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

Abstract

Fluorescence microscopy system comprising a nonlinear optical medium (ML) and comprising: a laser source (SL) adapted to deliver first laser pulses (IL1) having a first wavelength; an optical power modulator (MP) adapted to modulate a power of the first laser pulses (IL1), so as to form second laser pulses (IL2) having different optical powers;said optical medium (OM) being 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 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 output beam (EB), a microscope (M) adapted to: collect a first fluorescence radiation generated via the first wavelength by the illumination of a sample (Sample) with the output beam, then acquire, from the first fluorescence radiation collected, 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.;

Description

Title of the invention: Fluorescence microscopy system, laser diagnostic system and fluorescence imaging method. Technical field

[0001] The present invention relates to laser diagnostic systems and in particular to the field of fluorescence imaging systems. Previous technique

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

[0003] A light source 11 generates radiation 12, for example polychromatic, 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 makes it possible to acquire an image of a region of the sample illuminated by the monochromatic radiation 12 and, for example, to detect the presence and location of fluorescent particles in this region.

[0004] In order to obtain a "complete" two-dimensional image of the sample EC, the system of [Fig. 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 makes it possible to change the region of the sample illuminated by the monochromatic radiation 12 and whose fluorescence image 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] In a manner known per se, the radiation 12 is focused onto the sample in the focal plane 21 of the lens. Furthermore, the depth of field is determined by the distance between the plane of the nearest object in focus and the most distant plane

[0007]

[0008]

[0009]

[0010]

[0011]

[0012]

[0013]

[0014] also developed simultaneously. Otherwise, it corresponds to the dimension of the "slice" of the EC sample imaged along the longitudinal direction. This "slice" is centered on the focal plane 21. In microscopy, the depth of field is very shallow and is generally measured in microns. It can be calculated using the following equation: where DOF(X) represents the depth of field, 2 is the wavelength of the radiation 12, n is the refractive index of the medium between the sample and the front lens of the objective, and NA is the numerical aperture of the objective. 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. 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. 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"). 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. 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.

[0015] Thus, the fluorescence microscopy system makes it possible to image a plurality of slices of the sample in an ultra-fast manner. Summary of the invention

[0016] To this end, an object of the invention relates to an ultra-fast focusing assembly intended for a fluorescence imaging system or a diagnostic system comprising: - a laser source suitable for delivering initial laser pulses with a first wavelength A; - an ultra-fast 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 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.

[0017] Another object of the invention is a fluorescence microscopy system comprising: - an ultra-fast focusing system according to the invention - a microscope suitable for: - collect the output beam so that it can illuminate a sample - detect a first fluorescence radiation generated via the first wavelength 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 differ from the other images.

[0018] According to one embodiment of the fluorescence microscopy system, the latter includes a transverse focusing mechanical 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 transverse focusing mechanical system.

[0019] According to one embodiment of the fluorescence microscopy system, the latter comprises an optical coupling element disposed on an optical path of the output beam and adapted to collimate the output beam.

[0020] 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 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. Preferably, the interferometric optical device is a Fabry-Pérot interferometer or a Michelson interferometer.

[0021] 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.

[0022] According to one embodiment of the fluorescence microscopy system, the laser source and the optical power modulator are adapted so that a maximum power Pmax and a minimum power Pmin of the second pulses are such that Az 0.5.e£C and preferably such that &z - eEC, with eEc a thickness of the sample and with ^z' a longitudinal distance between the most downstream transverse observation plane of the optical path and the most upstream transverse observation plane of the optical path.

[0023] According to one embodiment of the fluorescence microscopy system, the optical medium is adapted to allow a nonlinear conversion of said first wavelength such that each second modulated laser pulse has at least one second wavelength 22, said nonlinear conversion being, for example, a second harmonic generation or a third harmonic generation, the third pulses thus having said at least one second wavelength 22. Preferably, the optical medium is adapted so that said nonlinear conversion makes it possible to induce a saturation effect on the laser pulses in order to limit their fluence in the optical medium to a value less than 10 JI cm 2. Preferably, the optical medium is a YAG, YV04, or BBO crystal.

[0024] 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 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.

[0025] 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.

[0026] According to one embodiment of the fluorescence microscopy system, the optical medium is adapted to allow amplification of the second pulses in the optical medium. Preferably, the optical medium is a multimode amplifying optical fiber.

