Device for locating an individual particle in a sample and associated method

Abstract

The invention relates to a device (100) for locating an individual particle (2) in a sample (3) having a plurality of regions (31), the particle being capable of emitting photons when it receives light, the device comprising: - a sample holder (110); - a light source arranged to produce an illumination field suitable for illuminating the sample placed on its holder, an intensity of the illumination field having a temporal and spatial variation such that the intensity has, in each region of the sample, a predetermined temporal pattern different from the temporal patterns of the intensity in the other regions; - a detector (140) capable of converting photons emitted by the particle in response to the illumination thereof by the illumination field into a signal representative of the intensity of the illumination field received by the particle; - a computer (150) programmed to determine, on the basis of a temporal change in the signal derived from the detector and the temporal and spatial variation in the intensity of the illumination field, the region of the sample in which the particle is located.

Description

TITLE OF THE INVENTION: DEVICE FOR LOCATING AN INDIVIDUALIZED PARTICLE IN A SAMPLE AND ASSOCIATED METHOD TECHNICAL FIELD OF THE INVENTION

[0001] The present invention relates generally to the field of microscopy.

[0002] It relates more particularly to a device for locating an individual particle in a sample.

[0003] It also relates to a method of locating an individual particle in a sample.

[0004] The invention finds a particularly advantageous application in the production of super-resolution microscopy devices. STATE OF THE ART

[0005] The 2014 Nobel Prize in Physics recognized the revolutionary ideas of super-resolution fluorescence microscopy, which allows us to overcome the diffraction limit to obtain images with nanometer resolution and thus reveal cellular architecture at the molecular level. One of the central principles of this super-resolution microscopy relies on the temporal control of fluorescent molecular markers. These are activated sequentially to maintain emitter densities low enough to be able to spatially isolate them and localize them individually with nanometer precision. In other words, this super-resolution microscopy allows us to locate individual particles in a sample with extreme precision.These locations on successive images, on which the particles appear sparse, are then combined to reform a super-resolved image of the sample.

[0006] Current techniques, however, require cameras with arrays of sensors (also called pixels) to enable wide-field localization of molecules. These cameras are relatively slow detectors, and the fastest ones are very expensive. Some applications, such as fluorescence lifetime measurements, are therefore inaccessible due to the low sampling frequency of these cameras. PRESENTATION OF THE INVENTION

[0007] One aim of the invention is therefore to enable the localization of light-emitting particles quickly and reliably.

[0008] In this context, the present invention provides a device for locating an individualized particle in a sample having several regions, the particle being adapted to emit photons when it receives light, the device comprising: - a sample holder; - a light source arranged to produce an illumination field adapted to illuminate the sample placed on its support, an intensity of the illumination field having a temporal and spatial variation, so that said intensity has, in each region of the sample, a predetermined temporal pattern different from the temporal patterns of said intensity in the other regions; - a detector adapted to convert photons emitted by the particle in response to its illumination by the illumination field into a signal representative of the intensity of the illumination field received by the particle; - a computer programmed to determine, on the basis of a temporal evolution of the signal from the detector and the temporal and spatial variation of the intensity of the illumination field, the region of the sample in which the particle is located.

[0009] Thus, thanks to the invention, the location of the particle is coded bijectively by means of the temporal patterns, that is to say by means of the illumination field which is spatially structured and which is variable in time. This coding thus allows a super-localization of the entire observation field with a single detector.

[0010] Indeed, the position information of the particle is contained in the variation of amplitude of the signal which is generated by the detector. Thus, although the detector cannot in itself spatially determine the origin of the photons which it receives, the temporal evolution of the signal which it generates is representative of the temporal pattern which illuminates the particle. The analysis of this temporal evolution therefore makes it possible to go back to the region of the particle.

[0011] It is therefore not necessary to image the sample in wide field with a camera to determine the position of the particle in the sample, which significantly increases the acquisition speed.

[0012] The invention thus advantageously makes it possible to overcome the limitations imposed by camera sampling frequencies.

[0013] Furthermore, the applicant has demonstrated, by means of a calculation of the Cramèr-Rao bound, that with such a spatially structured and time-varying illumination field, a localization precision 2.4 times better in one dimension and 1.7 times better in two dimensions than that obtained by traditional localization can be achieved.

[0014] In summary, remarkably, the localization of the particle by means of the device according to the invention is precise and rapid.

[0015] To illustrate the type of bijective coding proposed in the invention, it is for example provided, according to an advantageous and non-limiting characteristic of the invention, that each temporal pattern of the intensity of the illumination field produced by the light source is a periodic temporal variation of this intensity and has a specific frequency, the specific frequencies of the temporal patterns associated with all the regions being distinct from each other.

[0016] Thus, each region is associated with a unique specific frequency. There is therefore no ambiguity about the location of the particle, even when using a single detector instead of a camera.

[0017] Other advantageous and non-limiting characteristics of the device according to the invention, taken individually or in all technically possible combinations, are the following: - each temporal pattern of the intensity of the illumination field produced by the light source is a periodic temporal variation of this intensity and has a specific frequency, the specific frequencies of the temporal patterns associated with all the regions being distinct from each other; - the regions of the sample are aligned along a main direction of the support and said light source is arranged so that each specific frequency is linked by the same relationship to a distance between the position of the region associated with the corresponding time pattern and a reference position along the main direction; - the said relationship is a relationship of proportionality; - each temporal pattern of the intensity of the illumination field produced by the light source is a sinusoidal temporal variation of this intensity; - the illumination field is adapted to simultaneously illuminate several regions of the sample; - the light source comprises an optical beam splitting device and a converging element arranged to generate and interfere two light beams with each other so as to produce interference fringes at the sample placed on its support; - the optical device for separating light beams comprises at least one of the following: a corner mirror having two perpendicular reflecting surfaces, a mirror and a partially reflecting plate; a splitter cube having a partially reflecting internal diagonal surface; - the light source further comprises a movable optical element whose movement causes said interference fringes to scroll to produce each temporal pattern of the intensity of the illumination field; - said mobile optical element comprises a rotating device arranged to vary over time an angle of incidence of each light beam on the sample placed on its support, the angles of incidence of the light beams varying simultaneously and in opposite directions; - the rotating device is a rotating mirror; - the light source comprises a polarization modulator adapted to modify the polarization of the illumination field so that the illumination field is polarized, sequentially or simultaneously, according to: a first polarization direction; and a second polarization direction distinct from the first polarization direction; the computer being programmed to determine a position of the particle according to the first main direction and according to the second main direction as a function of the polarization of the illumination field; - when the illumination field is simultaneously polarized according to the first and second directions, the specific frequencies of temporal patterns associated with the first direction of polarization are distinct from the specific frequencies of temporal patterns associated with the second direction of polarization.

[0018] The invention also provides a method for locating an individualized particle in a sample having several regions, the particle being adapted to emit photons when it receives light, the method comprising the following steps: - arrangement of the sample on a support; - production of an illumination field, the illumination field illuminating the sample placed on the support, an intensity of the illumination field exhibiting a variation temporal and spatial, such that said intensity has, in each region of the sample, a predetermined temporal pattern different from the temporal patterns of said intensity in the other regions; - conversion of the photons emitted by the particle in response to its illumination by the illumination field into a signal representative of the intensity of the illumination field received by the particle; - determination, on the basis of a temporal evolution of the signal from the detector and the temporal and spatial variation of the intensity of the illumination field, of the region of the sample in which the particle is located.

[0019] Other advantageous and non-limiting characteristics of the method according to the invention, taken individually or in all technically possible combinations, are the following: - the particle is selected from the group comprising: diffusing particles, fluorescent particles, phosphorescent particles, quantum dots, fluorescent proteins, dye molecules; - the particle is fluorescent, the illumination field is produced by a pulsed laser at a frequency greater than 1 MHz and the detector is adapted to count single photons, the method comprising a step of determining a delay between a pulse of the laser and the emission of a photon by the particle. DETAILED DESCRIPTION OF THE INVENTION

[0020] The description which follows with reference to the appended drawings, given as non-limiting examples, will make it clear what the invention consists of and how it can be implemented.

