Fluorescence microscopy system, laser diagnostic system, and fluorescence imaging method

Abstract

The invention relates to a fluorescence microscopy system comprising a nonlinear optical medium (ML) and comprising: - a laser source (SL) suitable for delivering first laser pulses (IL1) having a first wavelength λ1; - a power optical modulator (MP) suitable for modulating a power of the first laser pulses (IL1) so as to form second laser pulses (IL2) having different optical powers; - said optical medium (MO) being suitable for the second pulses to propagate by undergoing self-focusing within the optical medium so as to define, for each second pulse, a focal point having a longitudinal position in the optical medium that is different from the other second pulses, laser radiation originating from the optical medium and having the second self-focused pulses, referred to as third pulses, being named the output beam (FS); - a microscope (M) suitable for: - collecting a first fluorescence radiation generated via the first wavelength λ1, by illuminating a sample (Ech) with the output beam; then - acquiring, from the first collected fluorescence radiation, at least one image of the sample for each of the third pulses illuminating the sample, each at least one image being associated with a transverse observation plane of the sample that is different from the other images.

Description

DESCRIPTION Title of the invention: Fluorescence microscopy system, Laser diagnostic system and Fluorescence imaging method Field

[0001] The present invention relates to laser diagnostic systems, and in particular to the field of fluorescence imaging systems. Prior art:

[0002] It is known to perform fluorescence imaging of a sample using an AA system as illustrated in Figure 1.

[0003] A light source 11 generates, for example, polychromatic radiation 12, which is spectrally filtered by a filter 13 to become monochromatic. The monochromatic radiation 12 is directed and then focused onto an EC sample using a dichroic mirror 14 and an objective lens 15, respectively, so as to generate fluorescence radiation 16. This fluorescence radiation 16 is collimated by the objective lens 15 and then optionally spectrally filtered by a filter 17 before being focused by an eyepiece 18 onto a matrix photodetector 19. The matrix photodetector 19 allows an image to be acquired of a region of the sample illuminated by the monochromatic radiation 12 and, for example, the presence and location of fluorescent particles in this region to be detected.

[0004] To obtain a "complete" two-dimensional image of the sample EC, the system in Figure 1 typically includes a translation system 20a on which the sample EC is mounted. The translation system 20a allows the sample EC to be moved relative to the focal point of the monochromatic radiation 12 along a transverse plane. Thus, the translation system 20a changes the region of the sample illuminated by the monochromatic radiation 12, the fluorescence image of which is acquired by the photodetector 19. By acquiring an image for each region scanned by the translation system 20a, it is possible to reconstruct a complete fluorescence image of the sample EC.

[0005] This displacement system can advantageously be replaced by a 2D scanner allowing the radiation (12) to be scanned transversely on the EC sample more quickly.

[0006] As is known, the radiation 12 is focused onto the sample in the focal plane 21 of the objective. Furthermore, the depth of field is determined by the distance between the plane of the nearest object in focus and the plane of the farthest object also in focus simultaneously. In other words, it corresponds to the size of the imaged "slice" of the sample EC along the longitudinal direction. This "slice" is centered on the focal plane 21.

[0007] In microscopy, the depth of field is very shallow and is generally measured in microns. It can be calculated using the following equation: where ^^^^^^(^^) represents the depth of field, ^^ is the wavelength of the radiation 12, ^^ is the refractive index of the medium between the sample and the front lens of the objective, and ^^^^ is the numerical aperture of the objective.

[0008] In order to image a plurality of different "slices" of the sample, it is possible to move the objective 15 longitudinally (i.e. along the optical axis) relative to the sample EC, for example using a translation system 20b. Thus, the longitudinal position of the focal plane 21 and its position within the sample are moved.

[0009] However, the position of the focal plane 21 in the sample can be adjusted at a speed limited by the movement of a mechanical element (the translation system 20b) or by adjusting the spatial phase using a similarly slow optoelectronic modulator. The characteristic time of these movements is typically on the order of milliseconds.

[0010] The invention aims to overcome some of the aforementioned problems of the prior art. More specifically, the invention aims to overcome the limitation imposed by the displacement of mechanical elements and on the speed of change of a longitudinal position of the focal point (also referred to hereafter as "longitudinal focusing speed").

[0011] To this end, the invention proposes a fluorescence microscopy system comprising a focusing system based on controlling the optical power of laser pulses via an optical power modulator. These power-modulated laser pulses are directed into an optical medium where they undergo self-focusing (a third-order nonlinear optical phenomenon) with an instantaneous response. Thus, the focal point in the optical medium has a longitudinal position that varies with the power of the laser pulses.

[0012] Coupling this self-focused radiation in a microscope allows for adjustable focusing within a sample, using the power of the laser pulses. Under these conditions, power control, which can be achieved ultra-fast using an electro-optical modulator, for example, accelerates focusing that is usually accomplished through slow mechanical means.

[0013] Thus, the fluorescence microscopy system allows for the ultra-fast imaging of multiple slices of the sample. Summary of the invention:

[0014] To this end, an object of the invention relates to an ultra-fast development assembly intended for a fluorescence imaging system or a diagnostic system comprising: - a laser source adapted to deliver first laser pulses having a first wavelength - an ultrafast optical power modulator adapted to modulate the power of the first laser pulses, so as to form second laser pulses with different optical powers; - an optical medium adapted so that the second pulses propagate by undergoing a self-focusing phenomenon within the optical medium, so as to define, for each second pulse, a focal point with a longitudinal position in the optical medium different from the other second pulses, a laser beam emanating from the optical medium and presenting the second self-focused pulses, called third pulses, being called the output beam.

