Imaging system with photon reallocation

Abstract

Photon reallocation imaging system comprising: - A polychromatic light source (SL) to generate an illumination beam (FE); - A first optical system (SO1) to disperse said illumination beam with a first angular dispersion DA1 and focus it onto a sample (E) so as to form a first spectral line (LS1); - A second optical system (SO2) configured to collect a light beam elastically scattered (FS) by said sample, called the signal beam, apply a second angular dispersion DA2 to it and focus it into a focal plane so as to form a second spectral line (LS2), of greater length than the first spectral line; - An image matrix sensor (CMI) arranged in said image focal plane; - A means (S1) for inducing a relative displacement between the first spectral line and the sample along a third direction (y), perpendicular to the first direction;and - A means for forming an image of the sample by reallocation of photons from one or more images acquired by said image matrix sensor corresponding to a plurality of different positions taken by the first spectral line on the sample. Figure for the abstract: Fig. 2;

Description

Title of the invention: Imaging system with photon reallocation

[0001] The invention relates to the field of optical microscopy, and more generally to imaging.

[0002] Optical microscopy plays an essential role in biology and micro- and nanotechnologies, as it allows samples to be observed at a high throughput, unlike, for example, electron microscopy which requires complex preparation operations. However, its resolving power is necessarily limited by light diffraction. According to Abbe's theory, for a conventional optical microscope the maximum resolution d is given by

[0003] J -

[0004] where 2 is the wavelength of the light used (between 380 nm and 780 nm for visible light) and ON the numerical aperture which can hardly exceed a value of 1.4 for biological samples. It follows that the resolution in visible light cannot exceed 135 nm, which is not suitable for the observation of very small structures such as viral particles. An improvement in spatial resolution can also be obtained by reducing the wavelength, but at the cost of considerable technical difficulties.

[0005] So-called "superresolution" techniques make it possible to exceed the Abbe resolution limit by exploiting fluorescent markers and / or non-linear effects. These techniques are complex to implement and are not suitable for all applications.

[0006] Furthermore, both in biology and in micro- and nanotechnologies, it is often necessary to also obtain a high spatial resolution in an axial direction (the Abbe limit concerns the lateral resolution, in a plane perpendicular to the optical axis). Confocal microscopy makes it possible to obtain images with a very low depth of field (of the order of a few hundred nanometers), and therefore to carry out a "sectioning" of the sample allowing access to its three-dimensional structure. This technique is most often associated with the use of fluorescent markers, but it is also possible to use it in reflectance, without markers.

[0007] A confocal microscope uses a point illumination source, an image of which is projected onto the sample to be observed by means of an objective. The light coming from the sample (backscattered light in the case of a reflectance confocal microscope; fluorescent emission when a fluorescent marker is used) is focused on a pinhole optically conjugated to the point source, then detected for example by means of a photomultiplier. The function of the pinhole is to suppress the radiation which does not come from the focal plane of the objective, thus achieving optical sectioning. An image of the sample is obtained point by point, by scanning. More precisely, a two-dimensional scan, in the two directions perpendicular to the optical axis, makes it possible to obtain an image of a slice of the sample centered around the focal plane of the objective. By adding an axial scan of this focal plane, a three-dimensional image is obtained.

[0008] The thickness of the slice is smaller, and therefore the axial resolution is better, the smaller the diameter of the pinhole, but the gain is low when we go below 1 Airy unit (AU). The Airy unit is the diameter of the Airy spot of the microscope, and is equal to —L—.

[0009] Confocal microscopy allows a gain in lateral resolution, compared to the Abbe limit, which can theoretically reach up to 30%, using a pinhole with a diameter of less than 1 AU. However, this is obtained at the cost of a degradation of the signal-to-noise difference.

[0010] The photon reallocation technique, first proposed in (Sheppard 1988) makes it possible to improve the lateral resolution of a confocal microscope by a factor of 2 in principle. The idea behind this technique is to replace the photomultiplier, or more generally the point radiation detector, with a matrix detector which makes it possible to acquire an elementary image for each acquisition point. The image is then resized (ideally, reduced by a factor of 2) by digital or optical means before moving on to the next scanning point. The final image is obtained by integrating the different successively acquired scanning images, each shifted by one scanning step relative to the previous image.

[0011] A purely optical implementation of the photon reallocation technique is described in (York 2013), (De Luca 2013) and (Curd 2015). It consists of applying a first angular scan to the illumination beam, applying an inverse angular scan to the beam coming from the sample, then applying to this same beam coming from the sample a second angular scan, synchronized with the first scan. The second angular scan has a larger amplitude normalized to the beam section, ideally by a factor of two, than the first scan. "Normalized to the beam section" means that if a is the ratio between the amplitude of the second angular scan and that of the first angular scan and M is the ratio between the section of the beam coming from the sample and that of the illumination beam, it is the quantity a / M which must be greater than 1 and ideally equal to 2.

[0012] As in classical confocal microscopy, it is possible to parallelize the acquisition using a pinhole matrix and microlens matrices, see for example the aforementioned article (York 2013).