[0021] On the attached drawings:

[0022] Figure 1 is a schematic representation seen from above of a device for locating an individualized particle in a sample according to a first embodiment,

[0023] Figure 2 is a top view schematic representation of a first example of an optical separation device and a movable optical element of the device of Figure 1,

[0024] Figure 3 is a top view schematic representation of a second example of an optical separation device and a movable optical element of the device of Figure 1,

[0025] Figure 4 is a schematic representation of a scrolling of interference fringes over time (t) produced by the device of Figure 1 in projection along a main direction (X) at the level of the sample,

[0026] Figure 5 is a first example of a frequency spectrum obtained for a particle at a first position along the main direction (X) of Figure 4,

[0027] Figure 6 is a second example of a frequency spectrum obtained for a particle at a second position along the main direction (X) of Figure 4,

[0028] Figure 7 is a graphical representation of the localization accuracy of the device of Figure 1,

[0029] Figure 8 is a schematic representation seen from above of a device for locating an individualized particle in a sample according to a second embodiment,

[0030] Figure 9 is a schematic perspective representation of interference fringes produced by the device of Figure 8 at the sample level,

[0031] Figure 10 is a schematic representation of the frequency processing of a signal from the detector in order to determine the trajectory of a particle,

[0032] Figure 11 is a graphical representation of scrolling frequencies of interference fringes produced by the device of Figure 8 as a function of the position (x, y) of a particle in a first direction and in a second direction,

[0033] Figure 12 is a block diagram of a sequence of steps of a method according to the invention allowing the localization of an individualized particle in a sample according to a main direction,

[0034] Figure 13 is a schematic representation in section in a plane (O, X, Z) of the light beams incident on the sample through an objective of a device according to the invention and of oblique interference fringes produced by these beams within the sample.

[0035] In the following description, corresponding or identical elements of each embodiment of the invention will be identified by the same reference signs, and will not be described in detail each time.

[0036] Two embodiments of a device 100; 300 for locating an individualized particle 2 in a sample 3 having several regions 31 according to the invention are respectively represented in FIGS. 1 to 6 and 8.

[0037] The device 100; 300 is an imaging device for locating the particle 2.

[0038] According to the invention, the device 100; 300 for locating the particle 2 comprises: - a support 110; 310 of sample 3, - a light source arranged to produce an illumination field 200; 400 adapted to illuminate the sample 3 placed on its support 110; 310, an intensity of the illumination field 200; 400 having a temporal and spatial variation, so that said intensity has, in each region 31 of the sample 3, a predetermined temporal pattern different from the temporal patterns of said intensity in the other regions; - a detector 140; 340 adapted to convert photons emitted by the particle 2 in response to its illumination by the illumination field 200; 400 into a signal representative of the intensity of the illumination field 200; 400 received by the particle 2 ; - a computer 150; 350 programmed to determine, on the basis of a temporal evolution of the signal from the detector 140; 340 and the temporal and spatial variation of the intensity of the illumination field 200; 400, the region 31 of the sample 3 in which particle 2 is located.

[0039] Particle 2 is for example a molecule, a group of molecules or a group of identical atoms. Particle 2 is here adapted to emit photons when it receives light. Particle 2 for example has a linear response to the light it receives, which means that its photon emission is proportional to the intensity of the light it receives. Particle 2 can also have a non-linear response to the light it receives, in particular when an excitation level of particle 2 is close to the values ​​associated with a saturation of particle 2. This is for example the case when the particle used is a fluorophore and a photon absorption rate by particle 2 is of the same order of magnitude as the inverse of a fluorescence lifetime of particle 2.

[0040] Particle 2 is for example selected from the group comprising: diffusing particles (such as a gold particle), fluorescent particles, also called fluorophores, phosphorescent particles, quantum dots, fluorescent proteins, dye molecules. Particle 2 here has a size of less than one micrometer. The device 100 is therefore more specifically a microscopy device. Generally, the particle 2 preferably has a size between 1 nm and 40 nm.

[0041] The particle 2 is conventionally located in the volume of the sample 3 or on the surface of the sample 3. The sample 3 is at least partially transparent to light, in particular to the wavelengths generated by the device 100 and described below, so that the light reaches the particle 2.

[0042] As shown in Figures 1 and 8, sample 3 is here thin in the sense that its thickness is significantly less than its width and length. For example, sample 3 is a slice or section of a biological tissue. Sample 3 therefore extends substantially around a mean plane. Sample 3 has, for example, a thickness along the Z axis of Figure 1 of between 10 micrometers (pm) and 100 pm and a length and / or width in the plane (OXY) of Figure 1 greater than 1 centimeter (cm).

[0043] The regions 31 of the sample 3 are distinct portions of the sample 3, the set of portions forming the sample 3. The regions 31 may be point-like or extended. They may be extended in one, two or three dimensions. Each region 31 may, for example, correspond to a particular position within the sample 3. Thus, the term “region” includes in particular the notion of point-like position defined by specific coordinates (x,y,z) in the reference frame (O, X, Y, Z) of Figure 1. The term “region” may also designate, for example, a plane extending in the directions Y and Z and intersecting the axis X at a particular position.

[0044] The fact that particle 2 is individualized here means that the particle is spatially separated from other particles by a distance greater than the resolution limit of the device according to the invention. In practice, here, this means that two neighboring particles 2 are at least 2 times, and preferably 5 times, apart from the resolution limit given by diffraction which is approximately 1 pm. Two neighboring particles 2 are therefore, for example, at least 2 pm apart. This means, for example, that the region 31 containing particle 2 does not include any other particles.

[0045] The support 110; 310 of the device 100; 300 is adapted to receive the sample 3. Here, the support 110; 310 more specifically comprises a plate 111; 311 extending along an extension plane.

[0046] Furthermore, the holder 110; 310 is also suitable for moving the sample 3 with respect to the optical elements of the device 100; 300, in particular with respect to a lens 170; 370 bringing the light produced by the light source towards the support 110; 310. For this purpose, the support 110; 310 comprises actuators 112; 312, for example piezoelectric, adapted to move the plate 111; 311 in its extension plane.

[0047] Sample 3 is placed on plate 111; 311 so that the mean plane of sample 3 extends parallel to the extension plane of plate 111; 311.

[0048] The regions 31 of the sample 3 may be defined discretely, for example so as to grid the sample 3 into rows and columns perpendicular to its thickness. Each region 31 of the sample 3 may be associated with a corresponding area of ​​the plate 111; 311 forming the support 110; 310, which is located opposite the region 31 when the sample 3 is placed on the support.

[0049] The regions 31 of the sample 3 can also be defined continuously. In this case, they are for example identified by coordinates, for example Cartesian, in a reference frame linked to the support 110; 310. The reference frame linked to the support 110; 310 comprises for example an orthogonal reference frame referenced OXYZ in FIGS. 1 and 8. In this orthogonal reference frame OXYZ, the thickness of the sample 3 is defined as the dimension of the sample 3 along the Z axis, the length of the sample 3 is defined as the dimension of the sample 3 along the X axis and the width of the sample 3 is defined as the dimension of the sample 3 along the Y axis. The origin O of the orthogonal reference frame OXYZ is for example placed at one of the corners of the plate 111; 311 when the latter has the shape of a rectangle.

[0050] The light source of the device 100; 300 is partially shown in Figures 2, 3. It is also presented in full in Figure 8 in the context of the second embodiment of the device 300. It is arranged to produce an illumination field 200; 400 adapted to illuminate the sample 3 placed on the support 110; 310. The light source is therefore configured to illuminate the particle 2 which, in return, emits photons.

[0051] The term "illumination field" 200; 400 denotes here the spatial distribution of the intensity of the light (i.e. the separate beams described later) emitted by the light source and conveyed to the sample 3. The intensity of the illumination field 200; 400 produced by the light source exhibits a temporal and spatial variation.

[0052] Such a temporal and spatial variation of the intensity of the illumination field 200 is for example represented in FIG. 4 in the context of the first embodiment of the device according to the invention. As shown in FIG. 4, at each given instant t, the intensity of the illumination field 200 produced by the light source of the device 100 according to the invention has different values ​​at different locations in space corresponding here to different distances from the origin O of the orthogonal reference frame OXYZ (FIG. 1) along the X axis. In particular, here, the intensity of the illumination field 200 has one or more maxima (represented by the white areas) and one or more minima (represented by the black areas) along this X axis.

[0053] The spatial variation of the intensity of the illumination field is different at each instant t, which leads to the temporal variation of this intensity.