[0015] Another object of the invention is a fluorescence microscopy system comprising: - an ultra-fast focusing assembly according to the invention - a microscope adapted to: - collect the output beam so that it can illuminate a sample - detect a first fluorescence radiation generated via the first wavelength ^^1, by illuminating the sample with the output beam, then - acquire, from the first detected fluorescence radiation, at least one image of the sample for each of the third pulses illuminating the sample, each at least one image being associated with a transverse observation plane of the sample differing from the other images.

[0016] According to one embodiment of the fluorescence microscopy system, the latter includes a mechanical transverse focusing system, said microscope being configured so that at least one image of the sample is acquired by a transverse spatial scan of the sample by the output beam via the mechanical transverse focusing system.

[0017] According to one embodiment of the fluorescence microscopy system, the latter includes an optical coupling element disposed on an optical path of the output beam and adapted to collimate the output beam.

[0018] According to one embodiment of the fluorescence microscopy system, the latter comprises an interferometric optical device disposed in the optical path upstream of the optical medium, and adapted to divide each first or second laser pulse into a plurality of replicas having different optical powers so that the microscope acquires so-called interlaced images of the sample for each of the self-focused replicas in said optical medium illuminating the sample, each image being associated with a transverse observation plane of the sample different from the other simultaneous images, the interlaced images acquired from a given first or second laser pulse are acquired by the microscope and form said at least one image of the sample. Preferably, the interferometric optical device is a Fabry-Pérot interferometer or a Michelson interferometer.

[0019] According to one embodiment of the fluorescence microscopy system, the optical power modulator is adapted to perform ultrafast modulation with a modulation frequency between 100 kHz and 1000 GHz. Preferably, the optical power modulator is an interferometric optical assembly or an electro-optical modulator, for example, an electro-optical or acousto-optical modulator.

[0020] According to one embodiment of the fluorescence microscopy system, the laser source and the optical power modulator are adapted to achieve maximum power ^^ ^^^^^^ and minimal power ^^ ^^^^^^ of the second impulses are such that Δ^^′ ≥ 0.5. ^^^^^^ and preferably such that Δ^^ ′ = ^^ ^^^^ with ^^ ^^^^a sample thickness and with Δ^^′ a longitudinal distance between the downstreammost transverse observation plane of the optical path and the upstreammost transverse observation plane of the optical path.

[0021] According to one embodiment of the fluorescence microscopy system, the optical medium is adapted to allow a non-linear conversion of said first wavelength such that each second modulated laser pulse exhibits at least one second wavelength √2, said nonlinear conversion being, for example, a second harmonic generation or a third harmonic generation, the third pulses thus exhibiting said at least one second wavelength √2. Preferably, the optical medium is adapted so that said nonlinear conversion induces a saturation effect on the laser pulses in order to limit their fluence in the optical medium to a value less than 10 √2. Even more preferably, the optical medium is a YAG, YVO4, or BBO crystal.

[0022] According to an embodiment compatible with the three preceding embodiments, the microscope is further adapted for: - detect a second fluorescence radiation generated via the second wavelength ^^2, by illuminating the sample with the output beam, then - acquire, from the second detected fluorescence radiation, at least one so-called additional image of the sample for each of the third pulses illuminating the sample, each at least one additional image being associated with a transverse observation plane of the sample differing from the other additional images.

[0023] According to one embodiment of the fluorescence microscopy system, the microscope includes a processor adapted to reconstruct a three-dimensional depth image from said at least one image of the sample associated with different transverse observation planes of the sample.

[0024] According to one embodiment of the fluorescence microscopy system, the optical medium is adapted to allow amplification of the second pulses within the optical medium. Preferably, the optical medium is a multimode amplifying optical fiber.

[0025] According to one embodiment of the fluorescence microscopy system, the latter includes an additional optical element adapted to allow additional amplification or attenuation of the first pulses upstream of the optical medium.

[0026] Another object of the invention is a laser diagnostic system for a sample comprising: - an ultra-fast focusing assembly according to the invention - a diagnostic device adapted to collect the output beam reflected, transmitted or scattered by a sample so as to determine a physical parameter of the sample.

[0027] According to one embodiment of the diagnostic system, the latter is adapted to collect the output beam reflected or scattered by the sample and then determine a variation in the shape of the sample, which generally has a plate-like shape, by comparing the intensity of the third pulses reflected or scattered by the sample, said variation in the shape of the plate forming said physical parameter of the sample.

