Classifications

• • G—PHYSICS
  • G01—MEASURING; TESTING
  • G01B—MEASURING LENGTH, THICKNESS OR SIMILAR LINEAR DIMENSIONS; MEASURING ANGLES; MEASURING AREAS; MEASURING IRREGULARITIES OF SURFACES OR CONTOURS
  • G01B11/00—Measuring arrangements characterised by the use of optical techniques
  • G01B11/14—Measuring arrangements characterised by the use of optical techniques for measuring distance or clearance between spaced objects or spaced apertures
• • G—PHYSICS
  • G01—MEASURING; TESTING
  • G01B—MEASURING LENGTH, THICKNESS OR SIMILAR LINEAR DIMENSIONS; MEASURING ANGLES; MEASURING AREAS; MEASURING IRREGULARITIES OF SURFACES OR CONTOURS
  • G01B9/00—Measuring instruments characterised by the use of optical techniques
  • G01B9/02—Interferometers
  • G01B9/02097—Self-interferometers
  • G01B9/02098—Shearing interferometers
• • G—PHYSICS
  • G01—MEASURING; TESTING
  • G01B—MEASURING LENGTH, THICKNESS OR SIMILAR LINEAR DIMENSIONS; MEASURING ANGLES; MEASURING AREAS; MEASURING IRREGULARITIES OF SURFACES OR CONTOURS
  • G01B9/00—Measuring instruments characterised by the use of optical techniques
  • G01B9/04—Measuring microscopes
• • G—PHYSICS
  • G01—MEASURING; TESTING
  • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
  • G01N15/00—Investigating characteristics of particles; Investigating permeability, pore-volume or surface-area of porous materials
  • G01N15/10—Investigating individual particles
  • G01N15/14—Optical investigation techniques, e.g. flow cytometry
  • G01N15/1429—Signal processing
  • G01N15/1433—Signal processing using image recognition
• • 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
  • G02B21/0076—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
  • G01B—MEASURING LENGTH, THICKNESS OR SIMILAR LINEAR DIMENSIONS; MEASURING ANGLES; MEASURING AREAS; MEASURING IRREGULARITIES OF SURFACES OR CONTOURS
  • G01B2290/00—Aspects of interferometers not specifically covered by any group under G01B9/02
  • G01B2290/30—Grating as beam-splitter
• • G—PHYSICS
  • G01—MEASURING; TESTING
  • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
  • G01N15/00—Investigating characteristics of particles; Investigating permeability, pore-volume or surface-area of porous materials
  • G01N15/01—Investigating characteristics of particles; Investigating permeability, pore-volume or surface-area of porous materials specially adapted for biological cells, e.g. blood cells
• • G—PHYSICS
  • G01—MEASURING; TESTING
  • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
  • G01N15/00—Investigating characteristics of particles; Investigating permeability, pore-volume or surface-area of porous materials
  • G01N15/10—Investigating individual particles
  • G01N2015/1006—Investigating individual particles for cytology

Definitions

• the present invention relates to a method and an optical telemetry device for determining the three-dimensional position of an object, and is particularly applicable to three-dimensional microscopic imaging in biology but also to passive optical telemetry. (without measurement of flight time).
• the impulse response shape (PSF) control techniques of the imaging system including the microscope objective aim to break the axial symmetry of the PSF so that there is a one-to-one relationship between the section. of the PSF and the axial position of the transmitter relative to the focal plane of the imaging system.
• PSF impulse response shape
• the multi-plane approach consists of simultaneously imaging the signal of a transmitter in axially separated planes.
• the article by S. Abrahamsson et al. For example, "fast multicolor 3D imaging using aberration-corrected multifocus microscopy", Nature Methods, Vol.10 No. 1 (2013) describes the arrangement of a particular network for generating nine images on a single detector corresponding to nine orders of diffraction.
• a limitation of multi-plane techniques is the division of the "photon budget" of the transmitter according to a given number of images, resulting in a loss of sensitivity and therefore of accuracy.
• Axial precision in super-resolution microscopy can be significantly improved by interfering the emitted waves from a transmitter at the focus of two microscope objectives (see G. Shtengel et al., "Interferometric fluorescent super-resolution microscopy resolves 3D cellular ultrastructure” Proc Natl Acad Sci USA 106, 3125 (2009)).