[0054] The temporal variation of the intensity of the illumination field 200 is illustrated in FIG. 4 by a displacement of the maxima(s) and minima(s) over time. The intensity of the illumination field 200 is for example defined at an upper surface 32 of the sample 3 which faces the light source. The intensity of the illumination field 200 can also be defined at the plate 111.

[0055] As mentioned previously, whatever the embodiment of the device considered, the light source is more particularly arranged so that the intensity of the illumination field 200; 400 has, in each region 31 of the sample 3, a predetermined temporal pattern different from the temporal patterns of the intensity in the other regions 31 of the sample 3. In other words, each region 31 is associated bijectively with a temporal pattern of the illumination field 200; 400. The temporal variation of the illumination field 200; 400 assigns a specific temporal code to each zone of the plate 111; 311 forming the support 110; 310 and therefore to each region 31 of the sample 3.

[0056] For this, the light source is here arranged precisely relative to the support 110; 310 of the sample 3, for example relative to the areas of the plate 111; 311 or relative to the OXYZ reference frame. Once the sample 3 is placed on the support 110; 310, the light source is then arranged relative to the sample 3 itself by the correspondence between the support 110; 310 and the sample 3.

[0057] Each temporal pattern is a variation of intensity according to a predetermined pattern, or structure. Each temporal pattern is thus characterized by its temporal variation. Thus, for each region 31 of the sample, the intensity of the illumination field 200; 400 varies temporally in a specific and predetermined manner.

[0058] Each temporal pattern comprises, for example, a periodic variation in intensity. For example, each pattern may more specifically be a sinusoidal temporal variation in intensity, as described in detail later.

[0059] Advantageously, as will be clearly shown below, several regions 31 are illuminated concomitantly by the illumination field 200; 400, as opposed to a sequential illumination of each of the regions (one after the other, in the manner of a scan). In other words, each region 31 is illuminated by its temporal pattern, in the sense that it actually receives photons, at the same time as other regions 31 are illuminated by their respective temporal patterns. When the size of the sample allows it (because it is sufficiently small), all the regions 31 of the sample 3 are illuminated simultaneously by the illumination field 200; 400, which means that the sample 3 is illuminated in its entirety. In general, all the regions 31 of the sample 3 located in the field of view of the objective 170; 370 are illuminated simultaneously. Localization is therefore fast since encoding is carried out in parallel for all regions.

[0060] To produce this temporal and spatial variation of the illumination field 200; 400, the light source more particularly comprises a light emitter 130; 330 adapted to produce light and a set of optical elements arranged to generate the variation of the illumination field 200; 400 from the light produced by the light emitter 130; 330. In the example of the first and second embodiments described here, this set of optical elements notably comprises an optical device for separating light beams 122; 322 adapted to divide the beam emitted by the light emitter 130; 330 into two light beams, a mobile optical element and a converging optical element adapted to superimpose the two separated beams to make them interfere.

[0061] The light emitter 130; 330 (Figures 2, 3 and 8) is for example a laser, for example a laser diode. The laser here emits an initial light beam 210; 410 whose central wavelength (i.e. highest amplitude) is for example between 405 nm and 800 nm. The light emitter 130: 330 can be adapted to produce light continuously with a constant intensity. It can also be a pulsed light source, producing light at regular time intervals.

[0062] The detector 140; 340 (figures 1 and 8) of the device 100; 300 according to the invention is adapted to convert the photons emitted by the particle 2 in response to its illumination by the illumination field 200; 400, into a signal. The signal is thus representative of the intensity of the illumination field 200; 400 received by the particle 2. The signal is here an electrical, digital or analog signal. The temporal variation of the signal includes information on the temporal pattern of the light beam which illuminated the particle 2.

[0063] The amplitude of the signal produced by the detector 140; 340 is more particularly representative of a number of photons emitted by the particle 2 in response to its illumination by the illumination field 200; 400 per unit of time. For example, when the particle 2 exhibits a linear response to the light it receives, the amplitude of the signal is then proportional to the intensity of the illumination field 200; 400 received by the particle 2.

[0064] The detector 140; 340 here has a sampling frequency greater than 1 MHz. The detector is for example selected from the group comprising: single-detectors, single-detector arrays, detectors comprising single-photon avalanche diodes, event detectors whose pixels generate signals in response to a change in received light intensity, neuromorphic detectors. A single-detector is defined as a detector comprising a single photosensitive element (for example a single pixel), it therefore does not have intrinsic spatial resolution.

[0065] The device 100; 300 may also comprise a set of optical elements for shaping and directing the light beam(s).

[0066] As shown more particularly in Figure 8, the optical shaping elements comprise for example one or more diaphragms 331, as well as one or more optical lenses 332 and a beam expander 334. Each diaphragm 331 makes it possible to eliminate stray light from the light beam. The optical lenses 332 make it possible to shape the beam: adjust its diameter, focus it at a precise location or create a parallel beam. The optical elements for orienting the beam(s) comprise for example mirrors 333.

[0067] These optical elements being well known to those skilled in the art, they will not be described in detail in the remainder of the description.

[0068] The device 100; 300 further comprises a dichroic mirror 141; 341 (figures 1 and 8) arranged to, on the one hand, reflect the light produced by the light emitter 130; 330 to direct it towards the support 110; 310 and the sample 3 and, on the other hand, transmit the photons emitted by the particle 2 towards the detector 140; 340.

[0069] The device 100 also comprises a recovery lens 142; 342 which focuses the photons emitted by the particle 2 towards the detector 140.

[0070] In the first embodiment shown in Figure 1, the light beam from the recovery lens 142 is directly received by the detector 140. In the second embodiment shown in Figure 8, the device 300 further comprises an optical fiber 343. The recovery lens 342 focuses the photons emitted by the particle 2 at the input of this optical fiber 343, the optical fiber 343 guiding them to the detector 340.

[0071] In Figures 1 and 8, the path of the photons emitted by particle 2 to the detector 140; 340 is represented schematically by a wide light beam whose envelope is represented by short and long dotted lines (two short one long) while the light beams produced by the light source have an envelope represented by regular dotted lines. This representation of the light beams going towards the sample 3 in short and long dotted lines also appears in Figures 2, 3, 9, and 13.

[0072] The device 100; 300 according to the invention finally comprises a computer 150; 350 programmed to analyze the signals produced by the detector 140; 340.

[0073] The computer 150; 350 here comprises at least one memory and at least one processor. The memory is a computer-readable recording medium comprising instructions which, when executed by the processor, make it possible to locate the particle 2. Locating the particle means in particular determining the region 31 of the sample 1 in which the particle 2 is located.

[0074] The computer 150; 350 is more particularly programmed to determine, on the basis of a temporal evolution of the signal and the temporal and spatial variation of the intensity of the illumination field 200; 400, that is to say on the basis of the predetermined temporal patterns, the region 31 of the sample 3 in which the particle 2 is located.

[0075] Indeed, as explained above, each region 31 of the sample 3 is associated bijectively with a temporal pattern of the illumination field 200; 400. The signal emitted by the detector 140; 340 is representative of the intensity of the illumination field 200; 400 received by the particle 2. The temporal evolution of the signal emitted by the detector 140; 340 is therefore associated with a temporal pattern of the illumination field 200; 400 and consequently with a region 31. The computer 150; 350 is thus programmed to analyze the temporal evolution of the signal emitted by the detector 140; 340, to recognize one of the predetermined temporal patterns therein, and to determine the region 31 associated with this temporal pattern, that is to say the region 31 in which the particle 2 is located.The calculator 150; 350 here identifies the regions 31 of the sample relative to the support 110, for example via the different zones of the plate 111; 311 of the support 110; 310, or, as in the embodiments described later, by means of the OXYZ reference linked to the support 110; 310.

[0076] Here, the computer 150; 350 is also programmed to control the light source. Alternatively, the light source comprises a dedicated computer. This dedicated computer then transmits the relevant information, in particular about the predetermined time patterns, to the computer so that the latter can locate the particle 2.

[0077] The implementation of the device 100; 300 here makes it possible to implement a method for locating the particle 2. As shown in Figure 12, this method according to the invention comprises the following steps: E1 - arrangement of sample 3 on support 110;310; E2 - production of the illumination field 200, 400 by the light source, and illumination of the sample 2 by the illumination field 200; 400; E3 - conversion, by the detector 140; 340, of the photons emitted by the particle 2 in response to its illumination by the illumination field 200; 400 so as to generate the signal; E4 - determination, on the basis of the temporal evolution of the signal and the temporal and spatial variation of the intensity of the illumination field 200, of the region 31 of the sample 3 in which the particle 2 is located.