[0028] A final object of the invention is a method for imaging a sample by fluorescence comprising the following steps: - generating first laser pulses having a first length - modulate the power of the first laser pulses, so as to form second laser pulses with different optical powers;- to generate, in an optical medium, a self-focusing phenomenon of the second pulses so as to define, for each second pulse, a focal point having a longitudinal position in the optical medium different from the other second pulses, a laser beam emanating from the optical medium and having the self-focused second pulses, called third pulses, being called the output beam, - to detect a first fluorescence beam generated via the first wavelength ^^1, by illuminating a sample with the output beam, then - to acquire, from the first detected fluorescence beam, at least one image of the sample for each of the third pulses illuminating the sample, each at least one image being associated with a transverse observation plane of the sample different from the other images. Brief description of the figures: ;

[0029] Other features, details and advantages of the invention will become apparent from the description provided with reference to the accompanying drawings given by way of example, which represent, respectively:

[0030] [Fig.1], a fluorescence imaging system known from the prior art,

[0031] [Fig.2], a fluorescence imaging system according to an embodiment of the invention,

[0032] [Fig. 3], a schematic illustration of the self-focusing of the second pulses in the optical medium in a fluorescence imaging system according to an embodiment of the invention,

[0033] [Fig. 4], a fluorescence imaging system according to an embodiment of the invention,

[0034] [Fig. 5], a fluorescence imaging system according to an embodiment of the invention,

[0035] [Fig. 6], a fluorescence imaging system according to an embodiment of the invention,

[0036] In the figures, unless otherwise indicated, the elements are not to scale and identical references designate identical elements.

[0037] Furthermore, unless otherwise indicated, the optional features described in the description and figures can be combined. Detailed description:

[0038] The invention relates to a laser-based diagnostic system for a sample, enabling ultra-fast focusing of the diagnostic laser beam. To clarify, "ultra-fast" here means that focusing is performed at a frequency greater than or equal to 10 kHz. The invention thus overcomes the focusing speed inherently limited by the use of a mechanical (or optoelectronic, such as SLM) element, as notably present in the fluorescence microscopy device shown in Figure 1, known from the prior art.

[0039] In the invention, this ultra-fast focusing is enabled by a focusing assembly based on a modulation of the optical power of self-focused laser pulses within an optical medium.

[0040] In the following description, we will detail various embodiments of a fluorescence microscopy system according to the invention, but it is understood that the elements described apply more generally to any laser diagnostic system comprising the focusing assembly according to the invention. Furthermore, the various structures detailed in this description and the figures are given by way of example and are intended to illustrate the invention. They do not These provisions shall in no way be considered as limiting the scope of the invention. Various modifications and variations of the systems described will be apparent to those skilled in the art without departing from the scope and spirit of the invention. In particular, all equivalent means known to those skilled in the art for implementing the microscope, the optical power modulator, and the interferometric optical device (when present) are covered by the scope of the invention.

[0041] Figure 2 schematically illustrates a fluorescence microscopy system 1 according to the invention, including in particular the ultra-fast focusing assembly SMP according to the invention.

[0042] The ultrafast focusing assembly SMP according to the invention comprises a pulsed SL laser source adapted to generate an incident beam FI having first laser pulses IL1. The IL1 pulses delivered by the SL laser source are preferably (but not necessarily) monochromatic with a wavelength and are typically generated with a repetition rate ranging from kilohertz to several hundred megahertz. The pulsed SL laser source is not specific to the invention, and an exhaustive description of its operation or its various embodiments would fall outside the scope of the invention.

[0043] The ultrafast focusing assembly SMP according to the invention further comprises an ultrafast optical power modulator MP adapted to modulate the optical power of the first laser pulses IL1. Thus, the optical power modulator MP makes it possible to form second laser pulses IL2 having different optical powers from the laser pulses IL1 which typically have equal or nearly equal power.

[0044] To clarify, by "different optical powers" we mean here that the MP power optical modulator is adapted so that at least two IL2 laser pulses have a power of ^^ ^^ and a power ^^ ^^ such as | ^^^^−^^^^ | | ^^^^−^^ ^^^^ ≥ 10%, preferably ^^ | ^^^^ ≥ 30%. Indeed, this condition ≥ 10% was determined by the inventors as being the condition minimum to allow a satisfactory difference in focus under conventional operating conditions (see below).

[0045] Preferably, the MP optical power modulator is suitable for performing ultrafast modulation of optical power with a modulation frequency between 100 kHz and 1000 GHz.

[0046] The MP power optical modulator is, for example, an amplitude-division interferometric optical assembly such as a Mach-Zehnder or Michelson interferometer.

[0047] According to another embodiment, the MP power optical modulator is an electro-optical modulator, for example based on a Pockels cell, or an acousto-optical modulator, or even a thermo-optical modulator.

[0048] According to another embodiment, the MP power optical modulator is resonant at the first wavelength. For example, in one embodiment, the ultrafast MP modulator comprises a strongly coupling resonant optical microcavity including quantum well stacks as described in WO 2020 / 193786 A1, the contents of which are considered included by reference. Such a device makes it possible to obtain a very good response time (i.e., a high modulation frequency) while also allowing excellent modulation depth.

[0049] In addition to the elements described above, the ultrafast focusing assembly SMP according to the invention comprises a nonlinear MO optical medium adapted to induce a self-focusing effect on IL2 pulses during their propagation within the MO optical medium. Thus, the MO optical medium allows for a variation in the longitudinal position (along the direction of the optical axis) of the focal point of the IL2 pulses when their optical power changes.

[0050] As a reminder, the self-focusing effect is induced by the optical Kerr effect, a third-order nonlinear process that appears in media exposed to intense electromagnetic radiation and produces a variation in the refractive index ^^ = ^^0 + ^^2^^^^ with ^^0 and ^^2 the linear and nonlinear refractive indices of the optical medium MO and ^^ ^^ the intensity of IL2 laser pulses within the MO optical medium.

