104, the various elements of the platform, 600, 800 and 900.
• the control system, 1 100 will also use, if available information external, 1 105, relayed by the computer support. Note: 1,105 is not present in Figure 1 1
• Microscopy modalities For example, measurements in confocal or wide-field microscopy, at the same lateral or axial positions, or at different positions may be performed to calibrate certain parameters of the scene model.
• the direct model is enriched by the fact that
• FIG. 9 is a simplified schematic illustration 900 of a fluorophore data super-resolution algorithm method, in accordance with one embodiment of the present invention.
• An algorithm procedure shown in the figure. 9, quantifies the number of fluorophores, retrieves the attributes of each fluorophore and quantifies the accuracy of each output parameter.
• the pretreatment procedure, 11, rearranges the spatio-temporal information, 1 10, in sets of superresolution images, 112. This operation can be performed using a filter bank procedure.
• the intermediate data set is then a small series of small images, typically 16 * 16 pixels.
• the pretreatment procedure applies to a small number of space-time elements of the order of a few thousand, it can be performed in real time and using existing computer equipment.
• the descriptors include, but are not limited to: the intensity of each image, the presence, like a distribution of light and its characterization as a regular distribution or a vortex, its center of gravity and its moments of order one and more.
• the third step is a filtering operation, 1 15, in which only the descriptors, which are statistically relevant, are retained.
• the classification operation, 116 is the last step of the algorithm.
• the algorithm is able to recognize, on the basis of the set of descriptors, 1 14, and a base of
• the algorithmic SRCDA can use the classical techniques of the inverse problems.
• three new algorithmic approaches, the modified MAP algorithm, the E-LSE algorithm and the ICE algorithm are described in this patent application and are deemed to be part of this invention.
• the optical module, LatSRCS, 700 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.
• compact light distributions of different topologies are created by interference, with varying amplitudes and phases between a regular wave and a singular wave.
• 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.
• the incident intensity at a specific position on the sample 1 will be proportional, for each light distribution, to a specific combination of Stokes parameters.
• the LatSRCS Optical Module, 700 utilizes an inherent feature, described previously, 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 emerging light distribution, recoverable by a detection assembly 65.
• 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 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 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.
• 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 set of Enhanced detection consists of a small area of pixels and also recovers the spatial distribution of fluorescent light. All of the retrieved information consisting of a plurality of images, designated as the superresolution side images.
• the contribution of a nanoemitter 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.
• 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.
• FIG. 6a is a simplified schematic illustration of a LatSRCS optical 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 FIG. the light source of the scanning confocal microscope is 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 sub-assembly achromatically achieving the
• 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 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, a Pockels or Kerr cell, 72, and an analyzer.
• Stokes parameter information can be transformed into sequential information, through a sequence of spatially differentiated light distributions, and bearing sequential information, as described above.
• the modified LongSRCS module an optical module for shaping the emission beam, for the axial and / or lateral location of nanoemitters.
• This module implements a single-channel PDOS method, in which the variation of one of the parameters describing the created distribution is used to measure the axial or lateral parameters.
• the parameters used can be either an angle parameter for a helical axial variation distribution, or the ratio between the lobes of the distribution - for a two-lobe distribution (or more) with axial variation.
• This module has some similarity with the original LongSRCS module, described by one of the inventors in [37], but differs in that the variation of the topology of the created distribution is used to measure the axial or lateral parameters and not by a ratio of intensity between two detectors, as described in the original version of the LongSRCS module, [37].
• This module like the original module, has applications as an add-on module of the LatSRCS module, but also for the axial location of nanoemitters, for example for the localization modalities, such as, for example, the PALM, STORM or GSDIM modes or the like.
• the "dark helix" distribution has a rotation effect of the axis connecting the two zeros along the Z axis.
• the position of the transmitter can be deduced with great axial precision by a measurement of the axis connecting the two zeros.
• This 3D location can be used either in projection, that is to say by projecting one or more half-moon distributions offset on the object and using a suitable algorithm, for example the algorithms described in this invention, or in emission by passing the light emitted through an optical module creating this distribution and by analyzing the PSF back.
• the illumination beam can be shaped using conical diffraction and its phase and polarization effects.