[0078] In order to better understand the invention, a first embodiment of the device 100 for locating the particle 2 along a main direction is first described. This first embodiment is described with reference to Figures 1 to 6.

[0079] This main direction here corresponds to the direction of movement of the interference fringes which create the predetermined temporal patterns, as explained later. Thus, according to this first embodiment, the illumination field 200 produced by the light source has predetermined temporal patterns aligned along the main direction.

[0080] In this first embodiment, the main direction is parallel to the X axis of the OXYZ reference frame. The main direction is therefore substantially parallel to the mean plane of the sample 3. Each region 31 thus extends over the entire width of the sample 3, i.e. over its entire dimension along Y, and over the entire thickness of the sample 3, i.e. over its entire dimension along Z.

[0081] In this first embodiment, the regions 31 are thus also aligned along the main direction. The regions 31 are defined continuously. Each region 31 corresponds to an abscissa, referenced x, along the X axis and which can take a real value. Each region 31 of the sample 3 is therefore characterized by a distance between this region 31 and a reference position along the main direction. The reference position is preferably the origin O of the OXYZ reference frame.

[0082] Thus, the location of particle 2 along a principal direction corresponds here to the location of particle 2 along the X axis. Subsequently, reference is therefore made to the location of particle 2 along the X axis, i.e. to the determination of the abscissa of particle 2.

[0083] The length of the sample 3 (therefore along the X axis) along which the particle 2 is detectable is here greater than 500 nm and preferably greater than 1 pm. In other words, the regions 31 extend over a length greater than 500 nm and preferably greater than 1 pm. This length depends in particular on the portion of the sample 3 located in the field of observation of the detector 140.

[0084] According to a first exemplary embodiment of the optical separation device 122 of the light source of the first embodiment, the latter comprises a device of the interferometer type arranged to separate the initial light beam 210 into two separate light beams 221, 222 as shown in FIG. 2. These two separate light beams 221, 222 are superimposed using an objective 170, to make them interfere with each other so as to form interference fringes 230.

[0085] The illumination field 200 is then formed by these interference fringes 230. The interference fringes 230 are schematically represented in FIG. 4. In this figure (as well as in FIGS. 8, 9 and 13), the half-period of a given interference fringe which includes the maximum intensity of said fringe contains points while the half-period which includes the minimum intensity does not contain any points. The numerical references indicating the interference fringes point to one type of half-period as well as the other. The interference fringes 230 extend here along the Y axis, i.e. perpendicular to the X axis. As FIG. 4 clearly shows, the illumination field 200 simultaneously illuminates several X abscissae (i.e. several regions 31).

[0086] In the example shown in Figure 2, the optical device for separating light beams 122 comprises a wedge mirror 123 having two perpendicular reflecting surfaces, a plane mirror 124 and a semi-reflecting plate 125 which are arranged as in a Michelson interferometer used in air wedge mode.

[0087] As shown in Figure 2, the initial beam 210 is divided, here in equal parts, by the semi-reflecting plate 125 to form the two separate light beams 221, 222. The separate light beam 210 is, on the one hand, transmitted by the semi-reflecting plate 125 into a first separate light beam 221. This first separate light beam 221 is reflected in parallel by the corner mirror 123 then by the semi-reflecting plate 125 towards the support 110. The initial beam 210 is, on the other hand, reflected by the semi-reflecting plate 125 into a second separate light beam 222. This second separate light beam 222 is reflected by the plane mirror 124 to be transmitted towards the support 110 by the semi-reflecting plate 125.

[0088] In the example shown in Figure 3, the optical device for separating light beams 122 is formed by a splitter cube 126 having a semi-reflecting internal diagonal surface 127. The splitter cube 126 is for example made of glass. As shown in Figure 3, the splitter cube 126 is oriented so that the semi-reflecting internal diagonal surface 127 is parallel to the initial beam 210 before it enters the splitter cube 126. The initial beam 210 is divided, here equally, by the semi-reflecting internal diagonal surface 126 to form two separate light beams 22T, 222'. These then emerge parallel to each other from the splitter cube 126.

[0089] In all cases, the converging optical element of the light source here comprises the objective 170 which is provided with a converging lens 128 arranged to converge the two separate beams 221, 222; 22T, 222' towards the support 110 to cause the two separate light beams 221, 222; 22T, 222' to interfere so as to produce the interference fringes 230 (figure 4) at the level of the sample 3 placed on its support 110.

[0090] The mobile optical element is arranged so that its movement causes the interference fringes 230 to scroll. The movement of the mobile optical element is here controlled by the computer 150.

[0091] This scrolling of the interference fringes 230 then forms the temporal patterns. Thus, the temporal variation of the illumination field 200 results from the movement of the mobile optical element.

[0092] The movable optical element is here a rotating mirror and more particularly a galvanometric mirror 129. The movement of the movable optical element is therefore a rotation of the galvanometric mirror 129, which is shown diagrammatically by curved arrows in figures 2 and 3.

[0093] The galvanometric mirror 129 is adapted to vary over time the angle of incidence a of each separate light beam 221, 222; 221'; 222' on the sample 3 placed on its support 110. This is advantageous because the cost of a galvanometric mirror 129 is high.

[0094] For this, the galvanometric mirror 129 is placed upstream of the beam splitter device 122. In other words, as illustrated in FIGS. 2 and 3, the galvanometric mirror 129 makes it possible to orient, i.e. to modify the direction and / or to translate, the initial light beam 210. The galvanometric mirror 129 is for example arranged so that the initial light beam 210 is incident at the level of the axis of rotation of the galvanometric mirror 129.

[0095] Particularly advantageously, a rotation of the galvanometric mirror 129 varies the angles of incidence a of the separate light beams 221, 222; 22T, 222' simultaneously and in opposite directions. Thus, the position of the optical contact at the level of the sample 3 is invariant over time despite the rotation of the galvanometric mirror 129.

[0096] In the example of Figure 2, this effect is obtained in particular thanks to the corner mirror which makes it possible to return the first separated light beam 221. In this example, when the galvanometric mirror 129 rotates in the direction clockwise, the separate light beams 221, 222; 22T, 222' deviate from each other symmetrically.

[0097] In the example of Figure 3, a lens 143 is placed between the galvanometric mirror 129 and the splitter cube 126 to fix the angle of incidence of the initial light beam 210 on the splitter cube 126 despite the rotation of the galvanometric mirror 129. In this example, when the galvanometric mirror 129 rotates in the trigonometric direction, for example to move from the position referenced R1 to the position referenced R2, the separated light beams 221, 222; 22T, 222' move away from each other symmetrically, which is represented by arrows of the same length.

[0098] The operation of the first embodiment of the device 100 makes it possible to implement the method of locating the particle 2 along the X axis by determining its abscissa.

[0099] In this first embodiment, step E2 of producing the illumination field 200 and illuminating the sample 3 by the illumination field 200 comprises the rotation of the galvanometric mirror 129.

[0100] This rotation causes an identical variation in the angles of incidence of the separate light beams 221, 222; 22T, 222' on the sample 3 and therefore a scrolling of the interference fringes 230. At a given rotation speed, the interference fringes 230 scroll more specifically with a scrolling speed which depends on the distance from the optical contact, at which the phase shift between the separate light beams 221, 222; 22T, 222' is zero, along the X axis. The further a region 31 is from the optical contact, the faster the interference fringes 230 which illuminate it scroll.

[0101] In Figure 4, the optical contact is positioned at the abscissa referenced xo. The optical contact thus defines the reference position along the X axis (therefore along the main direction). As explained previously, the abscissa xo of the optical contact is invariant during rotation because the angles of incidence a of the separate light beams 221, 222; 22T, 222' vary simultaneously and in opposite directions. This is schematically represented in Figure 4 by a maximum and constant light intensity over time at the abscissa xo of the optical contact.

[0102] The galvanometric mirror 129 here rotates at a constant rotation speed (apart from a start phase and a stop phase). This regular rotation implies that each temporal pattern is a periodic, and more specifically sinusoidal, temporal variation of the intensity of the illumination field 200 at a specific frequency. In other words, each region 31 sees the interference fringes 230 scroll regularly. For each region 31, the intensity of the illumination field 200 received therefore varies sinusoidally.