[0051] In the invention, to allow the appearance of this optical Kerr effect (and therefore the self-focusing of the IL2 pulses), it is necessary that the SL laser source and the MP power modulator be adapted so that the optical power ^^ ^^∈[1;^^] of each pulse ^^^^2 ^^∈[1;^^] either greater than the critical autofocus power ^^ ^^^^ of the optical medium MO which is:

[0052] As illustrated in Figure 3, this self-focusing effect causes the IL2 pulses to focus at a focal point with a distance ^^ ^^^^ measured from the EO inlet of the optical medium MO, which is equal to: with ^^0 the waist (or smallest radius) of the incident FI beam.

[0053] For IL2 pulses of sufficiently high power compared to ^^ ^^^^ , we can write:

[0054] By way of non-limiting example, according to one embodiment, the laser source SL is such that the wavelength ^^1 equals 1064 ^^^^ and ^^0 = 50 ^^^^, the optical medium MO is in YAG and ^^ 0 = 1.82 and ^^2 = 6 × . Using equation (1), we then calculate the critical power, which is ^^^^^^ = 1.5 ^^^^ . Thus, for an IL2 impulse of power ^^ = 6 ^^^^, we calculate using equation (3) a self-focusing distance of ^^^^^^ = 4.2 ^^^^.

[0055] Thus, we understand that optical power ^^ ^^∈[1;^^] of each of the impulses ^^^^2 ^^∈[1;^^] directly influences the longitudinal position of its focal point within the optical medium MO. More precisely, the greater the optical power ^^ ^^∈[1;^^] of the impulse ^^^^2 ^^∈[1;^^] The higher the focal point of this impulse will be close to the entrance of the optical medium (that is to say, will have a ^^ ^^^^ weak).

[0056] Ainsi, comme illustré dans la figure 2, pour ^^ impulsions ^^^^21, … , … , ^^^^2^^of decreasing power, we obtain ^^ focal point presenting a longitudinal position ^^1, … , ^^^^, … , ^^^^ increasingly further from the EO entrance of the optical medium.

[0057] More generally, in the SMP ultrafast focusing assembly of the invention, the second pulses IL2 propagate by undergoing a self-focusing phenomenon within the MO optical medium so as to define, for each second pulse ^^^^2 ^^∈[1;^^] , a focal point with a longitudinal position ^^ ^^ in the optical medium different from the other second impulses ^^^^2^^∈[1;^^],^^≠^^given their different optical power.

[0058] In the following description, the third pulses IL3 are the second pulses, exiting the optical medium MO, that have undergone self-focusing IL2, and the output beam FS is the laser radiation exiting the optical medium MO and exhibiting the third pulses IL3. Furthermore, Δ^^ denotes the longitudinal dimension in which it is possible to generate focal points with different longitudinal positions in the optical medium MO. This longitudinal dimension Δ^^ is subsequently called the "focusing range" and corresponds to the difference between the longitudinal position ^^ ^^ from the focal point furthest from the EO entrance of the optical medium (and therefore associated with the IL2 pulse of lowest optical power ^^ ^^^^^^ ) and the longitudinal position ^^1 of the focal point closest to the EO entrance of the optical medium (and therefore associated with the IL2 pulse of highest optical power ^^ ^^^^^^From equation (3), we can write Δ^^ in the following form:

[0059] As a non-limiting example, the inventors have demonstrated that a focusing range variation Δ^^ = 5 ^^^^ was possible for an enYAG MO optical medium and having a length (dimension along the optical axis) of 7 ^^^^.

[0060] Thus, by controlling the optical power of the IL2 pulses via the ultrafast MP modulator, the ultrafast focusing assembly SMP of the invention allows for ultrafast control of the longitudinal position of the IL2 pulse focal point. The ultrafast focusing assembly SMP of the invention is therefore particularly well-suited for coupling with a diagnostic device designed to collect the reflected, transmitted, or scattered FS output beam from an EC sample in order to ultrafastly determine a physical parameter in different transverse observation planes z1', …zN' of the EC sample.Indeed, an optical system, comprising for example an SC coupling optical assembly and an OB objective as illustrated in Figure 2, makes it possible to translate the ultrafast variation of the longitudinal position of the IL2 pulse focal point into a variation of the longitudinal position of the IL3 pulse focal point in the EC sample. More precisely, this optical system is adapted to form, in the EC sample, the image of the self-focused focal points in the MO optical medium. Thus, the SMP ultrafast focusing assembly of the invention makes it possible to diagnose different transverse observation planes z1', …zN' of the EC sample in an ultrafast manner.

[0061] The SMP ultra-fast focusing assembly of the invention thus allows a notable improvement compared to known prior art devices in which the longitudinal focusing speed is intrinsically limited by the use of a mechanical / electro-mechanical / opto-mechanical element as notably present in the fluorescence microscopy device of Figure 1.

[0062] Let us now return to the description of the fluorescence microscopy system 1 according to the invention. As illustrated in Figure 2, the output beam FS from the optical medium MO, exhibiting pulses that have undergone IL3 autofocus, is collected within a fluorescence microscope M in order to image a sample EC. More specifically, the output beam FS is collected by an objective lens OB of the microscope M so as to illuminate the sample EC, which then generates, from the absorption of the first wavelength a first RF1 fluorescence radiation.

