Imaging system with photon reallocation

Abstract

The invention relates to a photon-reallocation imaging system comprising: - a polychromatic light source (SL) for generating an illumination light beam (FE); - a first optical system (SO1) for dispersing said illumination light beam with a first angular dispersion DA1 and focusing it on a sample (E) so as to form a first spectral line (LS1); - a second optical system (SO2) configured to collect a light beam (FS) that has been elastically scattered by said sample, referred to as signal beam, to apply thereto a second angular dispersion DA2 and to focus it in a focal plane so as to form a second spectral line (LS2) with a length greater than that of the first spectral line; - a matrix image sensor (CMI) arranged in said image focal plane; - a means (S1) 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 through photon reallocation based on one or more images acquired by said matrix image sensor in correspondence with a plurality of different positions adopted by the first spectral line on the sample.

Description

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 a vital role in biology and micro- and nanotechnologies, as it allows for the observation of samples at 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]

[0004] where is the wavelength of the light used (between 380 nm and 780 nm for visible light) and ON is 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 observing very small structures such as viral particles. An improvement in spatial resolution can also be achieved by reducing the wavelength, but at the cost of considerable technical difficulties.

[0005] So-called "superresolution" techniques make it possible to overcome 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, in both biology and micro- and nanotechnologies, it is often necessary to also obtain 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 perform 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 smaller the pinhole diameter, the smaller the slice thickness, and therefore the better the axial resolution, but the gain is low when going below 1 Airy unit (AU). The Airy unit is the diameter of the microscope's Airy spot, and is equal to .

[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 achieved at the cost of a degradation of the signal-to-noise difference.

[0010] The photon reallocation technique, first proposed in (Sheppard 1988), can 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 that allows an elementary image to be acquired 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 scan 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 a second angular scan to this same beam coming from the sample, synchronized with the first scan. The second angular scan has a larger amplitude normalized to the beam cross-section, ideally by a factor of two, than the first scan. "Normalized to the beam cross-section" means that if α 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 cross-section of the beam coming from the sample and that of the illumination beam, it is the quantity α / 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 array and microlens arrays, 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 microscope, but 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 in the order of tens of micrometers.

[0014] Document EP 4012476 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 that 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 aim 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 image acquisition time. Furthermore, in some embodiments of the invention, this allows the use of resonant mirrors oscillating at a very high frequency – of the order of several tens of kHz – which further reduces 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 (Tearney 1998) and (Hwang 2015). More In particular, (Tearney 1998) concerns a fiber-reinforced confocal microscope intended to be incorporated into an endoscope, while the microscope of (Hwang 2015) replaces scanning in the 2 edirection by a variable frequency modulation in space. None of these systems allows to exceed 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 illumination light beam in a range of illumination wavelengths; - A first optical system configured to disperse said illumination light beam with a first angular dispersion DA1 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, apply a second angular dispersion DA2 to it, and 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 of 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 DA1 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 A; G =MG DA2 / DA1>1, M G 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] The 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; - a focusing optical system configured to receive as input the illumination 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, to spectrally disperse 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 perform 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 for forming 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 scan applied by the second variable orientation mirror being synchronous and having an amplitude equal to A S / M S times the amplitude of that applied by the first mirror, AS being a factor between 1.8 and 2.2 and M S 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 DA1 at the first diffraction order and said angular dispersion DA2=2DA1 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 (Tearney 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 render possible reallocation of photons.

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

[0033] [Fig.1], 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. 1], a polychromatic SL light source generates a spatially – but generally not temporally – coherent illumination beam FE. The SL source comprises, for example, a broad spectrum source SLS, e.g., 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 FSC filter determines, together with the angular dispersion DA1, the extent of the first spectral line scanning the sample.Its sizing results from a compromise – a large spectral range reduces constraints on the angular dispersion DA1 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 a spectral width Δλ / λ of the order of 10%. The resolution across the field of view also becomes variable in case of large spectral range (optical resolution proportional to the local wavelength).

[0043] A converging lens L 110 and a P1 pinhole camera 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 SO1, intended to illuminate the sample E. Said optical system SO1 comprises, in addition to the splitter plate BS, a first converging lens L 105 which collimates the beam FE; a planar mirror M 102 (non-essential), two other converging lenses L 104 , L 103 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 P1 at the exit of the light source SL. The illumination beam exits the lens L 103 collimated and is directed towards a diffraction grating G1 having an angular dispersion DA1 in a first direction x. The beam diffracted by the grating G1 passes through an afocal system consisting of two other converging lenses L 1 01 , L 102and is reflected by a planar mirror M 102 (non-essential) before being focused on the sample E by a microscope objective MO. The spectral dispersion operated by the grating G1 and the focusing by the objective MO lead to the formation of a spectral line LS1 on the sample, that is to say an elongated focal spot, oriented in the x direction, in which the wavelength of the light varies monotonically with the position in said x direction.