[0103] All specific frequencies are related by the same relationship to the distance between the position of their associated time pattern, i.e. the abscissa x of their associated region 31, and the optical contact along the main direction. The specific frequencies are therefore all related here by the same relationship to the value X-Xo.

[0104] Here, this relationship is more particularly a relationship of proportionality. This means that each region 31 sees the interference fringes 230 pass at a specific frequency which is proportional to the abscissa x of said region 31. Thus, by noting I the intensity of the illumination field 200, and by assigning the value zero to the abscissa xO, for a given region 31 of abscissa x, the intensity I varies over time t according to the formula (1): l(x,t) oc 1 +cos(atx), where a is a coefficient of proportionality which depends in particular on the speed of rotation of the galvanometric mirror 129.

[0105] Thus, the rotation of the galvanometric mirror 129 at a constant rotation speed implies that each temporal pattern is a sinusoidal variation of the intensity of the illumination field 200 at a specific frequency which is proportional to the abscissa of the region 31 associated with said temporal pattern.

[0106] Such scrolling is illustrated in Figure 4 in which a first region 31 of abscissa xi is located closer to the optical contact (abscissa xo) than a second region 31 of abscissa X2, which means that X2 > xi. In the time interval shown in Figure 4, the first region 31 sees approximately four interference fringes 230 scroll by and the second region 31 approximately six interference fringes 230. The intensity I2 received by the second region 31 therefore varies at a specific frequency higher than that of the intensity 11 received by the first region 31, the specific frequencies being respectively proportional to the abscissas X2 and xi.

[0107] As illustrated in Figure 4, the distance measured along the X axis on which the particle 2 is detectable is here greater than the fringe distance L at at least one given instant, during the rotation of the galvanometric mirror 129. Preferably, the distance measured along the X axis on which the particle 2 is detectable is greater than the interfringe L during a given time interval, for example, at all times beyond a reference time. This distance is for example here greater than the distance X2-X0 along the X axis of Figure 4. In other words, the regions 31 extend over a length greater than the interfringe L at at least one given time during the rotation of the galvanometric mirror 129, preferably over a given time interval. This means that at said given time or in the given time interval, the regions 31 are illuminated by more than one interference fringe 230, preferably by several interference fringes.

[0108] Here, during step E2, the galvanometric mirror 129 more particularly performs oscillations, that is to say rotations in one direction and in the other. In one direction of rotation, new interference fringes 230 appear on the sample 3 over time, as is the case in FIG. 4, in the other direction of rotation, interference fringes 230 come out of the sample 3 over time. In both cases, the interference fringes 230 pass at specific frequencies.

[0109] The amplitude of these oscillations is for example less than 5 degrees. A rotation of the galvanometric mirror 129 is for example carried out in a time interval between 10 ms and 100 ms. The oscillation frequency of the galvanometric mirror 129 is then between 10 Hz and 5000 Hz.

[0110] The scrolling frequency of the interference fringes 230 is therefore several orders of magnitude higher than the oscillation frequency of the galvanometric mirror 129, which makes it possible to locate particle 2 very quickly. Indeed, as appears below, it is preferable for particle 2 to be illuminated by several periods of the time pattern to obtain a precise determination of its location.

[0111] The specific frequencies, i.e. the frequencies of the interference fringes 230, can for example be as high as 1 MHz.

[0112] After the conversion of the photons emitted by particle 2 to form the signal, in step E3, the computer 150 analyzes the temporal evolution of the signal in step E4.

[0113] Here, step E4 more specifically comprises the performance by the computer 150 of a Fourier transform of the signal. This makes it possible to determine the frequencies contained in the signal.

[0114] As a reminder, the temporal evolution of the signal is representative of the temporal pattern that illuminated particle 2. For example, when particle 2 is located at abscissa xi, the temporal evolution of the signal is representative of intensity 11 in the sense that the signal varies mainly at the same frequency as intensity 11.

[0115] The Fourier transform thus makes it possible to determine the specific frequency associated with said temporal pattern.

[0116] In practice, the Fourier transform presents a peak, i.e. a maximum M1, M2, corresponding to the specific frequency of the temporal pattern which illuminates particle 2.

[0117] The computer 150 then determines the region 31 in which the particle 2 is located. In this first embodiment, it can be deduced from the formula (1) that the specific frequency, noted f, of the pattern which illuminates the particle located at the abscissa x is given by: f = a x. The abscissa x of the region 31 in which the particle 2 is located is therefore given by x = f / a. The coefficient a is for example determined by moving the sample 3, and therefore the particle 2, in translation along the X axis by means of the actuators 112. This in fact makes it possible to determine the coefficient a by solving a system of two equations with two unknowns.

[0118] In other words, on the basis of the specific frequency, the calculator 150 therefore recognizes the temporal pattern which illuminates the particle 2. Then, each region 31 being associated bijectively with a temporal pattern, the calculator 150 determines the region 31 in which the particle 2 is located.

[0119] For example, when particle 2 is located at abscissa xi in Figure 4, the Fourier transform of the signal can in practice exhibit a frequency spectrum as illustrated in Figure 5. In comparison, when particle 2 is located at abscissa X2 in Figure 4, the Fourier transform of the signal can in practice exhibit a frequency spectrum as illustrated in Figure 6. The frequency spectrum in Figure 6 exhibits a maximum M2 at a frequency approximately twice that of the maximum M1 of the spectrum in Figure 5, which derives from the fact that abscissa X2 is approximately twice that of abscissa xi (here because the relationship between specific frequencies and abscissas is a relationship of proportionality). In figures 5 and 6, the peaks at mid-height of the maxima referenced M1 and M2 correspond here to a second particle positioned at another abscissa.

[0120] The specific frequency can be determined very precisely thanks to the high sampling frequency of the detector 140. Preferably, several periods of variation of the intensity of the illumination field 200, for example more than four, are detected by the detector 140 in order to increase the determination of the specific frequency. Thus, the faster the interference fringes 230 pass, the more precise the determination of the specific frequency in a given time (since more periods are detected).

[0121] This very precise determination of the specific frequency therefore allows a near-precise determination of the abscissa x of particle 2 by the formula x = f / a mentioned above. In this sense, the process of localizing particle 2 is therefore a super-resolution process.

[0122] In Figure 7, experimentally obtained particle localization accuracies, expressed by standard deviations o as a function of the number of photons N detected, are graphically compared to the theoretical Cramer-Rao limit (solid line) for a signal-to-noise ratio equal to that of the experimental measurements (here one). The experimental accuracies are represented by circles or stars depending on the collection angle used to capture the light reflected by sample 3 (see top right of Figure 7). As shown in Figure 7, the experimental accuracies are very close to the theoretical lower limit, which clearly illustrates the super-resolution of the localization method.

[0123] The method implemented in this first embodiment also makes it possible to locate several particles present in the sample 3 along the X axis. For example, in the case where two particles 2 are positioned at the abscissas xi and X2, as shown in FIG. 4, the signal is representative of both the intensity 11 and the intensity I2. The frequency spectrum of the signal then has two peaks: one at the variation frequency of 11 and the other at the variation frequency of I2. The computer 150 then determines the two abscissas corresponding to the two specific frequencies detected. When the number of particles is not predetermined, the peaks are for example distinguished from the noise by appropriate thresholding.

[0124] As a variant of this first embodiment, the galvanometric mirror can rotate at a non-constant speed. The time patterns are then not sinusoidal variations. A pattern could for example be a sinusoid whose specific frequency increases over time. Many temporal patterns can be achieved by adapting the rotation of the galvanometric mirror. The computer is then also programmed to recognize these temporal patterns, for example by having in memory all the temporal patterns that are predetermined, in order to determine the region in which the particle is located.

[0125] Up to now it has been described how to locate the particle 2 in one dimension (here along the X axis). The device 100, however, makes it possible to locate the particle in two dimensions, i.e. along the main direction and along a secondary direction distinct from the main direction.

[0126] For this, it is for example possible to implement another light source operating alternately with the light source, which means that they illuminate sample 3 in turn. The other light source is adapted to locate particle 2 according to the secondary direction by means of another illumination field having another spatial and temporal variation.

[0127] Advantageously, it is also possible to locate particle 2 in two dimensions, without using two light sources alternately illuminating the sample.

[0128] According to the second embodiment of the device 300 shown in figure 8, the localization of the particle 2 is carried out in two dimensions by means of a single light source.