[0063] Given that the IL3 pulses of the output beam FS have focal points within the optical medium at different positions z1, …zN, the IL3 pulses are focused into different transverse observation planes z1', …zN' of the sample EC. This implies that each IL3 pulse will be focused by the objective lens OB within the sample into a specific observation plane, different from the other pulses, depending on the longitudinal position of self-focusing in the optical medium MO. This will therefore allow fluorescence imaging of this respective observation plane. Thus, by successively detecting the first generated fluorescence RF1 radiation in these different transverse observation planes z1', …zN', it is possible to successively image a plurality of different "slices" of the sample EC.

[0064] For this purpose, the microscope M of system 1 of the invention comprises an eyepiece OC adapted to form the image of the transverse observation planes z1', …zN', on a matrix photodetector DT capable of detecting the first fluorescence radiation RF1. In addition, a processing unit UT connected to the matrix photodetector DT is configured to acquire, from the first detected fluorescence radiation, an image ^^ ^^ of the sample for each of the IL3 pulses illuminating the sample. As explained above, each image ^^ ^^ is acquired from a respective IL3 pulse of different power than the others and therefore allows imaging of a transverse observation plane zi' of the respective sample and differs from the other images

[0065] Il est entendu que ces images ^^1, … , ^^^^, … , The different transverse observation planes z1', …zN' of the sample are acquired successively over time because the modulation of the IL1 pulses by the MP power modulator is also performed successively over time. However, as discussed in detail above, the modulation performed by the MP power modulator is ultrafast. This implies that the variation in the longitudinal position of the IL2 pulse focal point in the MO optical medium (controlled by the MP power modulator) and the variation in the longitudinal position of the IL3 pulse focal point in the sample are also performed ultrafast.

[0066] In summary, system 1 of the invention allows for ultra-fast longitudinal "scanning" of the sample by performing imaging of a plurality of different "slices" of the EC sample (corresponding to the different transverse observation planes z1', …zN'). In other words, the fluorescence microscopy system 1 allows ultra-fast longitudinal focusing in the sample.

[0067] Also, the fluorescence microscopy system 1 allows a clear improvement over known prior art fluorescence microscopy devices in which longitudinal focusing was carried out by a mechanical element and whose speed was intrinsically limited.

[0068] Preferably, as illustrated in Figure 2, the fluorescence microscopy system 1 includes an SC coupling optical assembly to optimize the coupling of the output beam in the fluorescence microscope M.

[0069] The SC coupling optical assembly is known from the prior art, and a detailed description of its various embodiments would fall outside the scope of the invention. According to a preferred embodiment, the SC coupling optical assembly is a lens or a plurality of lenses for collimating the output beam FS and forming, with the objective OB of the microscope M, an afocal optical assembly for forming an image of the different focal points at positions z1, …zN in different transverse observation planes z1', …zN' of the sample EC.

[0070] More generally, in system 1 of the invention, the objective OB allows—alone or with the entire set of coupling optics SC—the formation of an image of the different focal points at positions z1, …zN in different transverse observation planes z1', …zN' of the sample EC as illustrated in Figure 2. The longitudinal distance between the first observation plane z1' (the observation plane furthest upstream in the optical path) and the last observation plane z1' is called the "object focusing range Δz'". ^^ ′ (the observation plane furthest downstream of the optical path).

[0071] The relationship between the position of the transverse observation planes z1', …zN' within the EC sample and the position z1, …zN of the IL2 pulse focal points within the MO optical medium is governed by geometric optics in a manner known per se. Furthermore, regardless of the optical design of the objective OB (and the SC coupling system where applicable), there is a factor ^^ ^^ such that Δ^^′ = ^^^^ . Δ^^ , the factor ^^^^ corresponding to the transverse magnification of the optical assembly (formed by the objective OB and the coupling system SC in the embodiment of figure 2) producing the image of the focal points of positions z1, …zN in focal points arranged in the transverse observation planes z1', …zN'.

[0072] It is generally preferable to image the entire thickness ^^ ^^^^ of the sample. We call it "thickness ^^ ^^^^ of the sample, the size of the sample along the longitudinal direction ^^. To image the entire sample, the object focus range Δ^^′ must be greater than or equal to ^^ ^^^^ As mentioned above, Δ^^′ = ^^^^ . Δ^^ and Δ^^ depend (among other things) directly on the maximum power ^^ ^^^^^^ and minimal power ^^^^^^^^ of the second pulses IL2 (see equation (4)). Thus, preferably, the laser source and the optical power modulator are matched so that a maximum power ^^ ^^^^^^ and minimal power ^^ ^^^^^^ of the second impulses are such that Δ^^′ ≥ 0.5. ^^ ′^^^^ and preferably such that Δ^^ ≥ ^^^^^^.

[0073] Alternatively, it is preferable to image the majority of the sample thickness, thus achieving maximum power. ^^^^^^ and minimal power ^^ ′ ^^^^^^ second impulses such that Δ^^≥ 0.5. ^^^^^^

[0074] Preferably, as illustrated in the embodiment of Figure 2, the SMP ultrafast focusing assembly includes an OF optical assembly for focusing the IL2 pulses within the optical medium. The OF optical assembly increases the illumination of the IL2 laser pulses focused within the optical medium. This, in turn, increases the change in the refractive index induced by the Kerr effect, leading to an increase in the self-focusing effect. Indeed, as explained above and described by equation (2), an increase in illumination ^^ ^^ Thanks to the OF optical assembly, the autofocus distance can be reduced ^^ ^^^^ .