• the sample is then illuminated with a non-uniform light distribution, which can also be a black sphere (or top hat) so that the 3D distribution has two lobes of the same intensity below and above the focal plane. In the presence of spherical aberration in the system, these two lobes are not of the same intensity.
• By then analyzing the intensity ratio of the two images we can deduce the amount of spherical aberration of the system.
• Another approach to measuring spherical aberration uses another variant of systems based on conic diffraction.
• the shaping of a light beam by conical diffraction through a cascade of crystals makes it possible to obtain an intensity distribution directly connected to the spherical aberration.
• This distribution is obtained between crossed linear polarizers and two biaxial crystals whose optical axes are aligned. A half-wave plate is inserted between the two crystals. Due to its characteristic form, this distribution will be called “four-leaf clover” or “four-leaf-clover” in English. In the absence of spherical aberration, this distribution consists of four perfectly equal lobes and all in the same focus.
• Figure 12 describes the symmetry break created in a "Four-leaf-clover" distribution in the presence of spherical aberration.
• Figure 12 shows the distributions simulated under Matlab for different values of defocus and spherical aberration.
• Spherical aberration induced by an optical system breaks the symmetry of this distribution both in the focus and intensity distribution in the four lobes as can be seen in the figures below.
• below representing the "Four-leaf-clover” distribution with different spherical aberration values.
• a simple qualitative observation allows a binary test presence / absence of spherical aberration.
• a fine measure of the spherical aberration value is possible by a lobe focus offset estimator and intensity ratios at different focus planes.
• the optical system described in this invention can incorporate a module for tracking in real time on a camera a scanned point by a beam scanning system (galvanometric mirrors, bidirectional piezoelectric mirror or any other system).
• a beam scanning system galvanometric mirrors, bidirectional piezoelectric mirror or any other system.
• multi-image system the set of optical and optoelectronic systems, in which a set of different and differentiated images, coming from the same spatial region of the object, two-dimensional or three-dimensional - 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 illumination; it can also be due to a natural movement or imposed from outside objects.
• the proposed algorithm makes it possible, 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 image. 'sample.
• This algorithm is based on the combination of several principles:
• the E-LSE algorithm uses the same Bayesian formulation as the modified MAP algorithm described previously, but exploits the posterior law differently (in a more complete way): in fact, we calculate the average of the posterior distribution and not its maximum value point (which is known to be more suitable for large-scale problems).
• This approach considered by Besag in 1984 [30], has recently been implemented numerically in the case of image denoising with a total variation type priori [31,32];
• parsimony a priori by the use of a small number of transmitters is not used and the average of the posterior distribution is calculated on all possible images as in [31,32].
• Z is a normalization constant - not the light propagation axis - which is not involved in the algorithm
• Each quantity of type u, (x, y) is determined during the calibration step: it represents the intensity emitted at the pixel y of the camera by an emitter located at the pixel x of the high resolution image, in response to an index illumination i (i therefore encodes here 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 from both the sample (diffuse fluorescence for example) but also from the sensor.
• the quantities rm (y) simply correspond to the measurements (images recorded by the camera): m, (y) is the intensity measured at the pixel y of the index image, that is to say the image recorded after index illumination).
• the proposed algorithm consists of changing the emitters represented by the vectors x and X in accordance with the law given by the density ⁇ ( ⁇ , ⁇ ).
• the algorithm is iterative: at each iteration, one of the transmitters is perturbed (in position or in intensity) and this perturbation 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 j respectively correspond to the position and the intensity of the emitters at the iteration j of the algorithm, then the image / reconstructed after N iterations is given by
• 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 can be used in a second version including a set of additional parameters describing global parameters of spatial region of the object is known a priori is determined a posteriori.
• This algorithm in both versions, can be used in all multi-image systems, in which a set of different and differentiated images, from the same spatial region of the object, two-dimensional or three-dimensional - are recorded and analyzed.
• the speed of the transmitters can be taken into account.
• the ICE algorithm has recently been introduced for image denoising in the case of full variation regularization [39].
• the ICE algorithm can also be used to solve more complex inverse problems (deblurring, interpolation). This fixed point type algorithm converges very quickly, and the resulting solution is extremely close to that associated with the LSE algorithm for this problem.