[0013] Photon reallocation has been applied in particular to confocal fluorescence microscopy, see for example the aforementioned articles (York 2013), (De Luca 2013) and (Curd 2015), where a gain in lateral resolution of a factor of 1.5 was observed. To the inventor's knowledge, the only application in reflectance of photon reallocation was described in (DuBose 2019). In this article, however, it was not a question of a microscope, but of an ophthalmoscope, in which the objective is replaced by the lens of a patient's eye, thus having a low numerical aperture, and consequently a resolution of the order of tens of micrometers.

[0014] Document EP 4 012 476 and the article (Aguilar 2020) describe a method and apparatus for confocal microscopy by reflectance or transmission with photon reallocation with a spatial resolution optimized by an opportune dimensioning of the pinhole.

[0015] Confocal photon reallocation microscopy is a point-by-point scanning imaging technique. Consequently, it requires long acquisition times which are detrimental, or even prohibitive, for certain applications such as in-situ characterization in non-destructive testing for rapid diagnosis in the biomedical, materials, and micro- and nanotechnology industries.

[0016] The invention aims to overcome this drawback of the prior art.

[0017] According to the invention, this object is achieved by dispersing a light beam in a first direction so as to form a “spectral line” which illuminates the sample. Thus, a plurality of points of the sample aligned in said first direction are illuminated simultaneously with different wavelengths. A portion of the surface of the sample can therefore be inspected by scanning only in a second direction which is not parallel (and preferably perpendicular) to the first.

[0018] Having to scan along a single direction instead of two significantly reduces the image acquisition time. Furthermore, in certain embodiments of the invention, this makes it possible to use resonant mirrors oscillating at a very high frequency - of the order of several tens of kHz - which makes it possible to further reduce the acquisition time. The use of such mirrors would be difficult to envisage if, in accordance with the prior art, it was necessary to synchronize two scans along the first and second directions.

[0019] The use of a spectral line to replace scanning along a spatial direction has already been proposed, for example in (Teamey 1998) and (Hwang 2015). More In particular, (Teamey 1998) concerns a fiber-reinforced confocal microscope intended to be incorporated into an endoscope, while the microscope of (Hwang 2015) replaces the scanning in the 2nd direction by a variable frequency modulation in space. Neither of these systems allows to overcome the Abbe limit, and in particular is not compatible with the principle of photon reallocation.

[0020] An object of the invention is therefore a photon reallocation imaging system comprising: - A polychromatic light source configured to generate at least one spatially coherent illuminating light beam in a range of illuminating wavelengths; - A first optical system configured to disperse said illuminating light beam with a first angular dispersion DAi and focus it on a sample so as to form a first spectral line oriented in a first direction; - A second optical system configured to collect a light beam elastically scattered by said sample, called a signal beam, to apply a second angular dispersion DA2 to it, and to focus it in a focal plane so as to form a second spectral line oriented in a second direction; - An image matrix sensor arranged in said image focal plane; - A means for inducing a relative displacement between the first spectral line and the sample in a third direction, perpendicular to the first direction; and - A means for forming an image of the sample by reallocation of photons from one or more images acquired by said image matrix sensor in correspondence with a plurality of different positions taken by the first spectral line on the sample; wherein the first optical system comprises a first dispersive optical device for dispersing said illuminating light beam with a first angular dispersion DAi and the second optical system comprises a second dispersive optical device for applying said illuminating light beam with said second angular dispersion DA2; the length of the second spectral line in the second direction being greater than that of the first spectral line in the first direction by a factor Ag=MgDA2 / DAi>1, Mg being the ratio between the dimension of the illumination beam on the first dispersive optical device measured in the first direction and the dimension of the signal beam on the second dispersive optical device measured in the second direction.

[0021] According to particular embodiments of the invention:

[0022] Said AG factor can be between 1.8 and 2.2.

[0023] The first optical system may comprise: - a first collimator system, configured to collimate said illumination light beam; - said first dispersive optical device; - an optical focusing system configured to receive as input the illuminating light beam dispersed by said first dispersive device and focus it on the sample.

[0024] The second optical system may comprise: - said or another focusing system for collecting said signal beam, collimating it and directing it towards said first dispersive optical device, or another dispersive optical device, such that it is spectrally recombined; - said first or said other dispersive optical device, configured to spectrally recombine said signal beam; - said second dispersive optical device having the second angular dispersion DA2, for spectrally dispersing said signal beam along said second direction; and - at least one optical element configured to focus in a focal plane, called the image focal plane, the signal beam spectrally dispersed by the second dispersive optical device.

[0025] In this case, the second optical system may comprise a pinhole arranged in a plane conjugate with a focal plane of the focusing system of the second optical system so as to carry out confocal filtering.

[0026] The means for forming an image of the sample by reallocation of photons from a plurality of images of the second spectral line acquired by said image matrix sensor may comprise a digital image processor. In this case, the means for inducing a relative displacement between the first spectral line and the sample may comprise a sample translation stage, or it may comprise a variable orientation mirror common to the first and second optical systems, configured to apply an angular scan in the third direction to the illumination light beam and an opposite angular scan to the spectrally recombined signal beam.