[0075] Thus, adding the OF optical assembly increases the maximum illumination ^^ ^^,^^^^^^IL2 pulses. This allows a displacement of the longitudinal position of the focal points towards the EO entrance of the optical medium and in particular a displacement of the first observation plane ^^1′ upstream of the Optical path. However, it is still possible to reduce the power of some of the IL2 pulses via the laser source or the MP modulator to obtain the final observation plane. ^^ ′ placed in the same longitudinal position with or without optical assembly OF. In other words, the optical assembly OF allows increasing the focusing range Δ^^ in which it is possible to generate focal points with different longitudinal positions and therefore allows an increase in the object focusing range Δ^^′.

[0076] Alternatively, adding an attenuator or amplifier before the OF optical assembly allows the power range of the second pulses after the OF optical assembly to be adjusted to adequately optimize the compatibility of the SL laser source and the MP power modulator with the operating regime of the MO optical medium's self-focusing. This makes the system more modular.

[0077] As explained above, the FS output beam is focused within the EC sample. This implies that the dimension of the focal spot in the observation plane ^^ ^^ is reduced. Thus, the fluorescence information of the observation plane ^^ ^^ determined via the image ^^ ^^ is intrinsically limited spatially by the dimension of this focal task.

[0078] To overcome this problem, in the embodiment of Figure 4, the fluorescence microscopy system 1 includes a translation system ST adapted to allow transverse spatial scanning (along the directions ^^ and ^^ in the example of Figure 4) of the sample by the output beam FS. Thus, a plurality ^^ of images ^^^^∈[1;^^],^^of the transverse observation planes ^^ are acquired. ^^∈[1;^^] ′ different from the same transverse region ^^ ^^ from ^^ laser pulses ^^^^2 ^^∈[1;^^] of power ^^ ^^∈[1;^^] different, then we scan different cross-regions ^^ ^^∈[1;^^] of the sample. In a single transverse scan, the UT processing unit is then adapted to reconstruct a "complete" fluorescence image ^^ ^^,^^ of each cross-sectional observation plan ^^ ^^ ′ of the sample by digital processing of (for example, by concatenating or adding the fluorescence intensities of the transverse regions ^^ ^^∈[1;^^] from the same cross-sectional observation plan ^^ ^^ ′ ).

[0079] In other words, the "complete" fluorescence image ^^ ^^,^^ of a cross-sectional observation plan ^^ ^^ The sample is obtained by a transverse spatial scan of the sample by the output beam via the ST translation system, for the same laser pulse power IL2 during the scan. The use of an ST translation system is compatible with all embodiments of the invention of the fluorescence microscopy system 1.

[0080] According to the embodiment illustrated in Figure 4, the sample EC is mounted on the translation system ST, which is, for example, a piezoelectric translation stage connected to the laser source SL to allow synchronization of the transverse spatial scanning with the repetition of the laser pulses IL1. Alternatively, according to another embodiment, a scanning system using rotating two mirrors (scanner) is positioned in the beam path to allow transverse spatial scanning of the sample by the output beam. These two systems are more generally grouped under the generic term of mechanical transverse focusing systems.

[0081] Preferably, the UT processing unit is suited to reconstruct a three-dimensional depth image of the sample from the "complete" fluorescence images. of the sample, each corresponding to a respective slice of the sample (a cross-sectional observation plan ^^ ^^ ′ respective). This type of digital 3D image reconstruction from a plurality of planar 2D images is known to those skilled in the art and is particularly used in the field of medical imaging (for example in MRI).

[0082] According to one embodiment of the invention, the MO optical medium is adapted to allow amplification of the first IL2 pulses during their propagation within the MO optical medium. For example, the MO optical medium is a bulk medium, or a multimode optical fiber doped with rare-earth ions and pumped by a pump beam (not shown in the figures). The amplification of the IL2 pulses within the MO medium allows additional control over the longitudinal position of the focusing point of the self-focused pulses. Indeed, the amplification of an IL2i pulse results in an increase in power. ^^ of this impulse and therefore a displacement of the longitudinal position of the focal point towards the entrance EO of the optical medium (see equation (3)). Thus, just like the optical assembly OF, an amplifying optical medium MO allows the object focusing range to be increased Δ^^′.

[0083] In a preferred embodiment, the optical medium comprises a structure adapted to allow better control of the self-focusing of IL2 pulses within the optical medium. A medium with a Bragg grating, for example, allows compression of a laser pulse, thereby increasing its peak power during propagation and decreasing the self-focusing distance from the point of entry into the nonlinear medium.

[0084] Figure 5 illustrates an embodiment of the invention for performing simultaneous imaging of several transverse observation planes ^^1′, … , … , ^^^^′ of the sample EC. For this purpose, the fluorescence microscopy system 1 comprises an interferometric optical device DOI arranged in the optical path of the laser beam, optically upstream of the optical medium MO (for example, downstream of the modulator MP as illustrated in Figure 5) and adapted to divide each first pulse IL1 (or each second pulse IL2 as appropriate) into a plurality of replicas IL11, …IL1 N exhibiting different optical powers. The impulse IL1 or IL2 from which a plurality of replicas are formed simultaneously is called the "initial impulse".