[0027] 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.

[0028] Another object of the invention is a laser-based diagnostic system for a sample comprising: - an ultra-fast focusing system according to the invention - a suitable diagnostic device to collect the output beam reflected, transmitted or disseminated by a sample in such a way as to determine a physical parameter of the sample.

[0029] 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 of a shape of the sample having a general plate-like shape by comparing the intensity of the third pulses reflected or scattered by the sample, said variation of the shape of the plate forming said physical parameter of the sample.

[0030] A final object of the invention is a method for imaging a sample by fluorescence comprising the following steps: - generate initial laser pulses with a first wavelength 2f; - 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, - detect a first fluorescence radiation generated via the first wavelength Jp by illuminating a 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 differ from the other images. Brief description of the drawings

[0031] Other features, details and advantages of the invention will become apparent from the description given with reference to the accompanying drawings provided by way of example, which represent, respectively:

[0032] [Fig. 1], a fluorescence imaging system known from the prior art,

[0033] [Fig.2], a fluorescence imaging system according to an embodiment of the invention,

[0034] [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,

[0035] [Fig.4], a fluorescence imaging system according to an embodiment of the invention,

[0036] [Fig.5], a fluorescence imaging system according to an embodiment of the invention,

[0037] [Fig.6], a fluorescence imaging system according to an embodiment of the invention,

[0038] In the figures, unless otherwise indicated, the elements are not to scale and identical references designate identical elements.

[0039] Furthermore, unless otherwise specified, the optional features described in the description and figures can be combined. Description of embodiments

[0040] The invention relates to a laser-based diagnostic system for a sample, enabling ultra-fast focusing of the diagnostic laser beam. For clarification, "ultra-fast" here means that focusing is performed at a frequency greater than or equal to 10 kHz. The invention thus makes it possible to overcome the focusing speed inherently limited by the use of a mechanical (or optoelectronic, SLM-type) element, such as that found in the fluorescence microscopy device of [Fig. 1] known in the prior art.

[0041] In the invention, this ultra-fast focusing is made possible by a focusing assembly based on a modulation of the optical power of self-focused laser pulses within an optical medium.

[0042] 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 should 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.

[0043] Figure 2 schematically illustrates a microscopy system 1 by fluorescence according to the invention including in particular the ultra-fast focusing assembly SMP according to the invention.

[0044] 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 IL. The IL1 pulses delivered by the SL laser source are preferably (but not necessarily) monochromatic with wavelength A 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.

[0045]

[0046] 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. To clarify, by "different optical powers" we mean here that the optical power modulator MP is adapted so that at least two laser pulses IL2 have respectively a power Pa and a power Pg such that n-Pa > 10%, preferably. Indeed, this condition > ^g% Pb ~ Pu ~ ' Pu ~ has been determined by the inventors as the minimum condition to allow a satisfactory difference in focus under conventional operating conditions (see below).

[0047] Preferably, the MP optical power modulator is adapted to perform ultrafast modulation of optical power with a modulation frequency between 100 KHz and 1000 GHz.

[0048] The optical power modulator MP is for example an amplitude-splitting interferometric optical assembly such as a Mach-Zehnder or Michelson interferometer.

[0049] According to another embodiment, the optical power modulator MP is an electro-optical modulator, for example based on a Pockels cell, or an acousto-optical modulator, or even a thermo-optical modulator.

[0050] According to another embodiment, the MP power optical modulator is resonant at the first wavelength A. For example, according to one embodiment, the ultrafast MP modulator comprises a resonant optical microcavity in the strong coupling regime comprising 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.

[0051] 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 of the IL2 pulses during their propagation within the MO optical medium. Thus, the MO optical medium allows a variation in the longitudinal position (along the direction of the optical axis ~) of the focal point of the IL2 pulses during a variation in their optical power.

[0052] As a reminder, the self-focusing effect is induced by the optical Kerr effect, a third-order nonlinear process which appears in media exposed to intense electromagnetic radiation and which produces a variation of the refractive index n = n^ + n^Ij with no and n2 the linear and nonlinear refractive indices respectively of the optical medium MO and with II the intensity of the laser pulses IL2 within the optical medium MO.