[0027] According to other embodiments - The means for inducing a relative displacement between the first spectral line and the sample may comprise a first variable orientation mirror common to the first and second optical systems, configured to apply an angular scan in the third direction to the illumination light beam and an opposite angular scan to the spectrally recombined signal beam; and - The means to form an image of the sample by reallocation of photons comprises a second variable orientation mirror, configured to apply an angular scan in a fourth direction, perpendicular to the second direction, to the signal beam spectrally dispersed by the second dispersive optical device; The angular scanning applied by the second variable orientation mirror being synchronous and having an amplitude equal to As / Ms times the amplitude of that applied by the first mirror, As being a factor between 1.8 and 2.2 and Ms and the ratio between the dimension of the illumination light beam on the second variable orientation mirror, measured along the fourth direction, and the dimension of the spectrally dispersed signal beam on the first variable orientation mirror, measured along the third direction. In this case, the first variable orientation mirror may be structurally independent of the second variable orientation mirror and the first dispersive optical device is distinct from the second dispersive optical device.

[0028] The first dispersive optical device may be a diffractive system having said angular dispersion DAi at the first diffraction order and said angular dispersion DA2=2DAi at the second diffraction order, the second optical system being configured to collect the light scattered at the second diffraction order by said diffractive system.

[0029] In other embodiments, the first and second variable orientation mirrors may be formed by two distinct reflective regions of a single variable orientation support. For example, said orientation support. The first dispersive optical device may be distinct from the second dispersive optical device.

[0030] The light source may comprise a first set of a plurality of microlenses aligned along the third direction to generate a plurality of said illumination light beams in parallel, whereby said first optical system forms on the sample a plurality of first spectral lines oriented along a first direction and arranged along said third direction, and wherein said second optical system may comprise a second set of a plurality of microlenses aligned along said third direction to form on the image matrix sensor a plurality of first spectral lines oriented along a first direction and arranged along a fourth direction, perpendicular to said second direction.

[0031] It should be noted that, contrary to what is taught for example by (Teamey 1998) and (Hwang 2015), an imaging system according to the invention performs a second spatial dispersion operation to form, on the image sensor, a second spectral line of greater length (ideally double) than that of the first spectral line projected onto the sample. This is necessary to make possible reallocation of photons.

[0032] Other characteristics, details and advantages of the invention will emerge on reading the description given with reference to the appended figures given by way of example and which represent, respectively:

[0033] [Fig.l], a diagram of a confocal microscope with photon reallocation according to a first embodiment of the invention;

[0034] [Fig.2], a diagram of a confocal microscope with photon reallocation according to a second embodiment of the invention;

[0035] [Fig.3], a diagram of a confocal microscope with photon reallocation according to a third embodiment of the invention;

[0036] [Fig.4] a diagram of a confocal microscope with photon reallocation according to a fourth embodiment of the invention;

[0037] [Fig.5], a diagram of a confocal microscope with photon reallocation according to a fifth embodiment of the invention;

[0038] [Fig.6], a diagram of a confocal microscope with photon reallocation according to a sixth embodiment of the invention; and

[0039] [Fig.7], a diagram of a confocal microscope with photon reallocation according to a seventh embodiment of the invention

[0040] [Fig.8], a diagram of a confocal microscope with photon reallocation according to an eighth embodiment of the invention.

[0041] In the figures, the same reference signs designate corresponding elements.

[0042] In the device of [Fig.l], a polychromatic light source SL generates a spatially - but generally not temporally - coherent illumination beam FE. The SL source comprises, for example, a broad spectrum source SLS, for example an arc lamp, a light-emitting diode or a pulsed laser. A spectral filter FSC selects a portion of the spectrum of the SLS source. The extent of the wavelength range selected by the filter FSC determines, together with the angular dispersion DAb, the extent of the first spectral line scanning the sample.Its sizing results from a compromise - a large spectral extent reduces the constraints on the angular dispersion DAi and allows to benefit from a larger fraction of the total luminous intensity emitted by the SLS source, but makes it more difficult to compensate for chromatic aberrations in the imaging system (axial and longitudinal chromatism) especially with an f-theta scan lens which typically tolerates an AX / X spectral width of the order of 10%. The resolution across the field of view also becomes variable in case of large spectral extent (optical resolution proportional to the local wavelength).

[0043] A converging lens Ln0 and a pinhole Pi arranged in the focal plane of this The latter perform spectral filtering which ensures the spatial coherence of the FE lighting beam.

[0044] The illumination beam FE at the output of the light source SL is reflected by a splitter plate BS which directs it towards the first optical system SOI, intended to illuminate the sample E. Said optical system SOI comprises, in addition to the splitter plate BS, a first converging lens L105 which collimates the beam FE; a planar mirror M102 (non-essential), two other converging lenses L104, Li03 forming an afocal system, in the common focal plane of which is arranged a second pinhole P2, which is thus optically conjugated with the first pinhole Pi at the output of the light source SL. The illumination beam exits the collimated lens L103 and is directed towards a diffraction grating Gi having an angular dispersion DAi in a first direction x.The beam diffracted by the grating Gi passes through an afocal system consisting of two other converging lenses L 101, L102 and is reflected by a planar mirror M102 (non-essential) before being focused on the sample E by a microscope objective MO. The spectral dispersion operated by the grating Gi and the focusing by the objective MO lead to the formation of a spectral line LSI on the sample, that is to say an elongated focal spot, oriented along the x direction, in which the wavelength of the light varies monotonically with the position along the said x direction.