[0085] Critically, due to its optical design, the DOI interferometric optical device is adapted to form these IL11, …IL1N replicas simultaneously from an initial IL1 impulse. For the same reasons mentioned above, each IL11, …IL1N replica Nis successively autofocused in the MO medium at a longitudinal position different from the other replicas, depending on its optical power. Similarly, each replica IL11, …IL1N is successively focused within the sample in a respective transverse observation plane, different from the other replicas (for example, by managing the respective travel times of the replicas using a delay line). Thus, for each replica of an initial pulse that is autofocused in the optical medium and then illuminates the sample EC, the DT photodetector acquires an interlaced image of the sample. The interlaced images associated with an initial pulse are acquired simultaneously (or almost simultaneously, depending on the interferometric optical setup), and each interlaced image associated with a The initial impulse corresponds to an image of a transverse observation plane of the sample that is different from the other interlaced images.

[0086] Thus, unlike the embodiments detailed previously, the introduction of the DOI interferometric optical device allows, through the creation of the IL11, …IL1N replicas, to image a plurality of "slices" of the sample (the transverse observation planes) simultaneously (or almost simultaneously).

[0087] As a non-limiting example, the DOI optical interferometric device is a Michelson interferometer, a Fabry-Pérot interferometer or more generally an amplitude-division interferometer.

[0088] According to a variant of the embodiment shown in Figure 5, the interferometric optical device DOI and the optical power modulator MP form a single unit. In other words, the modulation of the IL1 pulse power is performed almost instantaneously by the interferometric optical device DOI. In this variant, the DT photodetector therefore acquires images directly. … , ^^^^ temporally intertwined, respectively associated with the transverse observation planes ^^1′, … , … , ^^^^′ . The image acquired by the DT photodetector therefore corresponds to an image in which N transverse planes are in focus.

[0089] Figure 6 illustrates an embodiment of the invention in which the optical medium MO further enables a nonlinear conversion of the first wavelength into at least a second wavelength λ² so as to acquire additional fluorescence information on the sample EC. This embodiment is compatible with all the embodiments detailed previously (in particular the embodiment of Figure 5).

[0090] As a non-limiting example, the optical medium is suitable for inducing, from the first wavelength λ^1, a generation of second or third harmonics. For example, the optical medium is a YAG crystal, a YVO4 crystal, or a BBO crystal.

[0091] As is well known, the nonlinear conversion of the first wavelength is performed at the focal point of the self-focused IL3 pulse in the MO optical medium. This implies that the focal points of the IL3 pulses are identical for wavelengths λ1 and λ2. Thus, each IL3 pulses of the FS output beam present wavelengths ^^1 and ^^2 which are both focused within the sample in the same transverse observation plane ^^1′, … , … , ^^^^′ as a function of the power of the IL2 pulse.

[0092] The sample is adapted to absorb the second wavelength ^^2 in order to emit a second RF2 fluorescence radiation.

[0093] In addition to detecting the first wavelength ^^1, the DT photodetector of microscope M is adapted to detect the second wavelength ^^2. As a non-limiting example, this multispectral detection by the DT photodetector is carried out by two separate sensors and a spectral optical element (for example a dichroic mirror or a prism) allowing a spatial separation of the first and second wavelengths and their detection by a respective sensor.

[0094] Thus, the processing unit UT is configured to acquire, from the second fluorescence radiation detected by the photodetector DT, an "additional" image of the sample for each of the third pulses. ^^∈[1;^^]illuminating the EC sample. Each additional image ^^^^,^^∈[1;^^] therefore corresponds to additional fluorescence information generated by the second wavelength ^^2 and is associated with a transverse observation plane ^^ ^^ ′ of the sample differ from the other additional images. As explained above, this transverse observation plane ^^^^′ for which the additional image ^^^^,^^∈[1;^^] is acquired via the wavelength ^^2 is identical to that imaged via the length

[0095] In the embodiment of Figure 6, in order to avoid the occurrence of laser-induced optical damage in the optical medium, the material of the optical medium MO is adapted so that the nonlinear conversion of the first wavelength allows a saturation effect to be induced on the laser pulses IL2. To this end, preferably, the optical medium and the laser source SL are adapted so that the laser pulses IL2 propagate in the optical medium with a fluence less than 10^^ / ^^^^².

[0096] The inventors determined that a YAG optical medium exhibits a satisfactory nonlinear conversion saturation effect, preventing the occurrence of optical damage.

Claims

Claim 1. Ultrafast focusing assembly (SMP) for a fluorescence imaging system or a diagnostic system comprising: - a laser source (SL) adapted to deliver first laser pulses (IL1) having a first wavelength - an ultrafast optical power modulator (MP) adapted to modulate the power of the first laser pulses (IL1), so as to form second laser pulses (IL2) with different optical powers; - an optical medium (MO) adapted so that the second pulses propagate by undergoing a self-focusing phenomenon within the optical medium so as to define, for each second pulse, a focal point having a longitudinal position (z1,…, zN) in the optical medium different from the other second pulses, a laser beam emanating from the optical medium and having the self-focused second pulses, called third pulses (IL3), being called the output beam (FS). 2.Fluorescence microscopy system (1) comprising: - an ultra-fast focusing assembly according to claim 1 - a microscope (M) adapted to: - collect the output beam (FS) so that it can illuminate a sample (EC) - detect a first fluorescence radiation (RF1) generated via the first length. by illuminating the sample (EC) with the output beam, then - acquiring, from the first detected fluorescence radiation, at least one image of the sample for each of the third pulses illuminating the sample, each at least one image being associated with an observation plane (z1', …z N') transverse of the sample differ from the other images.