• the principle of the ICE algorithm is to replace the calculation of the expectation of the posterior distribution performed by LSE by the iteration of the explicit calculation of the average of the posterior distribution of a pixel conditional on its neighbors.
• the application of the ICE algorithm to inverse problems for an image or a multi-image system must be demonstrated.
• the SRCDA algorithms use as input the measurements made on the sample, but also data inherent in the system that are based on either values theoretical, or on values obtained after a so-called calibration stage.
• measurements are performed on a reference sample to accurately measure the illumination functions and the return transfer function (PSF) of the optical system.
• PSF return transfer function
• the optical process composed according to at least one embodiment of the invention is a logical complement of 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. Resolution, parsimony, topology of enlightenment and positivity
• a microscopy system that does not use a nonlinear effect is limited, in the general case, to a superresolution factor of 2 with respect to the limit imposed by optical diffraction [38] and [41].
• a parsimonious scene real support of the sample substantially smaller than the size of the image area, for example in the case of scenes composed of filaments and / or point sources
• a continuous background too important (so that a positivity constraint on the reconstructed image is effective)
• this limit of 2 is no longer valid and can be exceeded [42-44].
• the achievable resolution for the reconstructed image is not limited by optical diffraction but by the quantity of photons (more precisely, by the signal-to-noise ratio of the measurement).
• the maximum superresolution factor essentially depends, in addition to the signal-to-noise ratio, on the properties of the scene: number of sources (relative to the number of measurements), distance minimum between two sources, etc. [43-44].
• the maximum superresolution factor will depend, in addition to the factors described previously, of the selected illuminations.
• the principle is based on the exploitation of one of the characteristics of the algorithms described above but not only, which is that the signal of interest is located mainly where the excitation light has been projected. It is possible to measure the proportion of signal that belongs to the region of interest (pinhole) for each image, ie each laser position, each light distribution and each orientation of this light distribution.
• Pinhole Ratio This measure, called Pinhole Ratio, consists in the method we describe, comparing in a spatial region of the object the proportion of photons that are re-emitted and imaged in the region of interest of each image from this region in relation to the total number of photons reemitted by the object in all the images considered.
• This ratio gives information locally on the nature of the imaged object, whether it verifies a predefined model (for example: planar object) in the sense of the Pinhole Ratio, or if it deviates strongly from this model.
• a predefined model for example: planar object
• the criterion defined here can take values between 0 and 1, as well as a nominal value PR ref in the case where the object satisfies the model. Values lower than PR ref are associated with an object that does not completely or not completely check the model according to a theoretical or empirical rule.
• a property of the Poisson noise is the possibility of generating, from a realization of a Poisson VAR of parameter I (the mean), two independent realizations of a Poisson VAR of parameter 1/2, thanks to a post-treatment.
• n is equal to the measurement of the initial Poisson variable
• p is an equal photon separation parameter. at 0.5 in our case.
• this property illustrates the known fact that if we put a 50/50 splitter mirror in front of the camera that images the fluorescence, and that we place a second camera to image the second fluorescence beam, then both cameras, under identical conditions of image acquisition, acquire a signal of average equal to half of the initial signal and always following a Poisson's law.
• the analysis or reconstruction of the two measurements thus generated provides two independent results in the sense of the probabilities, and the differences between these two results illustrate the dependence of the reconstruction algorithm on the noise of measurement, although the signal-to-noise ratio is lower in each generated measure than in the original measurement of a factor sqrt (2).
• the PSIT method can be used as a technique to measure the position of a nano-transmitter with high precision, using a different measuring mechanism complementary to the centroid method.
• the preprocessing procedure creates two images:
• a "top hat” image consisting of the sum of the three images of 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.
• the radial position p can be measured univocally by measuring a parameter, p a , equal to the normalized tangent arc by a factor ⁇ , of the intensity ratio between the normalized intensity emitted by the a nano-transmitter illuminated by the vortex wave, Iv, and the normalized intensity emitted by the nano-transmitter illuminated by the fundamental wave, IF.
• a parameter, p a equal to the normalized tangent arc by a factor ⁇ , of the intensity ratio between the normalized intensity emitted by the a nano-transmitter illuminated by the vortex wave, Iv, and the normalized intensity emitted by the nano-transmitter illuminated by the fundamental wave, IF.