[0045] The main function of the afocal system formed by the lenses L101 and L102 is to systematically place the diffraction grating Gi in a plane conjugate with the pupil of the microscope objective, located at the rear of the latter. In addition, it can allow the size of the illumination beam to be adjusted.

[0046] The sample E is mounted on a translation stage PT which allows it to be translated in a direction y perpendicular to x. In this way, the spectral line LS 1 scans a surface of the sample.

[0047] A second optical system SO2 collects the light beam FS (“signal beam”) elastically scattered by the sample - i.e. scattered without changing wavelength - to form a second spectral line LS2 on an image matrix sensor CMI. The imaging system of [Fig.l] operates in reflection, which allows a pooling of optical elements between the first and second optical systems. More particularly, all the elements of the first optical system SOI also belong to the second optical system, which also includes additional components. Also, the light backscattered by the sample E is collected and collimated by the microscope objective MO and spectrally recombined by the same diffraction grating Gi which had dispersed it during the outward path.The afocal system formed by the lenses Ln^ and L104 makes it possible to carry out confocal filtering, as in a confocal microscope according to the prior art, in addition to making it possible to modify the . FS beam size.

[0048] The beam FS passes through the splitter plate BS which had reflected the illumination beam FE from the source SL. The converging lenses L105 and Li06, placed on either side of said splitter plate, form an afocal system which makes it possible to place a second diffraction grating G2 in a conjugate plane of the pupil of the objective OM. This second diffraction grating disperses the light again in the x direction. A lens Li07 forms a second spectral line LS2 oriented along the x direction on the image matrix sensor CMI.

[0049] The image matrix sensor can be made, for example, in CMOS or CCD technology. It has a number of pixels in the x direction which is determined by the spatial extent of the spectral line that can be obtained by the optical system SO2, typically of the order of hundreds. The number of pixels in the y direction can be less - of the order of ten or a few tens - because these pixels are used only for the implementation, by a digital image processor PNI, of a photon reallocation algorithm. The implementation of this algorithm requires synchronization of the digital image processor PNI with the translation stage. The digital image processor PNI can be for example a microprocessor programmed in a timely manner or a dedicated digital circuit.

[0050] For this algorithm to achieve an improvement in spatial resolution, it is necessary for the second spectral line LS2 to have a length in the x direction that is greater, ideally by a factor of 2 or close to 2 (for example between 1.8 and 2.2). This is illustrated in the top left part of the figure; it is noted in particular that a segment corresponding to a wavelength interval AX is longer in the spectral line LS2 than in LSI. This elongation can be obtained by using a second diffraction grating G2 having an angular dispersion DA2 greater than that - DAi - of the first diffraction grating, by the magnification MG of the optical beam produced by the assembly consisting of the lenses L103, Li04 L105 and L006 or by a combination of the two.Generally speaking, if Mg is the ratio between the diameter dG2 of the beam incident on the grating G2 and the diameter dGi of the beam incident on the grating Gi (MG=dG2 / dGi), we obtain MGDA2 / DA i=length(LS2) / length(LSl)=AG, AG being a factor between 1.8 and 2.2 and ideally having a value of 2. For example, if MG=1 we will choose a second diffraction grating having an angular dispersion (approximately) double that of the first diffraction grating.

[0051] The embodiment of [Fig. 2] differs from that of [Fig. 1] only in that the mirror Mi02 of the first optical system is replaced by an oscillating mirror - or more generally one having a variable orientation - Si. More precisely, the variation in orientation of the mirror Si is controlled so as to induce a displacement of the first LSI spectral line on the sample E in the y direction; for this reason, Si will be called in the following "scanning mirror". In this way, the LSI spectral line scans the sample E without the need to translate the latter along the y direction. As in the embodiment of [Fig.l], the photon reallocation is carried out digitally by a PNI image processor, synchronized with the moving mirror Sp

[0052] The embodiment of [Fig.3] differs from that of [Fig.2] in that the second optical system SO2 also comprises an oscillating scanning mirror - or more generally one having a variable orientation - S2 arranged before the lens Li07 for focusing the beam FS onto the image matrix sensor CMI. An afocal system formed by the converging lenses L308 and L30g makes it possible to combine the mirror S2 with the pupil of the microscope objective OM.

[0053] The variation in orientation of the mirror S2 is controlled so as to induce a displacement of the second spectral line LS2 on the image matrix sensor in the y direction, thus carrying out the reallocation of photons in a purely optical manner. To obtain a gain in resolution, it is necessary for this scan to be synchronized with that carried out by the first spectral line LSI on the sample E, and to have a higher amplitude by a factor As / Ms, As being a factor between 1.8 and 2.2 and ideally having a value of 2 and Ms being the ratio between the diameter dS2 of the beam on the mirror S2 and the diameter dd of the beam on the mirror Si. In the embodiment of [Fig. 3], MG is the total magnification of the assembly constituted by the lenses L103, L104, L105, L106, L308 and L30g.

[0054] Unlike the case of the embodiments of [Fig.l] and [Fig.2], the number of pixels of the image matrix sensor CMI in the y direction is determined by the amplitude of the scanning carried out by the second spectral line LS2, and is typically of the same order of magnitude as the number of pixels in the y direction.