[0053] 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 laser source SL and the power modulator MP be adapted so that the optical power P^jy] of each IL2^.^ pulse is greater than the critical self-focusing power Pcr of the optical medium MO, which is:

[0054] » _ / 1 X Pcr~ n^i2 ( 1 )

[0055] As illustrated in Figure 3, this self-focusing effect causes the IL2 pulses to focus at a focal point with a distance zsf measured from the EO input of the MO optical medium, which is:

[0056] Zsf-œQ\2n1lL

[0057] with the wai st (or smaller radius) of the incident beam FI.

[0058] For IL2 pulses of sufficiently high power compared to Pcr, we can write:

[0059] M

[0060] By way of non-limiting example, according to one embodiment, the laser source SL is such that the wavelength Before 1064 nm and = 50 the optical medium MO is in YAG and - A82 and A 10-¾2 By equation (1), we then calculate «2 - OX. the critical power which is Pcr = 1.5 MW. Thus, for an IL2 pulse of power P- 6 MW, we calculate by equation (3) a self-focusing distance of zsf = 4.2 mm.

[0061] Thus, it is understood that the optical power / ¾^] of each of the IL2^.^ pulses directly influences the longitudinal position of its focal point within the optical medium MO. More precisely, the higher the optical power P^yy] of the pulse / £<2^^, the closer the focal point of this pulse will be to the entrance of the optical medium (i.e., it will have a small 's / ).

[0062] Thus, as illustrated in Figure 2, for N pulses of decreasing power, we obtain N focal points presenting a position longitudinal^ ■■■ increasingly further from the EO entrance of the middle optical.

[0063] More generally, in the ultrafast focusing assembly SMP of the invention, the second pulses IL2 propagate by undergoing a self-focusing phenomenon within the optical medium MO so as to define, for each second pulse, a focal point having a position longitudinal ZJ in the optical medium different from the other second impulses i^j given their different optical power.

[0064] In the following description, the third pulses IL3 are the second pulses, at the output of the optical medium MO, having 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, Az 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 Az is subsequently called the "focusing range" and corresponds to the difference between the longitudinal position zn of the focal point furthest from the entrance EO of the optical medium (and therefore associated with the impulse IL2 of lowest optical power P},ùn) and the longitudinal position zi of the focal point closest to the entrance EO of the optical medium (and therefore associated with the impulse IL2 of highest optical power Pmax)- A- from equation (3), we can write Az in the following form: .

[0065]

[0066] By way of non-limiting example, the inventors have demonstrated that a focus range variation of 5 cm was possible for a YAG MO optical medium with a length (dimension along the optical axis) of 7 cm.

[0067] Thus, by controlling the optical power of the IL2 pulses via the ultrafast modulator MP, the ultrafast focusing assembly SMP of the invention makes it possible to control the longitudinal position of the focal point of the IL2 pulses in an ultrafast manner. The ultrafast focusing assembly SMP of the invention is therefore particularly suitable for being coupled to a diagnostic device adapted to collect the output beam FS reflected, transmitted, or scattered by an EC sample in order to determine, in an ultrafast manner, a physical parameter in different transverse observation planes zf, ...zN' of the EC sample. Indeed, a An optical system, comprising for example an SC coupling optical assembly and an OB objective as illustrated in [Fig. 2], allows the ultrafast variation of the longitudinal position of the focal point of the IL2 pulses to be reported in a variation of a longitudinal position of the focal point of the IL3 pulses 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 ultrafast focusing assembly SMP of the invention makes it possible to diagnose different transverse observation planes z / , ...zN' of the EC sample in an ultrafast manner.

[0068] The ultra-fast SMP 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 [Fig. 1].

[0069] 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 self-focusing IL3, is collected within a fluorescence microscope M in order to perform imaging of 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, thereby generating, from the absorption of the first wavelength A, a first fluorescence radiation RFI.

[0070] Given that the IL3 pulses of the output beam FS have focal points within the optical medium at different positions zb, ..., zN, the IL3 pulses are focused into different transverse observation planes zf, ..., zN' of the sample EC. This implies that each IL3 pulse will be focused by the objective lens OB within the sample into a respective observation plane, different from the other pulses, depending on the longitudinal position of self-focusing in the optical medium MO, and will therefore allow fluorescence imaging of this respective observation plane. Thus, by successively detecting the first generated RFI fluorescence radiation in these different transverse observation planes zf, ..., zN', it is possible to successively image a plurality of different "slices" of the sample EC.