• This last technique which implements a measurement system called “4pi", that is to say using two objectives head to tail to collect the light in almost 4pi steradian, combined with a triple interferometric detection of photons emitted, is the one who offers today the best axial location accuracy, but at the cost of considerable experimental complexity that relegates to a very minor use in biology laboratories.
• This technique is also sensitive to differential aberrations resulting from the passage of the sample forwards or backwards in a 4pi assembly, for any type of sample.
• the present invention implements an interferometric technique which does not have the disadvantages of the previously described techniques; in particular, it allows a full-field imaging adapted to the detection of continuums of fluorescent emitters but requiring only one detector. It is applied in super resolution microscopy but also in conventional microscopy and also finds applications in passive optical telemetry, that is to say for determining the distance of an object in a scene without flight time analysis. .
• the invention relates to a device for measuring the distance, with respect to a reference plane, from a light point of an object, comprising:
• a two-dimensional detector comprising a detection plane
• an imaging system adapted to form an image of a luminous point situated on an object plane of interest in an image plane situated near the detection plane or a conjugate plane of the detection plane;
• a separating element making it possible to form, from a beam emitted by a luminous point of the object plane of interest and emerging from the imaging system, at least two coherent beams between them, having a region of spatial superposition in which the beams interfere with each other;
• signal processing means making it possible to determine from the interference pattern formed on the detection plane and resulting from optical interferences between said beams coherent with each other, the distance from the light point to a conjugate plane of the detection plane in the object space of the imaging system (10), said conjugate plane of the detection plane forming the reference plane.
• a source point may be a fluorescent emitter or "quantum dot” whose spatial dimensions are smaller than the diffraction task of the imaging system.
• a source point may include a larger and spatially coherent area of an object forming on the detection plane a "picture point” whose dimensions of which are much larger than those of the impulse response (or PSF) of the imaging system.
• the device thus described makes it possible in particular to reconstruct an object in 3D without the need to use controlled illumination means of the object; thus, the device described makes it possible, for example, to reconstruct in 3D an object emitting its own light (in the case of a fluorescent emitter in microscopy) or an object re-emitting light without having any control over its illumination (scene of everyday life).
• the separating element as defined in the device according to the present description makes it possible to print within the "image point", that is to say the image of a luminous point of the object formed on the plane of detection, a modulation whose period depends on the relative curvature of the wave coming from the luminous point, finally allowing to achieve a relative elevation mapping of the object.
• the separator element coupled to the detector thus behaves as a curvature sensor of the wave coming from the different light points of the object which prints a modulation within each image point without degrading the resolution.
• the separator element makes it possible to print a modulation within the image point whose period is sufficiently small to form at least two fringes at the image point and to obtain a sufficient measurement precision.
• the period of the fringes of the interference pattern formed on the detection plane (“interfrange”) is smaller than the diameter of the impulse response of the imaging system.
• imaging or PSF, which is the smallest image formed from a bright spot on the detection plane.
• the separating element comprises a diffraction grating close to the imaging plane, for example a two-dimensional diffraction grating.
• the diffraction grating is a transmission network, respectively in reflection, which does not transmit, respectively does not reflect, the zero order.
• the device is applied to three-dimensional imaging; the imaging system then includes a microscope objective.
• the device further comprises a relay optics for forming a conjugate plane of the detection plane in the image space of the imaging system.
• the invention relates to a method for measuring the distance, with respect to a reference plane, of a light point of an object of interest, comprising:
• the formation by means of a separating element, from a beam emitted by the light spot and emerging from the imaging system from at least two beams coherent with each other and having a region of spatial superposition in which the beams coherent between them interfere;
• the distance from the luminous point to the reference plane is obtained from the measurement of the period of the fringes of the interference pattern.
• FIGS. 1A and 1B diagrams illustrating two examples of telemetry device according to the present description
• Figures 2A to 2D diagrams illustrating the principle of the method implemented according to an example; 3, curves showing according to two particular examples the value of the interfrange (pseudo-period of modulation) as a function of the axial position of the source point relative to the reference plane;
• FIGS. 4A and 4B diagrams illustrating telemetry devices according to two other examples
• Figure 5 is a diagram illustrating an exemplary device according to the present description, applied to three-dimensional microscopic imaging
• FIGS. 6A to 6C images illustrating various steps of the telemetry method in a super-resolution microscopy application implemented with a montage of the type of FIG. 5 and with a biological sample comprising CHO cells ("Chinese Hamster Ovary ”) and a labeling of tubulin proteins of the cytoskeleton of these cells;
• FIG. 8A, 8B respectively a standard fluorescence image and an image of the biological sample obtained in a similar configuration (assembly and sample) to that used to obtain the images 6A to 6C;
• FIG. 9 a diagram of a device according to the present description, applied to passive optical telemetry in a scene.