3. Fluorescence microscopy system according to the preceding claim comprising a transverse mechanical focusing system (ST), said microscope being configured so that each at least one image of the sample is acquired by a transverse spatial scan of the sample by the output beam via the transverse mechanical focusing system (ST).

4. A fluorescence microscopy system according to claim 2 or 3, comprising a coupling optical element (SC) disposed on an optical path of the output beam and adapted to collimate the output beam.

5. A fluorescence microscopy system according to any one of claims 2 to 4, comprising an optical interferometric device (DOI) disposed on the optical path upstream of the optical medium (MO), and adapted to divide each first or second laser pulse into a plurality of replicas (IL11, …IL1). N) having different optical powers such that the microscope acquires so-called interlaced images of the sample for each of the self-focused replicas in said optical medium illuminating the sample, each image being associated with a transverse observation plane of the sample different from the other simultaneous images, the interlaced images acquired from a given first or second laser pulse being acquired by the microscope and forming said at least one image of the sample.

6. Fluorescence microscopy system according to the preceding claim, wherein the optical interferometric device (OID) is a Fabry-Pérot interferometer or a Michelson interferometer. 7.A fluorescence microscopy system according to any one of claims 2 to 6, wherein the optical power modulator is adapted to perform ultrafast modulation with a modulation frequency between 100 kHz and 1000 GHz.

8. A fluorescence microscopy system according to the preceding claim, wherein the optical power modulator is an interferometric optical assembly or an electro-optical or acousto-optical modulator.

9. A fluorescence microscopy system according to any one of claims 2 to 8, wherein the laser source and the optical power modulator are adapted to achieve maximum power. ^^^^^^ and minimal power ^^ ^^^^^^ of the second impulses are such that Δ^^′ ≥ 0.

5. ^^ ′^^^^ and preferably such that Δ^^ = ^^^^^^, with^^ ^^^^a sample thickness and with Δ′ a longitudinal distance between the downstreammost transverse observation plane of the optical path and the upstreammost transverse observation plane of the optical path.

10. Fluorescence microscopy system according to any one of claims 2 to 9, wherein the optical medium is adapted to allow nonlinear conversion of said first wavelength so that each second modulated laser pulse has at least one second wavelength ^^2, said nonlinear conversion being, for example, a second harmonic generation or a generation of third harmonic, the third pulses thus exhibiting said at least second wavelength ^^2.

11. Fluorescence microscopy system according to the preceding claim, wherein the optical medium is adapted so that said nonlinear conversion makes it possible to induce a saturation effect of the laser pulses in order to limit the fluence of the latter in the optical medium to a value less than 10 ^^ / ^^^^².

12. Fluorescence microscopy system according to the preceding claim, wherein the optical medium is a YAG, YVO4, or BBO crystal. 13.A fluorescence microscopy system according to any one of claims 10 to 12, wherein the microscope is further adapted to: - detect a second fluorescence beam (RF2) generated via the second wavelength λ2, by illuminating the sample (Ech) with the output beam, and then - acquire, from the second detected fluorescence beam, at least one so-called additional image of the sample for each of the third pulses illuminating the sample, each at least one additional image being associated with a transverse observation plane of the sample that differs from the other additional images. 14.

15. A fluorescence microscopy system according to any one of claims 2 to 13, wherein the microscope comprises a processor (UT) adapted to reconstruct a three-dimensional depth image from said at least one image of the sample associated with different transverse observation planes of the sample.

16. A fluorescence microscopy system according to any one of claims 2 to 14, wherein the optical medium is adapted to allow amplification of the second pulses in the optical medium.

17. A fluorescence microscopy system according to any one of claims 2 to 15, comprising an additional optical element adapted to allow additional amplification or attenuation of the first pulses upstream of the optical medium.A laser-based sample diagnostic system (EC) comprising: - an ultrafast focusing assembly according to claim 1 - a diagnostic device adapted to collect the output beam reflected, transmitted, or scattered by a sample so as to determine a physical parameter of the sample.

18. A method for imaging a sample by fluorescence comprising the following steps: A. generate first laser pulses (IL1) having a first wavelength ^^1; B. modulate the power of the first laser pulses (IL1), so as to form second laser pulses (IL2) having different optical powers; C. generate, in an optical medium, a self-focusing phenomenon of the second pulses so as to define, for each second pulse, a focal point having a longitudinal position in the optical medium different from the other second pulses, a laser radiation from the optical medium and having the self-focused second pulses, called third pulses, being called the output beam (FS), D. detect a first fluorescence radiation generated via the first wavelength by the illumination of a sample (EC) with the output beam, then E.acquire, from the first detected fluorescence radiation, at least one image of the sample for each of the third pulses illuminating the sample, each at least one image being associated with a transverse observation plane of the sample differ from the other images.

Images