[0071] 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 z / , .. .zN', on a matrix photodetector DT capable of detecting the first fluorescence radiation RFi. In addition, a processing unit UT connected to the matrix photodetector DT is configured to acquire, from the first detected fluorescence radiation, an image A of the sample for each of the IL3 pulses illuminating the sample. As explained above, each image li is acquired at starting from a respective IL3 pulse of different power from the others and therefore allows the imaging of a transverse z / observation plane of the respective sample and differ from the other images Ijj^.

[0072] It is understood that these images [I], -, 1? In the different observation planes The transverse z / , ...zN' values ​​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.

[0073] In summary, the system 1 of the invention allows for ultra-fast longitudinal scanning of the sample by imaging a plurality of different "slices" of the sample EC (corresponding to the different transverse observation planes z / , ...zN'). In other words, the fluorescence microscopy system 1 allows for ultra-fast longitudinal focusing within the sample.

[0074] Also, the fluorescence microscopy system 1 allows a clear improvement compared to known prior art fluorescence microscopy devices in which longitudinal focusing was carried out by a mechanical element and whose speed was intrinsically limited.

[0075] Preferably, as illustrated in [Fig.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.

[0076] 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 zb ...zN in different transverse observation planes z / , ...zN' of the sample EC.

[0077] 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 zb ... zN in different transverse observation planes z / , ... zN' of the sample EC as illustrated in Figure 2. The longitudinal distance between the first plane is called the "object focusing range Az'". observation plane Z^ (the upstream observation plane of the optical path) and the last observation plane z^1 (the downstream observation plane of the optical path).

[0078] The relationship between the position of the transverse observation planes zf, ..., zN' within the sample EC and the position zb, ..., zN of the focal points of the IL2 impulses within the optical medium MO is governed by geometric optics in a manner known per se. Furthermore, regardless of the optical design of the objective OB (and the coupling system SC, if applicable), there exists a factor Δz such that Az' = yz Az, the factor Δz corresponding to the transverse magnification of the optical assembly (formed by the objective OB and the coupling system SC in the embodiment of [Fig. 2]) producing the image of the focal points of positions zb, ..., zN onto focal points arranged in the transverse observation planes zf, ..., zN'.

[0079] It is generally preferable to image the entire thickness eEc of the sample. The "sample thickness eEc" is defined as the dimension of the sample along the longitudinal z-direction. To image the entire sample, the object focusing range ^z' must be greater than or equal to eEc. As mentioned above, Az — y Az and ^z depend (among other things) directly on the maximum and minimum power P„ùn 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 Pmax and a minimum power P„ùn of the second pulses are such that Az' > 0.5eEc and preferably such that Az' ≥ eEc.

[0080] Alternatively, it is preferable to image the majority of the sample thickness, and we then have a maximum power and a minimum power P„ûn of the second pulses such that Az' > 0.5.e£c

[0081] Preferably, as illustrated in the embodiment of Figure 2, the ultrafast focusing assembly SMP comprises an optical assembly OF for focusing the IL2 pulses within the optical medium. The optical assembly OF increases the illumination of the IL2 laser pulses focused within the optical medium. This, in turn, increases the variation in the refractive index induced by the Kerr effect, thereby enhancing the self-focusing effect. Indeed, as explained above and as described by equation (2), increasing the illumination through the optical assembly OF reduces the self-focusing distance zsf.

[0082] Thus, the addition of the OF optical assembly makes it possible to increase the maximum illumination Ilmox of the IL2 pulses. This therefore allows a shift in the longitudinal position of the focal points towards the EO entrance of the optical medium and, in particular, a shift of the first observation plane zf upstream of the path optically. However, it is still possible to reduce the power of some of the IL2 pulses via the laser source or the MP modulator to have the last observation plane zNr placed at the same longitudinal position with or without the OF optical assembly. In other words, the OF optical assembly increases the focusing range within which it is possible to generate focal points with different longitudinal positions, and therefore allows an increase in the object focusing range &z'.

[0083] 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 self-focusing. The system is thus made more modular.

[0084] As explained above, the output beam FS is focused within the sample EC. This implies that the size of the focal spot in the observation plane is reduced. Thus, the fluorescence information of the observation plane determined via the image I is intrinsically spatially limited by the size of this focal spot.