[0129] The idea of ​​this second embodiment is to polarize the illumination field 400 according to two distinct polarization directions.

[0130] In this second embodiment, the secondary direction is here parallel to the Y axis of the XYZ reference frame. Thus, the main and secondary directions are orthogonal.

[0131] In this second embodiment, each region 31 extends over the entire thickness of the sample 3, i.e. over its entire dimension along Z. The regions 31 are thus distributed so as to grid the surface of the sample 3. The regions 31 are defined continuously. Each region 31 is characterized by an abscissa, referenced x, along the X axis and an ordinate, referenced y, along the Y axis which can take a real value.

[0132] The localization of particle 2 along a secondary direction is here equivalent to the localization of particle 2 along the Y axis. Subsequently, reference is therefore made to the localization of particle 2 along the Y axis, i.e. to the determination of the y-ordinate of particle 2. Here, particle 2 is therefore located in the XY plane.

[0133] The area of ​​the XY plane in which particle 2 is detectable is here greater than 500x500 nm 2 and preferably greater than 1 pm 2 . In other words, the 31 regions extend over an area greater than 500x500 nm 2 and preferably greater than 1 pm 2 This surface depends in particular on the portion of the sample 3 located in the observation field of the detector 140. This surface is also here greater than the square of the fringe spacing at at least one given instant during the rotation of the galvanometric mirror 129.

[0134] In this second embodiment, the illumination field 400 can be polarized according to a first polarization direction, the illumination field 400 then makes it possible to locate the particle 2 according to the X axis. This means that, according to the first polarization direction, the illumination field 400 has a first intensity which varies spatially and temporally so as to allow the location of the particle according to the main direction in accordance with the first embodiment.

[0135] The illumination field 400 can also be polarized according to a second polarization direction, here orthogonal to the first, the illumination field 400 then makes it possible to locate the particle 2 according to the Y axis. This means that, according to the second polarization direction, the illumination field 400 has a second intensity which varies spatially and temporally so as to allow the location of the particle according to the second main direction in accordance with the first embodiment.

[0136] Here, the first polarization direction of the illumination field 400 is, at the level of the sample 3, orthogonal to the main direction. The first polarization direction is more specifically parallel to the Y axis. The second polarization direction of the illumination field 400 is, at the level of the sample 3, parallel to the main direction. The second polarization direction P2' is more specifically parallel to the X axis (Figure 9). Thus, each polarization direction, for example the second polarization direction P2' in Figure 9, is orthogonal to the plane in which the separated light beams 422 propagate, that is to say the YZ plane in the case of Figure 9.

[0137] Thus, the polarizations are parallel to the interference fringes 430. This maximizes the contrast of the interference fringes. In other words, the first intensity presents first temporal patterns generated by a scrolling of first interference fringes 431 extending rectilinearly parallel to the first direction of polarization, and, conversely, the second intensity has second temporal patterns generated by a scrolling of second interference fringes 432 extending rectilinearly parallel to the second direction of polarization (figure 9).

[0138] For this, in practice, the light source of the device 300 according to the second embodiment comprises, as in the first embodiment described previously, a light emitter 330, a beam splitting device 322 and a movable optical element.

[0139] The light emitter 330 comprises a laser source and the movable optical element comprises a galvanometric mirror 329.

[0140] The optical device for separating light beams 322 comprises, as in the first embodiment, a corner mirror 323 having two perpendicular reflecting surfaces, a plane mirror 324 and a semi-reflecting plate 325 arranged in a Michelson interferometer type configuration.

[0141] These elements are similar to those of the first embodiment and will not be described in further detail here.

[0142] As shown in Figure 8, the light source of the second embodiment further comprises: - a 360 polarization modulator; and - first polarizing cube 361 and a second polarizing cube 362.

[0143] The polarization modulator 360 is placed between the light emitter 330 and the galvanometric mirror 329. The polarization modulator 160 is adapted to polarize the initial beam 410 according to the first polarization direction and / or the second polarization direction. This modulator can be an electro-optical modulator such as a Pockels cell or a magneto-optical modulator such as a Faraday cell.

[0144] The first polarizing cube 361 is placed downstream of the optical light beam splitting device 322 so as to direct the separated light beams 421 polarized according to the first polarization direction towards a first optical path 371 and the separated light beams 422 polarized according to the second polarization direction towards a second optical path 375. By rotating the polarization of the initial beam 410 between the first polarization direction polarization and the second polarization direction, the polarization modulator 360 makes it possible to select respectively the first optical path 371 and the second optical path 375.

[0145] Along the first optical path 371, the polarization is initially orthogonal to the interference fringes 330. The light source comprises a half-wave plate 372 arranged on the first optical path 371 so as to rotate the polarization by 90 degrees perpendicular to the direction of propagation. Thus, at the level of the sample 3, the first direction of polarization is parallel to the first interference fringes 431. This rotation of the polarization is shown diagrammatically in FIG. 8 by the passage from a horizontal polarization P1 to a vertical polarization P2. As also shown in FIG. 8, along the first optical path 371, the polarization and the first interference fringes 430 are vertical after the half-wave plate 372.

[0146] Along the second optical path 375, it is necessary to rotate the second interference fringes 432 by 90 degrees so that they extend perpendicular to the first interference fringes 431 so as to allow localization along the Y axis. For this, the light source comprises a Dove prism type assembly 376 arranged on the second optical path 375 so as to rotate the second interference fringes 432. The polarization is rotated by the polarization modulator 360 when the latter polarizes the initial beam according to the second polarization direction. This rotation of the polarization, which also makes it possible to select the second optical path 375, is shown diagrammatically in FIG. 8 by the transition from a vertical polarization PT to a horizontal polarization P2'.

[0147] The Dove 376 prism setup here is a reflecting prism made using mirrors. This avoids introducing additional birefringence. Typically, two mirrors are arranged at 45 degrees to the direction of propagation and a third, between these two mirrors, is arranged parallel to the direction of propagation.

[0148] Finally, the second polarizing cube 362 recombines the two polarizations, i.e. the light beams from the two optical paths 371, 375, before the illumination field 400 illuminates the sample 3.

[0149] The operation of the second embodiment of the device 100 makes it possible to implement the method of localizing the particle 2 described previously according to the main direction and according to the secondary direction, which here amounts to determining its abscissa x according to the X axis and its ordinate y according to the Y axis.

[0150] For this, the illumination field 400 can be polarized according to the first polarization direction and according to the second polarization direction sequentially or simultaneously.

[0151] When the polarizations of the illumination field 400 are implemented sequentially, i.e. alternately, the sample 3 is illuminated either by the first interference fringes 431 or by the second interference fringes 432. The polarization modulator 360 therefore makes it possible to select the direction of localization of the particle 2 in the sense that it makes it possible to choose between a localization along the X axis and a localization along the Y axis. The information on an instantaneous polarization of the illumination field 400, i.e. the fact that the illumination field 400 is polarized along the first polarization direction or along the second polarization direction, is transmitted from the polarization modulator 360 to the computer 350.The computer 350 is then programmed to determine, on the basis of this information on the polarization of the illumination field at time t, that the specific frequency that it detects in the frequency spectrum at time t applies to the location along the X axis, i.e. to the determination of the abscissa of the particle 2, or that the specific frequency that it detects in the frequency spectrum at time t applies to the location along the Y axis, i.e. to the determination of the ordinate of the particle 2.

[0152] Figure 10 illustrates a two-dimensional tracking (along the X and Y axes) of a fluorescent nanoparticle in random Brownian motion in a liquid. Specific frequencies associated with each X, Y axis are alternately extracted from the signal (referenced D at the bottom left of Figure 10) coming from the detector. Time sections T1, T2 of the signal in fact alternately encode the position of the particle along each X, Y axis. In Figure 10, two successive time sections T1, T2 are framed, a first section T1 coding for the Y axis and the following T2 for the X axis (the T1 section is also shown enlarged). The frequency spectrum S1, S2 of each time section T1, T2 makes it possible to determine the specific frequency of this section (at the top left of Figure 10, pointed by an x ​​or y arrow). The time evolution of specific frequencies can then be represented by time-frequency curves C1, C2. At the top right of the figure 10, the time-frequency curve referenced C1 is relative to the Y axis while the time-frequency curve referenced C2 is relative to the X axis. The temporal evolution of the specific frequencies makes it possible to deduce the position of the particle over time along the X and Y axes, that is to say the trajectory of the particle, as represented at the bottom right of figure 10.