• FIGS. 1A and 1B illustrate two examples of telemetry device according to the present description, adapted for measuring the distance, with respect to a given reference plane, from a luminous point Pi (or "source point") of a object of interest O in a scene.
• the telemetry device 100 shown diagrammatically in FIG. 1A generally comprises a two-dimensional detector 30 with a detection plane P DET connected to signal processing means 50, and an imaging system 10 adapted to form an image of a light point. Pi of an object plane of interest 11, in an image plane 11 'located near the detection plane 31 of the detector.
• the image plane 11 ' is in the vicinity of a plane P' DET conjugated to the detection plane P DET , the device further comprising a relay optic 40 making it possible to form in the image space of the imaging system 10 a conjugate plane of the detection plane.
• the proximity of the image plane to the detection plane depends on the precision sought for the distance measurement.
• the detection plane P DET OR of the conjugate plane P ' DET of the detection plane by the relay optics 40
• the measurement zone corresponds to the zone in which the accuracy of measuring the distance of a light point relative to the reference plane is satisfactory depending on the intended application.
• the measurement zone when seeking a location accuracy of an emitter relative to the reference plane well below the object field depth of the imaging system, can be have a total length L m less than four times the object depth dz of the imaging system and advantageously less than twice the depth of field dz object of the imaging system to ensure a good measurement accuracy.
• the object depth of field dz can be determined by
• n 1 for an imaging system immersed in the air, for example for passive telemetry applications and n3 ⁇ 41.5 for an imaging system immersed in an immersion oil, for example for applications in microscopy of super -resolution.
• the detection plane (or the conjugate of the detection plane) and the image plane 11 'conjugate of the object plane of interest 11 by the imaging system 10 may be separated from each other by a distance less than twice the image field of view of the imaging system, and preferably by a distance less than once the image field depth of the imaging system, in order to benefit from a good accuracy in the measurement of the distance of the luminous point.
• the distance between the detection plane (or the conjugate of the detection plane) and the image plane 11 'conjugate of the object plane of interest 11 by the imaging system 10 can be lengthened to ten times or even twenty times the image depth of field, to the detriment of the localization accuracy which then becomes of the order of depth of field; this mode of operation is mainly interesting for passive telemetry measurement, where to find out where the object is located axially without super-resolution is sufficient.
• the telemetry device For the determination of the distance from a luminous point Pi to the reference plane P REF , the telemetry device comprises a separator element 20 making it possible to form from a beam B 'emitted by the luminous point Pi and emerging from the luminaire system. 10, at least two beams coherent with one another (not shown in FIGS. 1A and 1B) and having a region of spatial superposition in which said coherent beams interfere with one another.
• the separator element 20 will be described in more detail below and may comprise for example a network, advantageously a two-dimensional array, located near the detection plane P DET (example of FIG. 1A) or the plane P ' DET conjugate of P DET detection plane (example of Figure 1B).
• the separator element 20 may also comprise a separator blade or a separator cube, as will be illustrated by way of examples in the following description.
• the separating element is arranged in such a way that the detection plane or the conjugate plane of the detection plane is in the spatial superposition zone of the beams which are coherent with one another and come from the separating element.
• the detection plane of the detector is formed for each source point Pi an image which is the convolution of the response impulse of the imaging system (PSF) with an interference pattern resulting from the interference of beams from the separator element.
• PSF imaging system
• the parameters of the separator element for example the pitch of the grating in the case of a diffraction grating type separator element, or the optical index and the thickness of the separating plate in the case of a separator element. splitter separator element
• the parameters of the separator element for example the pitch of the grating in the case of a diffraction grating type separator element, or the optical index and the thickness of the separating plate in the case of a separator element. splitter separator element
• the period of the modulation (in other words the interfrange of the interference pattern) depends on the relative position of the source point with respect to the reference plane P REF which is the conjugate plane of the plane of detection P DET in the object space of the imaging system 10.
• P REF the conjugate plane of the plane of detection P DET in the object space of the imaging system 10.