[0085] To overcome this problem, in the embodiment of Figure 4, the fluorescence microscopy system 1 includes a translation system ST adapted to allow a transverse spatial scan (along the x and y directions in the example of Figure 4) of the sample by the output beam FS. Thus, a plurality N of images of different transverse observation planes of the same transverse region Rk are acquired from N laser pulses IL1^wj of different powers, and then different transverse regions R^-m] of the sample are scanned. In a single transverse scan, the processing unit UT is then adapted to reconstruct a "complete" fluorescence image Icj of each transverse observation plane z / of the sample by digital image processing. (P31 example by concatenating or adding the fluorescence intensities of the transverse regions Rk^M] of the same transverse observation plane Z / ').

[0086] In other words, the "complete" fluorescence image of a transverse observation plane of the sample is obtained by a transverse spatial scan of the sample with 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.

[0087] According to the embodiment illustrated in [Fig. 4], the sample EC is mounted on the translation system ST, which is, for example, a piezo translation stage An electrical connection is made to the SL laser source to allow synchronization of the transverse spatial scanning with the repetition of the IL1 laser pulses. Alternatively, in another embodiment, a scanning system using rotating two mirrors (a scanner) is positioned in the beam path to enable 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.

[0088] Preferably, the processing unit UT is adapted to reconstruct a three-dimensional depth image of the sample from the 'V' complete fluorescence images of the sample, each corresponding to a respective slice of the sample (a respective transverse observation plane Z;'). 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 (e.g., MRI).

[0089] According to one embodiment of the invention, the optical medium MO is adapted to allow amplification of the first IL2 pulses during their propagation within the optical medium MO. For example, the optical medium MO 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 focal point of the self-focusing pulses. Indeed, the amplification of an IL2 pulse leads to an increase in the power P of this pulse and therefore a shift in the longitudinal position of the focal point towards the EO inlet of the optical medium (see equation (3)). Thus, just like the OF optical assembly, an amplifying optical medium MO allows for an increase in the object focusing range As'.

[0090] According to a preferred embodiment, the optical medium comprises a structure adapted to allow better control of the self-focusing of IL2 pulses in the optical medium. A medium with a Bragg grating, for example, allows a laser pulse to be compressed, thus increasing its peak power during propagation and decreasing the self-focusing distance from the entrance into the nonlinear medium.

[0091] Figure 5 illustrates an embodiment of the invention allowing simultaneous imaging of several transverse observation planes Z^, ..., g / , .Zy' of the sample EC. For this purpose, the fluorescence microscopy system 1 comprises an interferometric optical device DOI disposed in the optical path of the laser beam, optically upstream of the optical medium MO (for example, downstream of the MP modulator as illustrated in [Fig. 5]) and adapted to divide each first IL1 pulse (or each second IL2 pulse as appropriate) into a plurality of IL1b...IL1N replicas with different optical powers. The IL1 or IL2 pulse from which a plurality of replicas are formed simultaneously is called the "initial pulse".

[0092] Critically, by virtue of its optical design, the DOI interferometric optical device is adapted to form these IL1b ... IL1N replicas simultaneously from an initial pulse IL1. For the same reasons mentioned above, each replica IL1b ... IL1N is successively autofocused in the MO medium at a longitudinal position different from the other replicas, depending on its optical power. Similarly, each replica IL1b ... 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 of the replicas of an initial pulse that are autofocused in the optical medium and then illuminate the sample EC, the DT photodetector acquires an interlaced image of the sample.The interlaced images associated with an initial impulse are acquired simultaneously (or almost simultaneously depending on the interferometric optical device) and each interlaced image associated with an initial impulse corresponds to an image of a transverse observation plane of the sample different from the other interlaced images.

[0093] Thus, unlike the embodiments detailed previously, the introduction of the DOI interferometric optical device allows, by the creation of the replicas IL1 b ...IL1n, to image a plurality of "slices" of the sample (the transverse observation planes) simultaneously (or almost simultaneously).

[0094] By way of non-limiting example, the DOI optical interferometric device is a Michelson interferometer, a Fabry-Pérot interferometer or more generally an amplitude-division interferometer.

[0095] 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 pulse power IL1 is performed almost instantaneously by the interferometric optical device DOI. In this variant, the photodetector DT therefore directly acquires temporally interlaced images 1t, --.1,, • ■ - associated respectively with the transverse observation planes z / , ..., z / , ..., zNr. The image acquired by the photodetector DT thus corresponds to an image in which N transverse planes are in focus.