[0055] Furthermore, if the scans performed by the scanning mirrors Si and S2 are sufficiently fast, the image sensor CMI does not necessarily have to acquire a separate image for each position of the spectral lines LSI and LS2: the same image can correspond to several positions. In a limiting case, the acquisition of a single image may be sufficient, which makes it possible to dispense with synchronization between the scanning mirrors Si, S2 and the image matrix sensor.

[0056] In the embodiments of [Fig.l], [Fig.2] and [Fig.3], the rate of the scanning(s) is limited by the need to perform synchronization: between the translation stage PT and the image matrix sensor CMI in the case of [Fig.l]; between Si and the image matrix sensor CMI in the case of [Fig.2]; and at least between Si and S2 in the case of [Fig.3]. This limitation can be overcome thanks to the embodiment of [Fig.4] in which the two scanning mirrors Si and S2 are made on two opposite faces of the same oscillating, or more generally variable orientation, SPO support. In the specific embodiment of the figure, this is made possible by the use of the two redirection mirrors M402 and M403. This eliminates any requirement in terms of synchronization, and therefore greatly facilitates the use of a high scanning frequency - of the order of kilohertz - obtained for example by means of a resonant SPO support or a polygonal scanner. Since the angular amplitude of the scanning carried out by the mirrors Si and S2 is, by construction, the same, the second optical system must provide a magnification Ms of the beam FS between the two scanning mirrors of the order of 2 (in other words dS2 / dSi=Ms=A s, with As=2 - at least approximately). In the embodiment of [Fig.4], this is ensured by the afocal system formed by the lenses Li05, Li06, L408 and L409.

[0057] The embodiment of [Fig. 5] is similar to that of [Fig. 4], except that the functions of the two diffraction gratings Gi and G2 are performed by two distinct regions of a single grating G, which is made possible by the use of the redirection mirrors M502, M503 and M504 and by the fact that, in the example of [Fig. 5], the illumination beam FE passes through the beam splitter BS while the beam FS is reflected from it. Given that the two distinct gratings Gi and G2 are replaced by two zones of a single grating, DAi=DA2, it is therefore necessary that the magnification MG be approximately 2, in other words dG2=2dGi at least approximately.

[0058] The embodiments of [Fig.4] and [Fig.5] require two diffraction gratings, or a single grating large enough to present two distinct areas that can be illuminated independently of each other. The embodiment of [Fig.6], on the other hand, comprises a single grating, the spatial extent of which does not necessarily have to be greater than that of the illumination beam FE.

[0059] The system of [Fig.6] comprises a light source SL as described above with reference to [Fig.l]. The illumination beam FE from this source is collimated by the lens L105 for collimating the illumination beam FE, is deflected by a first redirection mirror M60i (non-essential), and reaches the diffraction grating Gi which disperses it spectrally to the first diffraction order. The spectrally dispersed beam is spatially filtered by the slit FR located in the common focal plane of the two lenses L60i and L602 forming an afocal system. The length of the slit FR in the spectral dispersion direction x depends on the introduced angular dispersion of the grating, while the width in the y direction is determined as for the pinhole of a conventional confocal microscope. The main function of the afocal system formed by the lenses L602 and Lm3 is to optically conjugate the slit FR and the pinhole Pi of the source SL.Alternatively, the FR slot may be omitted.

[0060] The illumination beam dispersed by the network Gi and spatially filtered is directed towards the microscope objective MO by the scanning mirror Sb formed by a reflective surface of an oscillating or variable orientation support SPO, which makes it possible to obtain a spectral line LSI scanning the sample E, as described above with reference to other embodiments. The light backscattered by the sample is collected by the objective MO, reflected by Sb which compensates for the effect of the scanning introduced in the "forward path" and spatially filtered by the assembly L60i - FR- L602, before arriving again on the diffraction grating Gp The originality of this embodiment is that, instead of collecting the beam spectrally recombined by the first diffraction order of the grating, which propagates along the path of the illumination beam but in the opposite direction, the light diffracted to the second order is used (beam FS' in the figure).The angular dispersion of the second order being twice that of the first order; consequently, the diffracted beam FS' is not spectrally recombined, but has an angular dispersion of amplitude twice that of the illumination beam FE having been diffracted to the first order (in other words, the spectral dispersion introduced during the "forward path" is over-compensated). This beam FS' is magnified by a factor of Ms=2 approximately by the afocal system formed by the lenses L607 and L6os (also, dS2=2dSi), redirected (optionally) by the mirrors M6o2, M603, M6o4, reflected by the scanning mirror S2, formed on the same variable orientation support SPO as Si and finally focused by the lens L6o9 to form the spectral line LS2 on the image matrix sensor CMI. By construction, in this embodiment dG1=dG2. .

[0061] It will be noted that in the embodiments described above, the first optical system SOI for forming the first spectral line LSI on the sample E and the second optical system largely coincide, the same optical components being crossed by the light on the outward journey (formation of the first spectral line and illumination of the sample) and on the return journey (collection and processing of the signal beam). More particularly, in the embodiments of [Fig. 1] to [Fig. 5], the first optical system is a subset of the second. This is less true in the case of the embodiment of [Fig. 6]: the use of the 2nd diffraction order of the diffraction grating in fact separates the path of the signal beam from that of the illumination beam. In other embodiments, the first and second optical systems may be totally or substantially disjoint. This is the case, in particular, of imaging systems operating in transmission.