[0045] The main function of the afocal system formed by the L lenses 101 and L 102 is to systematically place the diffraction grating G1 in a plane conjugate to 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] 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 LS1 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 in [Fig. 1] operates in reflection, which allows for a pooling of optical elements between the first and second optical systems. More specifically, all the elements of the first optical system SO1 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 G1 that had dispersed it during the outward path. The afocal system formed by the lenses L 103 and L 104allows confocal filtering to be carried out, as in a confocal microscope according to the prior art, in addition to allowing modification of the FS beam size.

[0048] The beam FS passes through the beam splitter BS which had reflected the illumination beam FE from the source SL. The converging lenses L 105 and L 106 , placed on either side of said separating 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 L 107 forms a second spectral line LS2 oriented along the x direction on the CMI image matrix sensor.

[0049] The image matrix sensor can be implemented, for example, in CMOS or CCD technology. It has a number of pixels in the x direction that is determined by the spatial extent of the spectral line that can be obtained by the SO2 optical system, typically in the hundreds. The number of pixels in the y direction can be lower – in the order of ten or a few tens – because these pixels are only used 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 suitably programmed microprocessor 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 (e.g. between 1.8 and 2.2). This is illustrated in the top left part of the figure; we note in particular that a segment corresponding to a wavelength interval Δλ is longer in the spectral line LS2 than in LS1. This elongation can be achieved by using a second diffraction grating G2 with an angular dispersion DA2 greater than that – DA1 – of the first diffraction grating, at the magnification M G of the optical beam produced by the assembly consisting of the lenses L 103 , L 104 L 105 and L 006 or a combination of both. Generally speaking, if M G is the ratio between the diameter d G2of the incident beam on the G2 grating and the diameter d G1 of the incident beam on the network G1(M G =d G2 / d G1 ), we obtain M G DA2 / DA 1=length(LS2) / length(LS1)=A G , HAS G being a factor between 1.8 and 2.2 and ideally having a value of 2. For example, if M G =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 M 102 of the first optical system is replaced by an oscillating mirror – or more generally one with a variable orientation – S1. More precisely, the variation in orientation of the mirror S1 is controlled so as to induce a displacement of the first spectral line LS1 on the sample E in the y direction; for this reason, S1 will be called in the following "scanning mirror". In this way, the spectral line LS1 scans the sample E without needing to translate the latter along the y direction. As in the embodiment of [Fig.1], the photon reallocation is carried out digitally by an image processor PNI, synchronized with the moving mirror S1.

[0052] The embodiment of [Fig.3] differs from that of [Fig.2] in that the second optical system SO2 also includes an oscillating scanning mirror – or more generally one with a variable orientation – S2 arranged before the lens L 107 focusing the FS beam onto the CMI image matrix sensor. An afocal system formed by the converging lenses L 308 and L 309 allows the S2 mirror to be combined with the pupil of the OM microscope objective.

[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 realizing the reallocation of photons in a purely optical manner. To obtain a gain in resolution, it is necessary that this scan is synchronized with that carried out by the first spectral line LS1 on the sample E, and has a higher amplitude by a factor A S / M S , HAS S being a factor between 1.8 and 2.2 and ideally having a value of 2 and M S being the ratio between the diameter d S2 of the beam on the mirror S2 and the diameter d s1 of the beam on the mirror S1. In the embodiment of [Fig.3], M G is the total magnification of the assembly consisting of the lenses L 103 , L 104 , L 105 , L 106 , L 308 and L 309 .

[0054] Unlike the case of the embodiments of [Fig.1] 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 S1 and S2 are sufficiently fast, the CMI image sensor does not necessarily have to acquire a separate image for each position of the spectral lines LS1 and LS2: a single 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 S1, S2 and the image matrix sensor.

[0056] In the embodiments of [Fig.1], [Fig.2] and [Fig.3], the rate of the scan(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.1]; between S1 and the image matrix sensor CMI in the case of [Fig.2]; and at least between S1 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 S1 and S2 are made on two opposite faces of the same oscillating support, or more generally with variable orientation, SPO. In the specific embodiment of the figure, this is made possible by the use of the two redirection mirrors M 402 and M 403. This eliminates any requirement for synchronization, and therefore greatly facilitates the use of a high scanning frequency – in the order of kilohertz – obtained for example by means of a resonant SPO support or a polygonal scanner. Since the angular amplitude of the scan carried out by the mirrors S1 and S2 is, by construction, the same, the second optical system must provide a magnification M S of the FS beam between the two scanning mirrors of the order of 2 (in other words d S2 / d S1 =M S =AS, with A S =2 – at least approximately). In the embodiment of [Fig.4], this is ensured by the afocal system formed by the lenses L 105 ,L 106 , L 408 and L 409 .