• FIGS. 2A to 2D illustrate in greater detail the principle of the method implemented according to a particular example in which the separator element comprises a network 21.
• the network 21 is advantageously a two-dimensional diffraction grating. Two independent axial positioning measurements (eg distance measurements relative to the reference plane) (one along each of the axes of the network) can thus be obtained for a single source point, which increases the axial location accuracy while making possible measurements on images presenting any distribution of sources (continuum of fluorophores, scene of the everyday life ).
• the network 21 is, in a variant, a transmission network adapted to transmit all of the incident light energy, ie a phase grating, when the arrangement is in transmission, or may alternatively be a reflection grating adapted to reflect the entirety of the light energy incident when the assembly is in reflection.
• a transmission network adapted to transmit all of the incident light energy, ie a phase grating, when the arrangement is in transmission, or may alternatively be a reflection grating adapted to reflect the entirety of the light energy incident when the assembly is in reflection.
• a transmission or reflection grating will be chosen which does not transmit, respectively does not reflect, the order 0.
• To eliminate the zero order can be obtained for example by adjusting to ⁇ [2 ⁇ ] the modulation of the phase shift introduced over a period, for example by etching the network substrate or local modification of the substrate index.
• Another possibility to suppress the order 0 is to remove the order 0 in the Fourier space of the network, but in this case, there is introduced a loss of photons and therefore a loss of signal to noise ratio.
• Image point of a source point of the object, the image point being confused with the impulse response of the imaging system, or PSF, in super resolution microscopy applications for example.
• the pitch of the grating comprised between one third of the radius of the image point and three times the radius of the image point, in order to have sufficient lateral sampling of the fringes and to limit oversampling.
• a pitch p of the order of the diameter of the image point to have 2 fringes per image point; and in the case of the application of super resolution microscopy, a pitch p between r / 3 and 3r, advantageously of the order of 2r, where r is the radius of the PSF given by equation (2) ci - above.
• FIGS. 2A to 2D illustrate, by way of example and schematically, the wave propagation in the case of a measuring device comprising a diffraction grating 21 and the light intensity measured in a lateral direction in the plane of detection in the case of two source points positioned at two different axial positions.
• a one-dimensional network of pitch p and it is assumed that the network diffracts only the orders +1 and -1.
• a diffraction grating is, for example, a complex amplitude grating as described, for example, in J. Primot et al. ("Extended Hartmann test based on the pseudoguiding property of a Hartmann mask completed by a phase chessboard", Applied Optics, Vol 39, Issue 31, pp. 5715-5720 (2000)).
• ⁇ interfringe ⁇ as a function of the distance of the source point with respect to the reference plane (ie the conjugate plane of the detection plane in the object space of the system of imaging).
• the relationship between the interfrange (period of the modulation) and the distance from the source point considered to the reference plane can be determined theoretically from the parameters of the diffraction grating chosen, as is explained below according to an example. In the example shown in FIGS.
• the grating 21 is located at a distance d from the detection plane P DET -
• the grating makes it possible to form two diffracted beams coherent with each other, denoted ⁇ and B ' 2 and corresponding respectively to the orders +1 and -1.
• the grating 21 diffracts the light according to the order +1 with an angle ⁇ with respect to the propagation of the direct light (ie order 0 of the grating, indicated in simple dotted lines in the figures).
• the lateral shift between the point of impact on the detection plane of the diffracted order 1 and the point of impact (theoretical) of the order 0 is noted in FIGS. 2A and 2C.
• the lateral offset corresponds to half lateral shift between the points of impact the two "replicas" formed by the two coherent beams entre between each other and B ' 2 .
• the interferogram / (x) formed by the orders +1 and -1 on the detection plane located at a distance d from the network can be described by:
• the interfrange ⁇ measured is normalized by the half-period of the network and the normalized distance z as a function of the depth of field.
• curves show first of all that it is possible from the value of the interfrange to determine the distance z between the image of a source point and the detector and thus to deduce it in the object space. of the imaging system, the distance between a source point of the object and the reference plane.
• the curves of FIG. 3 further illustrate the shape of the curve as a function of the value of the lateral half-shift ⁇ between the replicas, the lateral half-offset being proportional to the distance d between the grating and the plane of detection.
• the curves show that by playing on ⁇ one can enlarge the zone along the optical axis where the measurement is possible but one loses in precision of localization.