[0062] Such a system is illustrated in [Fig.7]. Its operating principle is similar to that of [Fig.3], except that it operates in transmission and not in reflection. Similarly, the embodiments of [Fig.1]; [Fig.2], [Fig.4] and [Fig.5] can be modified for operation in transmission.

[0063] In the system of [Fig.7] the illumination beam FE emitted by the source SL is collimated by the converging lens L702 and reflected by the variable orientation mirror S10 which scans it in the y direction. After passing through an afocal system L703 - L704 (note the absence of a pinhole, useless in SOI) and being deflected by the mirror M70i (optional), the beam FE is spectrally dispersed in the x direction by the diffraction grating Gi0, located in the Fourier plane of the pinhole P i of the source SL thanks to said afocal system L703 - L704 and the lens L702. After passing through a second afocal system L705 - L706 and being deflected once again by a mirror M702 (also optional), the beam is focused by a first microscope objective MOi so as to form the spectral line LSI on the sample E. The function of the afocal system L706- L705 is to optically conjugate the rear pupil of the microscope objective and the grating Gb All these elements constitute the first optical system SOL

[0064] The light having passed through the sample (signal beam FS) is collected and collimated by a second microscope objective MO2 located opposite the first, then spectrally recombined by another diffraction grating Gu (it is noted that these two gratings perform the functions of dispersion and spectral recombination that, in the embodiments described above, were performed by the grating Go alone). If DAi0 is the angular dispersion of Gio and DAn that of Gu, we must have, ideally, Mgio-ghDAh = DAi0 with MGio-gh the magnification formed by L705, L 706, MOi, MO2, L707 and L708, in other words the ratio between the diameter dGn of the optical beam on the grating Gu and that (dGi0) on the grating Gi0. The M703 redirection mirror and the L707-L708 afocal system allow this network to be optically combined with the rear pupil of MO2.Equivalently, we can consider that the system formed by L705, L706, MOi, MO2, L707 and L708 optically conjugates Gio and Gu and that the dispersion DAi0 imaged in the plane of Gu is equal to DAn. The spectrally recombined light beam is spatially filtered by the assembly L709 - P2 - L7i0 and reaches the variable orientation mirror Su, operated synchronously with Si0 and with an oscillation amplitude divided by the total magnification MSio-sn between SiO and S h (ratio between dSn, diameter of the beam incident on Su and dSio, diameter of the beam incident on Si0), so as to compensate for the angular scanning introduced by Si0 (as in the case of the Gio and Gu arrays, here we have two variable orientation mirrors Sio and Su which replace a single orientation mirror Si in the embodiments operating in reflection).Equivalently, one can consider that Si0 and Su are optically conjugated to each other and that the angular displacement of Sio imaged in the plane of Su must be equal - or approximately equal - to the angular displacement of Su. Then, the FS beam is spectrally dispersed by yet another diffraction grating G2, exhibiting an angular dispersion DA2 such that MG2.GiiDA2 / DAn=AG=2 (at least approximately), where MG2 Gn is here the ratio between dG2 . (diameter of the incident beam on G2) and of the. Then the spectrally re-dispersed beam is reflected by another scanning mirror S2 which is synchronized with Si0 and Su and with an oscillation amplitude approximately equal to 2AS2MSio s2, with M s2 the magnification of the afocal system L7n, L7i2, L7n, L7i4 (in other words the ratio between dS2, diameter of the optical beam on the mirror S2 and dSn) and is focused by a lens L7i5 so as to form a spectral line LS2 oriented along the x direction and scanning the image matrix sensor CMI in the y direction. Afocal systems L7n- L7i2 and L7B - L7i4 ensure the optical conjugation between the different optical elements of the system SO2. It is noted that this system operating in transmission has more components than the systems of the embodiments described above, and requires the synchronization of three scanning mirrors.To alleviate these synchronization constraints, it is possible to replace Si0, Su, S[2 by a single scanning or oscillating system such as a resonant mirror or a polygonal scanner.

[0065] As explained above, to reduce the image acquisition time it is possible to accelerate the angular velocity of the scanning mirror(s), but this is technically difficult. An alternative approach, illustrated by [Fig.8], consists of replacing a single illumination beam with a plurality of MFE beams arranged in a y direction.

[0066] The embodiment of [Fig.7] is similar to that of [Fig.3], only the differences will be described in detail. First, the SL light source is replaced by an SLMF source comprising a set MMLi of microlenses aligned along the y direction, decomposing the single light beam emitted by the broad spectrum source SLS into the plurality of MFE beams. The spatial coherence of these beams is ensured by a plurality of MPi pinholes arranged in the focal plane of each microlens, replacing the single Pb pinhole. Similarly, the P2 pinhole ensuring confocal filtering is replaced by a plurality of MP2 pinholes aligned along the y direction. It is noted that the Li05 lens of [Fig.3] is replaced by a lens L8iO arranged at the output of the SLMF source to collimate the plurality of MFE illumination beams, as well as by a converging lens L805 and a second plurality of microlenses aligned along the y direction MML2 focusing the plurality of MFS signal beams after the latter have passed through the beam splitter BS. In this system, N>1 spectral lines PLS1, PLS2 aligned along the y direction (and oriented along the x direction) are generated on the sample E and on the image matrix sensor, respectively. Thanks to this, the same region of the sample can be imaged by carrying out, with the scanning mirrors Si and S2, an angular scan reduced by a factor N compared to the case of a single spectral line. The acquisition time of an image of said region of the sample is therefore reduced by this same factor N.