[0153] When the polarizations of the illumination field 400 are implemented simultaneously, the sample 3 is illuminated by both the first interference fringes 431 and the second interference fringes 432.

[0154] The signal detected by the detector 340 is then representative of both the scrolling of the first interference fringes 431 and the scrolling of the second interference fringes 432. The frequency spectrum then comprises two peaks: a first peak corresponding to the scrolling frequency of the first interference fringes 431 (i.e. a first frequency specific to the first pattern which illuminates the particle 2) and a second peak corresponding to the scrolling frequency of the second interference fringes 432 (i.e. a second frequency specific to a second pattern which illuminates the particle 2).

[0155] Preferably, the scrolling frequencies of the first interference fringes 431 (i.e., the first specific frequencies of the first time patterns) are distinct from the scrolling frequencies of the second interference fringes 432 (i.e., the second specific frequencies of the second time patterns). Thus, as shown in FIG. 11, the scrolling frequencies of the first interference fringes 431 belong to a first frequency range f1 that is disjoint (here higher) from a second frequency range f2 of the scrolling frequencies of the second interference fringes 432.

[0156] Thus, the computer 350 can unambiguously assign each of the two peaks of the frequency spectrum, i.e. the specific frequencies, either to a scrolling frequency of the first interference fringes 431 when the peak belongs to the first frequency range f1, or to a scrolling frequency of the second interference fringes 432 when the peak belongs to the second frequency range f2. The computer 350 can therefore determine for each peak whether the latter corresponds to an abscissa or an ordinate of the particle 2.

[0157] For this, each optical path 371, 375 can be adjusted independently so as to adjust the position of the optical contact for each polarization directions. Changing the position of the optical contact allows the frequency ranges to be shifted. This is achieved, for example, by moving the sample 3 using the actuators 112 or by changing the orientation of one of the mirrors 333 of the optical elements for orienting the light beam(s).

[0158] The method implemented by the device 300 according to the second embodiment can also make it possible to locate several particles present in the sample 3 in the XY plane. For example, with two particles, the computer 350 performs the matching of an abscissa with an ordinate on the basis of the time intervals during which the particles 2 emit photons. Indeed, the two particles illuminate at different times due to the stochasticity of appearance of the emission of the particles observed in sparse regime, that is to say when there is a single particle 2 in a diffraction volume which ensures that the diffraction spots corresponding to two different particles of the sample can be separated. This can be obtained either spontaneously, or by inducing optical and / or chemical control of the particles.In other words, the emission of photons by each particle occurs with a random time lag after its illumination. The photon emissions of the particles in the sample are therefore randomly shifted relative to each other, giving the impression that they are blinking.

[0159] Thus, when the polarizations of the illumination field 400 are implemented simultaneously, at a given instant, the frequency spectrum comprises only the specific frequencies of a single particle, which can therefore be located in the XY plane. Each frequency spectrum which is analyzed by the computer 350 is for example obtained by Fourier transform performed over a period of 50 ms of the signal. When the polarizations of the illumination field 400 are implemented sequentially, the alternation between the two polarizations is performed at a high rate to have an alternation at least once, and preferably several times, during the time interval during which a particle emits photons. The alternation is for example performed every 25 ms and each analyzed frequency spectrum is for example obtained by Fourier transform performed over a period of 50 ms of the signal.

[0160] Preferably, to individualize the particles in sample 3, sample 3 comprises a concentration of simultaneously emitting particles of less than 1 particle / pm 3 With such a concentration, the particles then appear sparse in sample 3 when they emit.

[0161] Up to now it has been described how to locate particle 2 in one dimension (first embodiment) or two dimensions (second embodiment). The invention, however, also makes it possible to locate the particle in three dimensions, i.e. along the main direction and the secondary direction and along a third direction that is not coplanar with the other two. The third direction is here parallel to the Z axis of the XYZ frame. Thus, the main direction, the secondary direction and the third direction are orthogonal. Here, particle 2 is therefore located in the XYZ frame.

[0162] For this, it is for example possible to implement a system combining the device 300 according to the second embodiment, so as to locate the particle 2 in the XY plane, with the device 100 according to the first embodiment but oriented so that its main direction is parallel to the Z axis, which means here that the latter illuminates the sample 3 through its edge with incident beams parallel to the XY plane.

[0163] Advantageously, it is provided here to adapt the device 300 according to the second embodiment by redividing one of the polarizations, i.e. by redividing one of the optical paths 371, 375. This makes it possible to use a single laser, a single galvanometric mirror and a single optical beam separation and convergence device.

[0164] For this, the first optical path 371 comprises for example a third polarizing cube dividing the polarization of this first optical path 371 in two. The two new polarizations are recombined before illuminating the sample 3. This allows localization along the Z axis (i.e. in thickness) by means of interference fringes oblique to the Z axis.

[0165] The idea is for example to tilt interference fringes in the Z+X direction and other interference fringes in the ZX direction. Figure 13 illustrates interference fringes 631 tilted in the Z+X direction. For this, the separate light beams 621, 622 of each new polarization (the one along Z+X in Figure 13) are spatially spaced from each other in the rear focal plane Pf of the objective 570. In practice, the separate light beams 621, 622 are off-centered with respect to the axis of the objective 570 in the rear focal plane Pf. Indeed, as illustrated in Figure 13, the angle of incidence of one of the two separate light beams 621, 622 on the sample 3 depends on its position in the plane rear focal plane Pf. Its deviation from the axis of the lens 570 is more particularly proportional to the sine of this angle of incidence. The interference fringes 631 are in practice inclined at a given angle (45° in Figure 13) equal to the average of the angles of incidence of the two separate light beams 621, 622. To maintain modulation along a given axis (for example along X+Z in Figure 13), the two separate light beams 621, 622 move in the rear focal plane Pf, moving away from and approaching the axis of the lens 570.

[0166] The device 100; 300 according to the invention can be implemented in numerous applications.

[0167] For example, the device 100; 300 according to the invention can be used to carry out lifetime imaging of fluorescent particles. In other words, the device 100; 300 then allows a more precise localization than the diffraction limit (thanks to the variation of the illumination field detailed above) of the particle(s) while measuring a relaxation time of the fluorescence of the particle 2. The relaxation time of the fluorescence of the particle 2, i.e. the time during which the particle emits fluorescence after illumination by the illumination field, provides information on its local chemical or biochemical intoxication.

[0168] For this, the light emitter 130; 330 is more particularly a laser pulsed at a frequency greater than 1 MHz. The detector 140; 340 is adapted to detect a single photon. The envelope of the signal is then representative of the scrolling of the interference fringes, which allows localization, and the individual detections of photons which form the signal can make it possible to measure a delay between a pulse of the laser and the emission of a photon by the particle in response to this pulse, as well as the duration of emission of the photons by the particle which makes it possible to determine the fluorescence lifetime of the particle, that is to say the duration of the emission of photons by the particle 2, or in other words a relaxation time of the particle 2.

[0169] As another example, the device 100; 300 makes it possible to follow the movement of the particle 2, typically when the sample 3 is a biological tissue, since the particle 2 is located at a high rate which is for example greater than 20 Hz.

[0170] The device 100; 300 according to the invention can be implemented in the context of multiplexing, i.e. detection of photons at wavelengths different. Multiplexing here means that sample 3 comprises at least two particles of different types: a first emitting photons predominantly at a first wavelength and a second particle emitting photons predominantly at a second wavelength distinct from the first wavelength. The emission spectra of the two particles may also be slightly shifted while partially overlapping, the particles preferably remaining excitable with the same wavelength.

[0171] In a first type of multiplexing, the device 100; 300 comprises a single light source (the light source), which means here that the illumination field 200: 400 has a single main wavelength. The device 100 then comprises a second detector adapted to detect photons at the second wavelength. The detector 140; 340 is for its part adapted to detect photons at the first wavelength.

[0172] In a second type of multiplexing, the device 100; 300 comprises two light sources each adapted to produce an illumination field whose main frequency is adapted to one of the particles and a single detector (i.e. the detector 140), which is for example a spectrometer, adapted to detect photons at the first and second wavelengths.