• the network will advantageously be placed at a distance sufficiently close to the detection plane so that the lateral shift introduced between the replicas is less than the diameter of the image point, for example the diameter of the PSF ( ⁇ , equation (2)) in the case of super resolution microscopy application.
• ⁇ the diameter of the PSF ( ⁇ , equation (2)) in the case of super resolution microscopy application.
• Inequency (6) is expressed as a function of the choice of the microscope objective at a ratio dlp typically less than a value between 10 and 50 depending on the choice of the microscope objective; thus for spas networks typically between 10 and 30 ⁇ , the distance d will be chosen less than a value that may vary between ten microns and 1 mm depending on the choice of the microscope objective.
• FIGS. 2A to 2D Although described in the case of a one-dimensional network having two diffraction orders, the principle illustrated by means of FIGS. 2A to 2D extends to other examples of diffraction gratings, and in particular a two-dimensional grating allowing two independent axial measurements. (one following each axis of the network) for each source point.
• the axial positioning of the object-forming source points can thus be obtained by measuring the period or local frequency of the interfering frequency formed on the detection plane.
• the positioning of each source point is determined by comparing the measured value of the period / local frequency of the interfering gram formed on the detection plane with a calibration curve of the period / frequency as a function of the axial positioning of the source point.
• the calibration curve can be obtained either theoretically (like the curve shown in FIG. 3 for example) or experimentally by frequency measurements obtained on images of source points whose axial positioning is known.
• the measurement of the local period / frequency of interference generated on the detector can be obtained in several ways.
• the measurement can be done in the direct space (ie directly on the image) by local adjustment (fitting) of the image by a function describing the interferogram (see equation (1) above for example) . This allows to go back to the local period of the interferogram.
• the measurement can also be done by local Fourier transform to find the main peak in the Fourier space, that is to say the local frequency in the image area considered.
• a diffraction grating as described above may be replaced by any system making it possible to generate an equivalent phase and / or absorption function, for example a spatial light modulator (SLM) or a deformable mirror.
• SLM spatial light modulator
• a deformable mirror With SLM, however, the need to work with polarized light results in detrimental photon loss, especially in microscopy applications. With a deformable mirror, the limit comes from the reduced number of actuators that results in too much period of the equivalent network.
• separator elements may be integrated in the device according to the present description to form the curvature sensor thus described. It suffices that such a separating element makes it possible to separate the incident beam emitted by the luminous point into at least two mutually coherent beams having a region of spatial superposition in which the beams interfere.
• FIGS. 4A and 4B thus illustrate two examples of measuring devices according to the present description.
• the separating element comprises a separating plate, for example a semi-reflecting plate, to form a Murty interferometer-type interferometer assembly and in the example of FIG. 4B, the separating element comprises a splitter cube to form a Mach-Zender type interferometric assembly.
• the telemetry device 300 shown diagrammatically in FIG. 4A comprises, as in the example of FIG. 1B, a two-dimensional detector 30 with a detection plane P D AND and an imaging system 10 adapted to form an image of a source point Pi an object plane of interest 1 1 in an image plane 1 1 'close to a plane P'DET conjugate PDET detection plane, the device further comprising a relay optics 40 to conjugate the plane P'DET with
• the telemetry device 300 furthermore comprises a semi-reflecting plate 22 of thickness e, for example a glass plate of index n, arranged to allow reflection.
• the semi-reflecting plate is arranged so as to form an angle of 45 ° with the optical axis of the imaging system, to form an interferometer known as the Murty interferometer. .
• ⁇ intergram / gram / (x) formed on the detection plane by the image of a point source at a distance z from the image by the imaging relay system (40) of the semireflecting plate (here confused with the plane of the detector) can be described by:
• the telemetry device 400 shown diagrammatically in FIG. 4B comprises, as in the example of FIG. 1B, a two-dimensional detector 30 with a detection plane P DET and an imaging system 10 adapted to form an image of a source point Pi d an object plane of interest 11 in an image plane 11 'close to a plane P' DET conjugated to the detection plane P DET , the device further comprising a relay optics enabling the plane P ' DET to be combined with the plane of detection P DET -
• the relay optics comprises two optics 41, 42, between which is arranged the separating element formed here of a Mach-Zender interferometer 23.