[0067] The invention has been described with reference to a number of embodiments, but variations are possible. For example:

[0068] - Optical beams do not necessarily have to be circular in cross-section. In In this case, the notion of diameter must be replaced by that of transverse dimension. More precisely, the notion of beam diameter on a grating must be replaced by that of beam dimension in the dispersion direction (x, in the examples described above) and the notion of beam diameter on a scanning mirror must be replaced by that of beam dimension in the scanning direction (y, in the examples described above).

[0069] - The internal structure of the light source SL is given for illustrative purposes only. for example, other structures are possible.

[0070] - All or some of the converging lenses can be replaced by others optical elements such as concave mirrors, diffractive systems such as holographic lenses, etc.

[0071] - The diffraction gratings, or some of them, can be replaced by other dispersive systems, for example three-dimensional diffractive structures, or even by refractive systems such as prisms (except, in the case of the embodiment of [Fig.6], which requires a diffractive system).

[0072] - The microscope objective(s) may be replaced by other types of focusing systems, for example concave mirrors or f-theta lenses. In this case, the dispersive elements will not necessarily be placed in a conjugate plane of a pupil of the focusing system but, more generally, in a conjugate plane of the Fourier plane of the sample

[0073] - The x and y directions do not necessarily have to be perpendicular, although it is advantageous. It is enough that they are not parallel.

[0074] - In embodiments in which two scanning mirrors Sb S2 are carried by the same structure with variable orientation, it is not necessary for them to be arranged on opposite faces of the structure.

[0075] - The notion of mirror or support “with variable orientation” covers both resonantly oscillating structures than structures whose orientation varies in a controlled manner, continuously or in discrete steps. References

[0076] (Sheppard 1988): CJR Sheppard, “Super-resolution in Confocal Imaging”, Optik 80, No. 2, pages 53, 54.

[0077] (York 2013) : A.G. York et al. « Instant super-resolution imaging in live cells and embryos via analog image processing », Nat. Methods 2013, Novembre, 10(11), pages 1122-1126.

[0078] (De Luca 2013) : G. M.R. De Luca « Re-scan confocal microscopy : scanning twice for better resolution » Biomédical Optics Express, Vol. 4, No. 11, Novembre 2013.

[0079] (Curd 2015) : A. Curd et al. « Construction of an instant structured illumination microscope », Methods 88 (2015) pages 37 - 47.

[0080] (DuBose 2019) : T. B. DuBose et al. « Super-resolution retinal imaging using optically reassigned scanning laser ophtalmology », Nature Photonics, Vol. 13, Avril 2019, pages 257 - 262.

[0081] (Sandison 1995) : D. R. Sandison et al. « Quantitative comparison of background rejection, signal-to-noise ratio, and resolution in confocal and full-field laser scanning microscopes » Applied Optics Vol. 34, No. 19, 1er juillet 1995, pages 3576 - 3588.

[0082] (Aguilar 2020) Aguilar, A., Boyreau, A., Bon, P. “Label-free super-resolution imaging below 90-nm using photon-reassignment”. Open Research Europe, 1(3), 3 (2021).

[0083] (Tearney 1998) G. J. Teamey et al. « Spectrally encoded confocal microscopy » Optics Letters, Vol. 23, No. 15, Août 1998.

[0084] (Hwang 2015) Hwang J. et al. « Frequency- and spectrally-encoded confocal microscopy » Optics Express, Vol. 23, No. 5, 9 mars 2015

Claims

Claims

1. 1. A photon reallocation imaging system comprising: - A polychromatic light source (SL) configured to generate at least one spatially coherent illuminating light beam (FE) in a range of illuminating wavelengths; - A first optical system (SOI) configured to disperse said illuminating light beam with a first angular dispersion DAi and focus it on a sample (E) so as to form a first spectral line (LSI) oriented in a first direction (x); - A second optical system (SO2) configured to collect a light beam elastically scattered (FS) by said sample, called a signal beam, apply a second angular dispersion DA2 to it, and focus it in a focal plane so as to form a second spectral line (LS2) oriented in a second direction (x); - An image matrix sensor (IMS) arranged in said image focal plane; - A means (PT, Si) for inducing a relative displacement between the first spectral line and the sample in a third direction (y), perpendicular to the first direction; and - A means for forming an image of the sample by reallocation of photons from one or more images acquired by said image matrix sensor in correspondence with a plurality of different positions taken by the first spectral line on the sample; wherein the first optical system comprises a first dispersive optical device (Gi, Gio) for dispersing said illuminating light beam with a first angular dispersion DAi and the second optical system comprises a second dispersive optical device (G2) for applying said illuminating light beam with said second angular dispersion DA2; the length of the second spectral line in the second direction being greater than that of the first spectral line in the first direction by a factor Ag=MgDA2 / DAi>1, MG being the ratio between the dimension of the illumination beam on the first dispersive optical device measured in the first direction and the dimension of the signal beam on the second dispersive optical device measured in the second direction.