• the normalized intensity emitted by the nanoemitter illuminated by the fundamental wave varies from 1 at the center for the fundamental wave to 0 at the radius of Airy,
• 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-moon distribution. , IVE- The relationship between these two intensities is a geometric law in square tangent:
• 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, requiring a large number of images and therefore a long measurement time and a strong 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 much higher resolution than the Airy disk, at rates that can reach the micro or nanoseconds allows the extension of stochastic techniques to many new applications.
• the resulting images of using the PSIT method can also be processed using the generalized Hough method, allowing to recognize structured objects, line, circle or others, in an image.
• centroid will measure the center of gravity of the light distribution, which will be at the origin
• the descriptors p and ⁇ enable us to measure the characteristics of two points at a resolution much higher 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 inclined at an angle ⁇ ; 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 ⁇ .
• the resolution of a system using the PSIT and / or PDOS techniques is therefore, in practice, dependent on the sample observed.
• the resolution obtained can substantially exceed the factor 2 (locally or even globally).
• 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;
• 82 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 reposition the element followed at a corrected position.
• the light distributions used by this technique are vortices generated by conical diffraction. However, this technique can also use other vortices or other distributions generated by conical diffraction.
• the molecule of interest has been labeled with one or more fluorophores which can be excited at ⁇ .
• the position of the molecule is first detected by a classical confocal image at ⁇ .
• 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 emitter.
• 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 important 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 fluorescent 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 the camera (up to 1kHz) or the detector (several MHz), allowing tracking of a molecule in real time over a period of time consistent.
• the localization can be conducted over a long period of time, bleaching being less likely to occur with such small doses of light.
• the accuracy of the location depends strongly on the signal-to-noise ratio of the image, therefore the background noise (camera noise and auto-fluorescence signal) must be taken into account.
• 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 particle bleaching.
• FIG. 6b is a simplified schematic illustration of a modified LatSRCS optical module, achromatic or otherwise, 700, according to an embodiment of the present invention.
• This module differs from the implementation of a LatSRCS optical module, 700, previously described and shown in Figure 6a, by adding several elements before the module:
• lasers 79a, 79b and 79c optionally at different wavelengths, optical fibers 78a, 78b and 78c from lasers 79a, 79b and 79c,
• polarization control sub-modules 77a, 77b and 77c for statically or dynamically changing the polarization of the lasers 79a, 79b and 79c, the polarization control submodules being positionable before or after the fiber.
• the polarization control sub-modules 77a, 77b and 77c may not be necessary and the polarization of light from the fiber is determined by the relative position of the laser and the fiber,
• a "laser combiner” (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.
• 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.
• the common fiber, 76b may not be necessary for some implementations.
• 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 able to realize the RESOLFT or STED techniques in all their different modalities.
• 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 described above.
• the excitation and depletion waves propagate along two orthogonal linear polarizations and a quarter-wave plate - optionally achromatic - is positioned at the entrance of the system to transform these polarizations into polarizations.
• a quarter-wave plate - optionally achromatic - is positioned at the entrance of the system to transform these polarizations into polarizations.
• the fiber is a birefringent fiber and creates a difference in operation greater than the coherence length of the depletion laser, creating an incoherent superposition of the two polarizations. orthogonal.
• a polarization submodule (not shown), consisting for example of a thick uniaxial crystal with high birefringence.
• a polarization submodule (not shown), intensity ratio control can be used. This polarization, intensity ratio control submodule is different from the previous polarization submodule, but can potentially be integrated with it.
• This intensity ratio control bias sub-module can be used to control the intensity ratio between the two polarizations, and thus, as will be described later, the intensity ratio between the 2D depletion beam (vortex ) and the 3D depletion beam (black sphere).
• the polarization control sub-modules, 77a, 77b and 77c can be actuated to simultaneously create sequences of light distributions, identical or different, for the excitation path and the depletion path.
• the intensity of the lasers can be modulated, creating a complex timing diagram of the intensity of each laser, the sequence of excitation light distributions and the sequence of light depletion distributions.
• the resulting emission image will be called the negative image.