• the interferometer comprises in this example a separating plate Si (or of a separator cube) making it possible to form, from the beam B 'coming from the source point Pi and emerging from the imaging system 10, two beams ⁇ and B' 2 coherent with one another. These beams propagate in two independent arms each containing a mirror with total reflection (M ls M 2 ). The beams are recombined thanks to the cube (or blade) separator S 2 . The beams are symbolized for the sake of clarity in Figure 4B by their optical axis.
• the diffraction grating makes it possible to obtain a greater measurement stability because the free space propagation lengths of the beams that are coherent with one another and intended to interfere are less important and, because of this, also the number of free parameters affecting the measurement are lower (notably the angle of the separating plate in the case of Murty's interferometer and angles of the mirrors and semi-reflective plates in the case of Mach Zender's interferometer).
• the diffraction grating also makes it possible to work with all the photons coming from the source point, which is not the case for the Mach Zender assembly, for example. It is therefore preferable to use the diffraction grating as a separating element.
• the network can also allow, in case of suppression of the order 0, to form achromatic interferences unlike the other separator elements.
• FIG. 5 shows a diagram illustrating an exemplary device according to the present description, applied to three-dimensional microscopic imaging, in particular for imaging biological specimens formed of molecular complexes.
• the molecular complexes whose average sizes are typically from a few nanometers for small complexes to about 100 nanometers for the most imposing structures, are marked according to known techniques by a probe capable of emitting a light signal, for example a fluorescent probe. , thus forming emitting particles smaller than the diffraction limit of the optical system used to form an image thereof.
• the particles that one seeks to locate evolve in a support medium that can be liquid or solid, for example in the form of a gel, for example a biological medium.
• the support medium may be arranged directly on a sample holder, deposited on a plate or held between two plates, for example glass plates.
• O object (FIG. 5) is the support medium and the emitting particles evolving therein, as well as the holding plate (s), if appropriate.
• the three-dimensional imaging device 500 comprises in the example of FIG. 5 an imaging system 10 able to form the emitting particle (ie the source point) an image on a detection plane P DET of a detector 30, advantageously a matrix detector, for example a CCD camera, CMOS, an amplified camera EMCCD type (abbreviation of the English expression "Electron Multiplying Coupled Charge Display”), a camera sCMOS, a matrix of photomultipliers.
• a matrix detector for example a CCD camera, CMOS, an amplified camera EMCCD type (abbreviation of the English expression "Electron Multiplying Coupled Charge Display"), a camera sCMOS, a matrix of photomultipliers.
• the imaging system 10 comprises a microscope objective 12, corrected for example for an optical configuration of focus-infinity work, associated with a lens 13, referred to as a tube lens, making it possible to form an image on an intermediate detection plane 11 '.
• the microscope objective assembly and tube lens forms a conventional optical microscope system.
• the imaging device 500 further comprises relay objectives 40 making it possible to form a plane P ' DET conjugate of the detection plane P DET of the detector 30 in the image space of the imaging system 10, the image plane 11 'being located near the conjugate plane P' DET .
• the image which is the convolution of the object with the impulse response of the imaging system or PSF, is here substantially confused with the impulse response.
• a motorized platform (not shown) may be present and allows the sample O to be moved in an XY plane perpendicular to the optical axis of the microscope objective.
• a mechanical axial focusing device (not shown) may be present and can adjust the axial position of the sample relative to the focal plane object of the microscope objective 12 and thus image the area of interest.
• the sample holder, the motorized platform, the axial focusing device, the microscope objective 12 and the tube lens 13 are arranged in a microscope body 60 of known type.
• the microscope body may furthermore comprise, in a conventional manner, an eyepiece, a source of illumination of the sample associated with a condenser.
• the microscope body is of inverted type (microscope objective positioned under the sample) but it could equally well be a right microscope (microscope objective above the microscope). 'sample).
• the imaging device 500 also comprises a separator element 20, for example a two-dimensional network close to the conjugate plane P ' DET for the formation of at least two coherent beams with a region of spatial superposition at the detector, for the implementing the method of determining the distance according to the present description.
• a separator element 20 for example a two-dimensional network close to the conjugate plane P ' DET for the formation of at least two coherent beams with a region of spatial superposition at the detector, for the implementing the method of determining the distance according to the present description.
• the network is for example a phase grating for suppressing the order 0 and whose pitch is chosen to form on the detector interference of period less than the lateral resolution of the microscope. It is arranged perpendicular to the optical axis so that the coherent replicas between them overlap and can interfere with each other.