[0057] The embodiment of [Fig.5] is similar to that of [Fig.4], except that the functions of the two diffraction gratings G1 and G2 are performed by two distinct regions of a single grating G, which is made possible by the use of the redirection mirrors M 502 , M 503 and M 504 and by the fact that, in the example of [Fig.5], the illumination beam FE passes through the splitter plate BS while the beam FS is reflected from it. Given that the two separate networks G1 and G2 are replaced by two zones of the same network, DA1 = DA2, it is therefore necessary that the magnification M G or about 2, in other words d G2 =2d G1 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.1]. The illumination beam FE from this source is collimated by the lens L 105 collimation of the illumination beam FE, is deflected by a first redirection mirror M 601 (non-essential), and reaches the diffraction grating G1 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 L601 and L 602 forming an afocal system. The length of the FR slit 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 L 602 and L 105 is to optically combine the FR slit and the P1 pinhole of the SL source. Alternatively, the FR slit can be omitted.

[0060] The illumination beam dispersed by the G1et spatially filtered network is directed towards the microscope objective MO by the scanning mirror S1, formed by a reflecting surface of an oscillating or variable orientation support SPO, which makes it possible to obtain a spectral line LS1 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 S1, which compensates for the effect of the scanning introduced in the “forward path” and spatially filtered by the assembly L 601 – FR– L 602, before reaching the diffraction grating G1 again. 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 is double that of the first order; consequently, the diffracted beam FS' is not spectrally recombined, but has an angular dispersion of amplitude double 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 M S =2 approximately by the afocal system formed by the L lenses 607 and L 608 (also, d S2 =2d S1), redirected (optionally) by M mirrors 602 , M 6 03 , M 604 , reflected by the scanning mirror S2, formed on the same variable orientation support SPO as S1 and finally focused by the lens L 609 to form the spectral line LS2 on the CMI image matrix sensor. By construction, in this embodiment of G1 =d G2 .

[0061] It will be noted that in the embodiments described above, the first optical system SO1 for forming the first spectral line LS1 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 2 ediffraction 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 separate. This is the case, in particular, for 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 L 702 and reflected by the variable orientation mirror S 10which applies a scan in the y direction. After passing through an afocal system L 703 - L 704 (note the absence of pinhole, useless in SO1) and having been deflected by the mirror M 701 (optional), the FE beam is spectrally dispersed in the x direction by the diffraction grating G 10 , located in the Fourier plane of the pinhole P 1 of the source SL thanks to said afocal system L 703 - L 704 and to the L lens 702 . After passing through a second afocal system L 705 - L 706 and have been deflected once again by a mirror M 702 (also optional), the beam is focused by a first microscope objective MO1 so as to form the spectral line LS1 on the sample E. The function of the afocal system L 706 - L 705is to optically combine the rear pupil of the microscope objective and the G1 grating. All these elements constitute the first SO1 optical system.

[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 G 11 (we note that these two networks perform the functions of dispersion and spectral recombination that, in the embodiments described above, were performed by the G0 network alone). If DA 10 is the angular dispersion of G 10 and DA 11 that of G 11 , we must have, ideally, M G10-G11 DA 11 = DA 10 with M G10-G11 the magnification formed by L 705 , L 7 06 , MO1, MO2, L 707 and L 708 , in other words the ratio between the diameter d G11of the optical beam on the G network 11 and that (d G10 ) on the G network 10 . The M redirect mirror 703 and the afocal system L 707 - L 708 allow this network to be optically combined with the rear pupil of MO2. Equivalently, we can consider that the system formed by L 705 , L 706 , MO1, MO2, L 707 and L 708 optically conjugates G 10 and G 11 and that the DA dispersion 10 imaged in the plane of G 11 is equal to DA 11 The spectrally recombined light beam is spatially filtered by the set L 709 – P2– L 710 and reaches the variable orientation mirror S 11 , operated synchronously with S 10 and with an oscillation amplitude divided by the total magnification M S10-S11 between S 10 and S 11 (ratio between d S11 , diameter of the beam incident on S 11 and d S10, diameter of the beam incident on S 10 ), so as to compensate for the angular sweep introduced by S 10 (as in the case of G networks 10 and G 11 , here we have two variable orientation mirrors S 10 and S 11 which replace a single-orientation mirror S1 in the embodiments operating in reflection). Equivalently, we can consider that S 10 and S 11 are optically conjugated to each other and that the angular displacement of S 10 imaged in the plane of S 11 must be equal – or approximately equal – to the angular displacement of S 11. Then, the beam FS is spectrally dispersed by yet another diffraction grating G2, exhibiting an angular dispersion DA2 such that M G2-G11 DA2 / DA 11 =A G =2 (at least approximately), where M G2-G11 here is the relationship between d G2 (diameter of the incident beam on G2) and d11 . Then the spectrally re-dispersed beam is reflected by another scanning mirror S2 which is synchronized with S 10 and S 11 and with an oscillation amplitude approximately equal to 2A S2 M S10-S2 , with M S2 the magnification of the afocal system L 711 , L 712 , L 713 , L 714 (in other words the relationship between d S2 , diameter of the optical beam on the mirror S2 and d S11 ) and is focused by a lens L 715 so as to form a spectral line LS2 oriented along the x direction and scanning the CMI image matrix sensor in the y direction. Afocal systems L 711 - L 712 and L 713 - L 714provide optical conjugation between the different optical elements of the SO2 system. It should be 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 S 10 , S 11 , S 12 by a single scanning or oscillating system such as a resonant mirror or polygonal scanner.