• the difference image consists of the weighted subtraction of these two images.
• this difference image may be a digital processing more evolved than a simple arithmetic difference and may incorporate a set of mathematical treatments known to those skilled in the art, making it possible to optimize the resulting image in function, for example, but non-limited, of the frequency content of the 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 lower intensity of depletion.
• 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.
• the depletion of the Airy or of the fundamental will be coupled 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.
• this embodiment may in some cases overcome the need to trigger the 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 vortex of depletion hardly differs from those emitted by the excitation vortex and can also be compensated.
• 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.
• coupling the depletion and the algorithmic should allow a suitable compromise between the intensity of the projected depletion beam and the resolution gain.
• an excitation beam is simultaneously projected in the form of an Airy and a depletion beam in the form of a vortex, the two beams differing in their polarization.
• This device makes it possible, without dynamic elements, to produce a totally achromatic STED device.
• STED or STED-3D systems uses an optical distribution of zero intensity at the center point, and remains zero along the axis, along a certain distance.
• STED-3D the use for STED-3D, of a distribution having a position of zero intensity, this position of zero intensity having a helicoidal spatial variation, as a function of the axial parameter.
• the preferred implementation of this distribution is the use of conic diffraction using the optical distributions that we have referenced as the Stokes distribution.
• SLM Spacial Light Modulator
• segmented mirrors that create phase and / or random distributions. amplitude in the pupil.
• helical topology distribution to create a zero intensity distribution having a helical motion is considered to be part of this invention.
• 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 one of the implementations described in this invention.
• the integrative two-polarization method the crystal parameters are chosen, such that the fundamental wave creates a black sphere and the vortex wave creates a vortex.
• the two distributions, orthogonally polarized do not interfere and an incoherent superposition of the two beams allows, with a single input beam to independently create the two beams necessary for STED 3D, the vortex and the black sphere.
• the ability to create with a single beam of depletion input, the two beams can greatly simplify the optical system. In addition, this system is common way, it can be implemented simply.
• a polarization sub-module (not shown), static or dynamic, positioned between the optical fiber and the crystal, can be used to control the intensity ratio between the two polarizations, and thus, the intensity ratio between the beam 2D depletion (vortex) and the 3D depletion beam (black sphere).
• the integrative single-polarization method, a birefringent fiber - or a polarization sub-module - has previously created, as described above, a difference in operation greater than the coherence length of the depletion laser. for a CW laser, and / or at the time of the pulsing, for a pulsed laser or for the gated STED, creating an incoherent superposition of the two orthogonal polarizations.
• the two distributions do not interfere; the two distributions being of orthogonal polarizations, will create, on the same polarization, the one a fundamental one - which by a choice of parameters of the crystal and the optical system, will be a black sphere, and the other a vortex.
• These two beams derived from two incoherent distributions, as previously described, do not interfere; this device makes it possible, with a single input beam, before the fiber, to independently create the two beams necessary for the 3D STED the vortex and the black sphere.
• the ability to create with one single input depletion beam, the two beams greatly simplifies the optical system. In addition, this system is common way, it can be implemented simply.
• the conical diffraction element is replaced by a sub-diffraction.
• module consisting of two conical diffraction crystals, of substantially equal conical diffraction parameter value, separated by a polarization submodule.
• the polarization sub-module is chosen such that at the depletion wavelength, the polarization submodule has no effect, and the action of the crystals are added, creating the vortex and / or the black sphere.
• the polarization submodule creates a rotation of the polarization of 90 ° or 180 °, the effect of the two crystals is subtracted, and the transmitted beam is identical to the incident beam.
• This device avoids creating too "exotic" distributions on the excitation beam.
• the crystals could have different conical diffraction parameter values, to also create an effect on the excitation beam, but with a conical diffraction parameter value different from that of the depletion beam.
• This variant can be made at several wavelengths of depletion and excitation, using the resources of the polarization modules well known to those skilled in the art.
• the conical diffraction element is replaced by a cascade of crystals having different spectral properties, such as LBO and KTP (KTA) so as to allow the realization of black sphere at several wavelengths of depletion.