[0173] The device 100; 300 according to the invention can also be used to produce a confocal or multi-confocal type system. The principle is then to replace the camera which is traditionally used in confocal or multi-confocal type systems by the detector 140; 340 (which is of course coupled to the light source and to the computer 150; 350). Advantageously, the frequency localization by means of the illumination field of the sparse particles in the samples makes it possible to replace the camera with a single-detector and therefore to increase the acquisition speed by at least one order of magnitude.

[0174] The present invention is in no way limited to the embodiments described and shown, but those skilled in the art will be able to provide any variation in accordance with the invention.

[0175] For example, interference fringes can be circular fringes such as Newton's rings or even present other more complex profiles.

[0176] For example, the variation of the illumination field can be achieved in a way other than by a scrolling of interference fringes. For example, the A light source may include multiple lasers, each illuminating a region of the sample according to its own temporal pattern. This temporal pattern corresponds, for example, to a laser pulse frequency. Thus, by using lasers with distinct pulse frequencies, it is possible to assign a specific frequency code to each region, thus making it possible to locate the particle. As another example, the patterns can be produced by wave-front shaping, which allows arbitrary shapes to be obtained. They can also be produced by the superposition of non-coherent laser beams.

[0177] Thus, it also appears that the variation in intensity of the illumination field can vary in ways other than sinusoidal. For example, it can have a notched structure when pulsed lasers are used.

[0178] Consequently, it also appears that localization can be achieved in reference frames that do not have a main direction as described in the examples above. Indeed, when several lasers each illuminate a region of the sample according to its own temporal pattern, the regions can be distributed in two dimensions in the plane of the sample according to various patterns that do not necessarily imply that the regions are aligned or crisscross the sample. For example, the regions can correspond to concentric rings. The regions can also be arranged along a curved line.

[0179] The main direction, as well as the secondary direction and the third direction can of course correspond to a reference frame other than the orthogonal reference frame XYZ.

[0180] Other beam splitting devices, other movable optical elements and other converging optical elements may be implemented.

[0181] In the second embodiment, the beams are selected on the basis of their polarization by optical means, in this case polarizing cubes. They can also be selected by mechanical means, such as galvanometric mirrors or rotating mirrors, or by acousto-optic means such as acousto-optic modulators.

[0182] Here, in order to locate one or more particles in a large sample, the sample 3 can be moved by means of the piezoelectric actuators 112. Different areas of the sample 3 are then treated one after the other. others as is the case in Figures 1 and 8. However, as a variant, the device may comprise several detectors. Each detector is then arranged towards a particular area of ​​the support (and therefore of the sample) to convert photons emitted by the particle(s) in this particular area. In other words, the support and the sample are divided into zones, each zone being associated with a detector. Within each zone, several regions are then defined in order to carry out the localization described above.

[0183] Finally, the localization method represented in figure 12 can of course be implemented by any suitable device and not exclusively by that represented in the figures.

Claims

CLAIMS

1. Device (100; 300) for locating an individualized particle (2) in a sample (3) having several regions (31), the particle (2) being adapted to emit photons when it receives light, the device (100; 300) comprising: - a support (110; 310) for a sample (3), - a light source arranged to produce an illumination field (200; 400) adapted to illuminate the sample (3) placed on its support (110; 310), an intensity (11, I2) of the illumination field (200; 400) having a temporal and spatial variation, so that said intensity (11, I2) has, in each region (31) of the sample (3), a predetermined temporal pattern different from the temporal patterns of said intensity (11, I2) in the other regions (31); - a detector (140; 340) adapted to convert photons emitted by the particle (2) in response to its illumination by the illumination field (200; 400) into a signal representative of the intensity (11, I2) of the illumination field (200; 400) received by the particle (2); - a computer (150; 350) programmed to determine, on the basis of a temporal evolution of the signal from the detector (140; 340) and the temporal and spatial variation of the intensity (11, I2) of the illumination field (200; 400), the region (31) of the sample (3) in which the particle (2) is located.

2. Device (100; 300) according to claim 1, wherein each temporal pattern of the intensity (11, I2) of the illumination field (200; 400) produced by the light source is a periodic temporal variation of this intensity (11, I2) and has a specific frequency, the specific frequencies of the temporal patterns associated with all the regions (31) being distinct from each other.

3. Device (100; 300) according to claim 2, wherein the regions (31) of the sample (3) are aligned along a main direction (X) of the support (110) and wherein said light source is arranged so that each specific frequency is linked by a same relationship to a distance between the position of the region associated with the temporal pattern corresponding and a reference position according to the main direction (X).

4. Device (100; 300) according to claim 3, wherein said relationship is a proportionality relationship.

5. Device (100; 300) according to one of claims 1 to 4, in which the illumination field (200; 400) is adapted to simultaneously illuminate several regions (31) of the sample (3).

6. Device (100; 300) according to one of claims 1 to 5, in which each temporal pattern of the intensity (11, I2) of the illumination field (200; 400) produced by the light source is a sinusoidal temporal variation of this intensity (11, I2).

7. Device (100; 300) according to one of claims 1 to 6, wherein the light source comprises an optical beam splitting device (122; 322) and a converging element (170; 370) arranged to generate and interfere two light beams (221, 222, 22T, 222'; 421, 422; 621, 622) with each other so as to produce interference fringes (230; 430, 431, 432; 631) at the sample (3) placed on its support (110; 310).

8. Device (100; 300) according to claim 7, wherein the optical light beam splitting device (122; 322) comprises at least one of the following: - a corner mirror (123; 323) having two perpendicular reflecting surfaces, a mirror (124; 324) and a partially reflecting plate (125; 325); - a separator cube (126) having a partially reflective internal diagonal surface (127).

9. Device (100; 300) according to claim 7 or 8, wherein the light source further comprises a movable optical element whose movement causes the scrolling of said interference fringes (230; 430, 431, 432; 631) to produce each temporal pattern of the intensity (11, I2) of the illumination field (200; 400).

10. Device (100; 300) according to claim 9, wherein said movable optical element comprises a rotatable device (129; 329) arranged to varying over time an angle of incidence (a) of each light beam (221, 222, 221 222'; 421, 422; 621, 622) on the sample (3) placed on its support (110; 310), the angles of incidence (a) of the light beams (221, 222, 22T, 222'; 421, 422; 621, 622) varying simultaneously and in opposite directions.

11. Device (100; 300) according to claim 10, wherein the rotating device (129; 329) is a rotating mirror.

12. Device (300) according to one of claims 1 to 11, wherein the light source comprises a polarization modulator (360) adapted to modify the polarization of the illumination field (400) so that the illumination field (400) is polarized, sequentially or simultaneously, according to: - a first direction of polarization; and - a second polarization direction distinct from the first polarization direction; the calculator (350) being programmed to determine a position of the particle (2) according to the first main direction and according to the second main direction as a function of the polarization of the illumination field (400).

13. The device (300) of claims 2 and 12, wherein when the illumination field (400) is simultaneously polarized in the first and second directions, the specific frequencies of temporal patterns associated with the first polarization direction are distinct from the specific frequencies of temporal patterns associated with the second polarization direction.

14. Method for locating an individualized particle (2) in a sample (3) having several regions (31), the particle (2) being adapted to emit photons when it receives light, the method comprising the following steps: - arrangement of the sample (3) on a support (110; 310); - production of an illumination field (200; 400), the illumination field (200; 400) illuminating the sample (3) placed on the support (110; 310), an intensity (11, I2) of the illumination field (200; 400) having a temporal and spatial variation, so that said intensity (11, I2) has, in each region (31) of the sample (3), a predetermined temporal pattern different from the temporal patterns of said intensity (11, I2) in the other regions (31); - conversion of the photons emitted by the particle (2) in response to its illumination by the illumination field (200; 400) into a signal representative of the intensity (11, I2) of the illumination field (200; 400) received by the particle (2); - determination, on the basis of a temporal evolution of the signal from the detector (140; 340) and the temporal and spatial variation of the intensity (11, I2) of the illumination field (200; 400), of the region (31) of the sample (3) in which the particle (2) is located.

15. Method according to claim 14, according to which the particle (2) is selected from the group comprising: diffusing particles, fluorescent particles, phosphorescent particles, quantum dots, fluorescent proteins, dye molecules.

16. A method according to claim 14, wherein: - particle (2) is fluorescent, - the illumination field (200; 400) is produced by a pulsed laser at a frequency greater than 1 MHz, and - the detector (140; 340) is adapted to count single photons, the method comprising a step of determining a delay between a pulse of the laser and the emission of a photon by the particle (2).

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