[0065] As explained above, to reduce 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 light source SL is replaced by a source SLMF comprising a set MML1 of microlenses aligned along the y direction, decomposing the single light beam emitted by the broad spectrum source SLS into the plurality of beams MFE. The spatial coherence of these beams is ensured by a plurality of pinholes MP1 arranged in the focal plane of each microlens, replacing the single pinhole P1. Similarly, the pinhole P2 ensuring confocal filtering is replaced by a plurality of pinholes MP2 aligned along the y direction. It is noted that the lens L 105 of [Fig.3] is replaced by a lens L 810 arranged at the output of the SLMF source to collimate the plurality of MFE illumination beams, as well as by a converging lens L 805and a second plurality of microlenses aligned along the y direction MML2 focusing the plurality of signal beams MFS 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 S1 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 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 SL light source is given only as an example, other structures are possible.

[0070] - All or some of the converging lenses can be replaced by other 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) can 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 this is advantageous. It is sufficient that they are not parallel.

[0074] - In the embodiments in which two scanning mirrors S1, S2 are carried by the same variable orientation structure, it is not necessary for them to be arranged on opposite faces of the structure.

[0075] - The concept of a mirror or support "with variable orientation" covers both resonantly oscillating structures and 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): AG York et al. “Instant super-resolution imaging in live cells and embryos via analog image processing”, Nat. Methods 2013, November, 10(11), pages 1122 – 1126.

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

[0079] (Curd 2015) : A. Curd et al. « Construction of an instant structured illumination mi- croscope », 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. Tearney 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 mi- croscopy » 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 illumination light beam (FE) in a range of illumination wavelengths; - A first optical system (SO1) configured to disperse said illumination light beam with a first angular dispersion DA1 and focus it on a sample (E) so as to form a first spectral line (LS1) 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 (CMI) arranged in said image focal plane;- A means (PT, S1) 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 (G1, G; 10) for dispersing said illuminating light beam with a first angular dispersion DA1 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 along the second direction being greater than that of the first spectral line along the first direction by a factor A G =M G DA2 / DA1>1, M G 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 factor A Gis between 1.8 and 2.

2.

3. 3. Photon reallocation imaging system according to one of the preceding claims in which the first optical system (SO1) comprises: - a first collimator system (L 105 , L 205 , L 305 , L 405 , L 505, L 605 ), configured to collimate said illumination light beam (FE); - said first dispersive optical device (G1, G 10); - a focusing optical system (MO, MO1) configured to receive as input the illumination light beam dispersed by said first dispersive device and focus it onto 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 (G1) or said other (G 11 ) 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 along said second direction (x); and - at least one optical element (L 107, L 207 , L 309 , L 409 , L 509 , L 609) 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 carry out 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 (PNI).

7. 7.A 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 stage. 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 (S1) 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 (S1) 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 scan applied by the second variable orientation mirror being synchronous and having an amplitude equal to A. S / M Stimes the amplitude of that applied by the first mirror, A S being a factor between 1.8 and 2.2 and M Sand 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.

10. 10. A photon reallocation imaging system according to claim 9 wherein the first variable orientation mirror (S1) is structurally independent of the second variable orientation mirror (S2) and the first dispersive optical device (G1) is distinct from the second dispersive optical device (G2).

11. 11. A photon reallocation imaging system according to one of claims 1 to 3, or 9 when dependent solely on claim 3, wherein the first dispersive optical device (G1) is a diffractive system having said angular dispersion DA1 at the first order of diffraction and said angular dispersion DA2=2DA1at 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. 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. 13. Imaging system according to claim 12 wherein said variable orientation support (SPO) is chosen from a resonant mirror support and a polygonal scanner.

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

15. 15.Imaging system according to one of the preceding claims wherein the light source (SLMF) comprises a first set of a plurality of microlenses (MML1) aligned along the third direction (y) to generate a plurality of said illumination light beams in parallel, whereby said first optical system (SO1) forms on the sample (E) a plurality of first spectral lines (LS1) 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 (LS1) oriented along a first direction (x) and arranged along a fourth direction (y), perpendicular to said second direction (x).

Images