• KTA KTP
• the dispersion of the characteristic parameter of the conical diffraction effect, po does not make it possible to carry out certain distributions at several wavelengths, in particular the black sphere, the shape of which depends strongly on this parameter. Compensation of the dispersion of the characteristic parameter of the conical diffraction effect, po, therefore allows the realization of a 3D STED with a common optical path, at two or more wavelengths.
• the dispersion of the characteristic parameter of the conical diffraction effect, po therefore allows the realization of a 3D STED with a common optical path, at two or more wavelengths.
• compensation of the characteristic parameter dispersion of the conical diffraction effect, po can be performed over a wide range of wavelengths either as previously described by a combination of crystals, or by spectrally correcting the second-order optical system, ie, by creating a wavelength-dependent digital aperture to either compensate for the dispersion of the characteristic parameter of the tapered diffraction effect, po, or to correct a dispersion compensation of the characteristic parameter of the tapered diffraction effect, po, created by a combination of crystals previously described.
• an excitation beam sequence, a depletion beam sequence are simultaneously projected, the two beam sequences being able to differ. by their polarization, creating light distributions of different topologies.
• the LatSRCS module makes it possible to produce 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.
• 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 to be created during the 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.
• 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 transmitters by means of a physical effect, which dilutes the density of the emitters capable of emitting fluorescence, so as to create regions in which the assumption of parsimony is valid, ie 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. These techniques include, for example, PALM STORM, DSTORM, FPALM and others.
• 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.
• the light will be detected either by a matrix detector or by a single 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.
• harmonic time distributions in which the electro-optical cells are actuated by a sinusoidal voltage.
• 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.
• Two cascaded crystals create an effect of adding the effects of the crystals (if they are oriented in the same direction), as described in Reference 46, or subtraction (if they are oriented at 180 °).
• using the effect of addition of the effects of the crystals one can have two crystals whose effects are added.
• a chromatic rotator which rotates at different angles, ideally at angles being multiples of 180 °, at different wavelengths, one can have a modulus in which the effects of the two crystals cancel each other out at a different angle. wavelength and add up at the other wavelength.
• the rotator in question can be a simple crystal (ex: quartz) whose the thickness is optimized according to the rotatory powers of the crystal at the two optimization wavelengths, in order to obtain a difference in the 180 ° rotation between the two beams.
• the energy ratio in the 3D STED created by a conical crystal is fixed, the vortex energy being carried by a polarization (circular) and the energetic of the black sphere being carried by the other polarization.
• the use of a dichroic element - in the first sense of dichroic is a selectively absorbing element polarizations - allows to modify this ratio.
• This element can be either a circular dichroic element - or a linear dichroic element requiring the addition of a polarization element before and optionally after the dichroic.
• Representative but non-limiting dichroic elements are Brewster's slides or certain dichroic glasses comprising homogeneously oriented elongate metal nanoparticles enclosed in glass surface layers.
• the described 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 modalities of microscopy.
• the superresolution system according to embodiments of the invention can just as easily be integrated on the other microscopy platforms.
• These microscopy platforms include but are not limited to: wide field microscopes, "wide field microscope”, dark field microscopes, "dark field microscope”, polarization microscopes, phase difference microscopes , Differential interference microscopes, microscopes
• 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 TEM (Transmission Electron Microscopy), or PEBM ( Electron Beam Microscopy), or the SE (Scanning Electron Microscopy)
• CLEM-Correlative Light Electron Microscopy or any other similar system such as TEM (Transmission Electron Microscopy), or PEBM ( Electron Beam Microscopy), or the SE (Scanning Electron Microscopy)
• the microscope platform is a complete SRCDP platform, and includes a LongSRCS module, implementing the PDOS method and using the SRCDA algorithm.
• the microscope platform is a partial SRCDP platform, and uses the algorithm SRCDA.
• the microscope platform is a partial SRCDP platform, and uses the control module.
• the microscope platform further comprises a LongSRCS module, implementing the PDOS method
• optical fibers The advantageous use of optical fibers is the transmission of the fundamental mode, the TEMoo mode and only it.
• certain configurations of optical fibers mainly but not exclusively based on fibers called "Photonic Crystal 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.
• 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 ⁇ .
• embodiments of the invention may be applied, as explained later, through an optical fiber. This allows many applications

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