[0096] 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 ^2 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 [Fig. 5]).

[0097] By way of non-limiting example, the optical medium is adapted to induce, from the first wavelength, a generation of second harmonics or a generation of third harmonics. For example, the optical medium is a YAG crystal, a YVO4 crystal, or a BBO crystal.

[0098] In a manner known per se, the nonlinear conversion of the first wavelength is carried out 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 A and U. Thus, each IL3 pulse of the output beam FS has wavelengths A and U, both of which are focused within the sample in the same transverse observation plane Zp, Zp, Zn', as a function of the power of the IL2 pulse.

[0099] The sample is adapted to absorb the second wavelength 22 so as to emit a second RF2 fluorescence radiation.

[0100] In addition to detecting the first wavelength, the DT photodetector of the microscope M is adapted to detect the second wavelength. By way of 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.

[0101] Thus, the processing unit UT is configured to acquire, from the second fluorescence beam detected by the photodetector DT, an "additional" image of the sample for each of the third pulses illuminating the sample EC. Each additional image [-] therefore corresponds to additional fluorescence information generated by the second wavelength 22 and is associated with a transverse observation plane z / of the sample that differs from the other additional images. As explained above, this transverse observation plane z / for which the additional image is acquired via wavelength 22 is identical to that imaged via wavelength A.

[0102] 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 to induce a saturation effect of the IL2 laser pulses. For this, preferentially, the optical medium and the SL laser source are adapted so that the IL2 laser pulses propagate in the optical medium with a fluence of less than 10 J! cm 2 .

[0103] The inventors have determined that a YAG optical medium exhibits a satisfactory nonlinear conversion saturation effect that prevents the occurrence of optical damage.

Claims

Demands

1. An ultrafast focusing assembly (SMP) for a fluorescence imaging or 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) having 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 (zb..., zN) in the optical medium different from the other second pulses, a laser radiation from the optical medium and presenting the self-focused second pulses, called third pulses (IL3), being called output beam (FS).

2. A fluorescence microscopy system (1) comprising: - an ultrafast 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 (RFi) generated via the first wavelength 2, by the illumination of the sample (EC) with the output beam, and 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 an observation plane (zf, ...zN') transverse of the sample differ from the other images.

3. Fluorescence microscopy system according to the preceding claim comprising a mechanical transverse focusing (ST) 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 (ST) system.

4. Fluorescence microscopy system according to claim 2 or 3, comprising an optical coupling 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 (OID) disposed on the optical path upstream of the optical medium (OM), and adapted to divide each first or second laser pulse into a plurality of replicas (IL1b .. .IL1N) 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. 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 a interferometric optical assembly or an electro-optical or acousto-optical modulator.

9. Fluorescence microscopy system according to any one of claims 2 to 8, wherein the laser source and the optical power modulator are adapted so that a maximum power Pmax and a minimum power P„ù„ of the second pulses are such that Az' > 0.5^£C and preferably such that Az - with cec a thickness of the sample and with Az' a longitudinal distance between the downstreammost transverse observation plane of the optical path and the upstreammost transverse observation plane of the optical path.

10. A fluorescence microscopy system according to any one of claims 2 to 9, wherein the optical medium is adapted to allow a nonlinear conversion of said first wavelength 2f such that each second modulated laser pulse has at least one second wavelength d2, said nonlinear conversion being, for example, a second harmonic generation or a third harmonic generation, the third pulses thus having said at least one second wavelength

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 J1 cm 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 22, by illuminating the sample (Ech) with the output beam, and then - acquire, from the detected second 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 differ from the other additional images.

14. Fluorescence microscopy system according to any one of claims 2 to 13, wherein the microscope includes 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.

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

16. 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.

17. A laser sample diagnostic system (EC) comprising: - an ultra-fast 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. generating first laser pulses (IL1) having a first wavelength λ; B. modulating the power of the first laser pulses (IL1) so as to form second laser pulses (IL2) having different optical powers; C. generating, 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 exhibiting the self-focused second pulses, called third pulses, being called the output beam (FB), D. detect a first fluorescence radiation generated via the first wavelength by illuminating 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 differing from the other images.

Images