• FIGS. 6A to 6C show images obtained at different stages of application of the method according to the present description, in a super-resolution microscopy application implemented with a device of the type of that of FIG. 5.
• Object O is a biological sample of CHO ("Chinese Hamster Ovary") cells fixed with paraformaldehyde.
• the tubulin proteins of the cellular cytoskeleton were fluorescently labeled with specific antibodies.
• the fluorescent probes are of the Alexa 647 type and the sample is observed by means of a CMOS type matrix detector 30 by the dSTORM technique, in which flashing is achieved through the use of the Vectashield mounting product (Vectro Labs) and the use of a 635 nm laser.
• Figure 6A shows a fluorescence image at the start of acquisition of dSTORM; the density of the fluorescent molecules is still significant, resulting in a spatial continuum of fluorescence emission. However, it is already possible to view the interference.
• Figure 6B shows a zoom on part of the image being acquired in which the density of emitters is lower; the response of a single fluorescent emitter (quantum dot) in which the modulation resulting from the interference of coherent replicas between them is distinguished. The periodicity of this modulation makes it possible to determine the axial positioning of the transmitter relative to the reference plane, as explained above.
• the lateral positioning is obtained by determining the centroid of the image (or image point) by barycentre or ideally by adjusting the image by a Gaussian describing the PSF of the imaging system 10, after suppression of the filtering modulation (pass low frequency) of the thumbnail.
• FIG. 6C shows the dSTORM 3D image reconstructed by 3D localization of each fluorescence emitter according to the present description (as in 6B) thanks to the stochastic flashing of the emitters, the gray scale representing the axial positioning of the emitters.
• FIGS. 7A, 7B, 7C show experimental measurements of the light distribution measured using a type 500 assembly in the presence of single fluorescent emitters (of the nanocrystal semiconductor type, or quantum dot) situated either in a conjugated plane the detector (7B) is before (7C) or after (7A) while remaining in the depth of field (ie no variation of the dimension of the image). It is clear that ⁇ interfringe ⁇ change depending on the axial positioning of the transmitter (i) although one is in the depth of field and (ii) without the size of the image of the transmitter is widened by compared to classical imagery (circle of confusion).
• FIGS. 8A and 8B represent experimental images of the same biological sample obtained by a standard epi-fluo (8 A) imaging and 3D STORM imaging technique with the technique proposed herein.
• the gain in lateral resolution (8B) with respect to 8A is clearly observed and also the axial positioning allowing to follow the 3D spatial evolution of the cyto skeleton.
• the distance measurement method according to the present description can also be applied to "passive" telemetry, that is to say without measurement of flight time, for the measurement. of distances of objects in a scene.
• FIG. 9 thus represents a scene with several objects O 1 , O 2 , situated at different distances from a distance measuring device 600.
• the objects are illuminated in natural light and retro diffuse the natural light so that each point of an object Oi forms a source point Pi.
• the device 600 comprises, as previously, a detector 30 with a detection plane P DET , an imaging system 10 adapted to form a scene in an image plane located near the detection plane P DET , and a separator element 20.
• a diffraction grating close to the detection plane P DET , and making it possible to form, from a beam emitted by the light point and emerging from the imaging system, at least two mutually coherent beams ("replicas") presenting a region of spatial superposition in which the beams interfere.
• the imaging system 10 comprises for example a photo or video lens adapted to work at infinity
• the reconstruction of the axial positioning of the source points can be done in a volume of a few times the depth of field ( ⁇ 10x) of the imaging system.
• Axial resolutions are of the order of depth of field and up to 1/100 of the latter.
• the absolute values of the axial resolution and the depth of field change. It may be interesting to adjust the aperture given a focal length so that the dynamic measurement of axial positioning of the various points of the object encompasses all the objects of interest in a given scene; that is, the objects of interest are included in the depth of field a few times.
• the modification of the lateral offset ⁇ allows to adjust the axial positioning measurement dynamic with the technique described in order to obtain a reconstruction of all the objects of interest of the scene.
• the measurement of the variation of the interfringe from one point to another makes it possible to go back to the 3D profile of the constituents of the scene.
• optical telemetry method according to the invention and the device for implementing said method comprise different variants, modifications and improvements which will be obvious to those skilled in the art, it being understood that these various variants, modifications and improvements are within the scope of the invention as defined by the following claims.

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