2. 2. A photon reallocation imaging system according to claim 1 wherein said AG factor is between 1.8 and 2.

2.

3. 3. Photon reallocation imaging system according to one of the preceding claims wherein the first optical system (SOI) comprises: - a first collimator system (Li05, L205, L305, L405, L505 L605), configured to collimate said illumination light beam (FE); - said first dispersive optical device (Gb Gi0); - a focusing optical system (MO, MOI) configured to receive as input the illumination light beam dispersed by said first dispersive device and focus it on the sample (E).

4. 4. Photon reallocation imaging system according to claim 3 wherein the second optical system (SO2) comprises - said or another focusing system (MO, MO2) for collecting said signal beam, collimating it and directing it towards said first dispersive optical device, or another dispersive optical device, such that it is spectrally recombined; - said first (Gi) or said other (Gu) dispersive optical device, configured to spectrally recombine said signal beam; - said second dispersive optical device (G2) having the second angular dispersion DA2, to spectrally disperse said signal beam in said second direction (x); and - at least one optical element (Li07, L207, L309, L409, L509, L609) configured to focus in a focal plane, called image focal plane, the signal beam spectrally dispersed by the second dispersive optical device (G2).

5. 5. Photon reallocation imaging system according to claim 4 wherein the second optical system comprises a pinhole (P2) arranged in a plane conjugate with a focal plane of the focusing system of the second optical system (MO) so as to perform confocal filtering.

6. 6. Photon reallocation imaging system according to one of claims 4 or 5 wherein the means for forming an image of the sample by reallocation of photons from a plurality of images of the second spectral line acquired by said image matrix sensor comprises a digital image processor (DIP).

7. 7. A photon reallocation imaging system according to claim 6 wherein the means for inducing relative displacement between the first spectral line and the sample comprises a stage of translation (PT) of the sample.

8. 8. Photon reallocation imaging system according to claim 6 wherein the means for inducing a relative displacement between the first spectral line and the sample comprises a variable orientation mirror (Si) common to the first and second optical systems, configured to apply an angular scan in the third direction to the illumination light beam and an opposite angular scan to the spectrally recombined signal beam.

9. 9. Photon reallocation imaging system according to one of claims 3 to 5 wherein: - The means for inducing a relative displacement between the first spectral line and the sample comprises a first variable orientation mirror (Si) common to the first and second optical systems, configured to apply an angular scan in the third direction to the illumination light beam and an opposite angular scan to the spectrally recombined signal beam; and - The means for forming an image of the sample by photon reallocation comprises a second variable orientation mirror (S2), configured to apply an angular scan in a fourth direction, perpendicular to the second direction, to the signal beam spectrally dispersed by the second dispersive optical device;The angular scanning applied by the second variable orientation mirror being synchronous and having an amplitude equal to As / Ms times the amplitude of that applied by the first mirror, As being a factor between 1.8 and 2.2 and Ms and the ratio between the dimension of the illuminating light beam on the second variable orientation mirror, measured along the fourth direction, and the dimension of the spectrally dispersed signal beam on the first variable orientation mirror, measured along the third direction.;

10. 10. Photon reallocation imaging system according to claim 9 wherein the first variable orientation mirror (Si) is structurally independent of the second variable orientation mirror (S2) and the first dispersive optical device (Gi) is distinct from the second dispersive optical device (G2).

11. 11. Photon reallocation imaging system according to one of claims 1 to 3, or 9 when it depends solely on claim 3, in which the first dispersive optical device (Gi) is a diffractive system having said angular dispersion DAi at the first order of

12.

13.

14.

15. diffraction and said angular dispersion DA2=2DAi at the second diffraction order, the second optical system being configured to collect the light scattered at the second diffraction order by said diffractive system.

12. Photon reallocation imaging system according to one of claims 9 or 11 wherein the first and second variable orientation mirrors are formed by two distinct reflective regions of the same variable orientation support (SPO).

13. Imaging system according to claim 12 wherein said variable orientation support (SPO) is selected from a resonant mirror support and a polygonal scanner.

14. Imaging system according to one of claims 12 and 13 in which the first dispersive optical device (G1) is distinct from the second dispersive optical device (G2).

15. Imaging system according to one of the preceding claims wherein the light source (SLMF) comprises a first set of a plurality of microlenses (MMLi) aligned along the third direction (y) to generate a plurality of said illumination light beams in parallel, whereby said first optical system (SOI) forms on the sample (E) a plurality of first spectral lines (LSI) oriented along a first direction (x) and arranged along said third direction (y), and wherein said second optical system (SO2) comprises a second set of a plurality of microlenses (MML2) aligned along said third direction (y) to form on the image matrix sensor (CMI) a plurality of first spectral lines (LSI) oriented along a first direction (x) and arranged along a fourth direction (y), perpendicular to said second direction (x).

Images