Photon redistribution imaging system

Abstract

The invention relates to a photon redistribution imaging system comprising:-a polychromatic light source (SL) for generating an illumination beam (FE); -a first optical system (SO1) for dispersing the illumination beam with a first angular dispersion DA1 and focusing it on the sample (E) to form a first spectral line (LS1); -a second optical system (SO2) configured to collect a light beam (FS) elastically scattered by said sample, referred to as a signal light beam, to which a second angular dispersion DA2 is applied and focused in the focal plane, to form a second spectral line (LS2) having a length greater than the length of the first spectral line; -a matrix image sensor (CMI) arranged in the image focal plane; -means (S1) for causing a relative displacement between the first spectral line and the sample in a third direction (y) perpendicular to the first direction; and-means for forming an image of the sample by photon redistribution based on one or more images acquired by said matrix image sensor corresponding to a plurality of different positions employed by the first spectral line on the sample.

Description

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

[0002] Optical microscopy plays a vital role in biology and micro- and nanotechnology because it allows for high-speed observation of samples without the complex preparation required by electron microscopy. However, the resolution of optical microscopy is necessarily limited by the diffraction of light. According to Abbe's theory, for a conventional optical microscope, the maximum resolution d is given by the following formula:

[0003]

[0004] Here, λ is the wavelength of the light used (380 to 780 nm for visible light), and ON is the numerical aperture, which is difficult to exceed a value of 1.4 for biological samples. Therefore, the resolution of visible light cannot exceed 135 nm, which is not suitable for observing very small structures such as virus particles. It is also possible to increase spatial resolution by reducing the wavelength, but at the cost of considerable technical difficulties.

[0005] So-called "super-resolution" techniques can exceed the Abbe diffraction limit by using fluorescent labels and / or nonlinear effects. These techniques are complex to implement and are not suitable for all applications.

[0006] In addition, in biology and micro-nanotechnology, it is often necessary to achieve high spatial resolution in the axial direction (the Abbe limit is related to the lateral resolution in a plane perpendicular to the optical axis). Confocal microscopy makes it possible to obtain images with a very shallow depth of field (on the order of a few hundred nanometers) and thus "slice" the sample to obtain its three-dimensional structure. This technique is most often associated with the use of fluorescent labels, but can also be used for reflection in the absence of labels.

[0007] Confocal microscopy uses a point illumination source, whose image is projected onto the sample to be observed through the objective lens. Light from the sample (backscattered light in the case of reflective confocal microscopy; fluorescence emission when fluorescent markers are used) is focused on a pinhole optically conjugate to the point source and then detected, for example, by a photomultiplier tube. The role of the pinhole is to eliminate radiation that does not come from the focal plane of the objective lens, thereby achieving optical sectioning. A point-by-point image of the sample is obtained by scanning. More specifically, two-dimensional scanning in two directions perpendicular to the optical axis provides an image of a slice of the sample centered on the focal plane of the objective lens. Adding axial scanning of this focal plane produces a three-dimensional image.

[0008] The smaller the pinhole diameter, the thinner the slice thickness, and thus the better the axial resolution. However, when the diameter is less than 1 Airy unit (AU, Airy unit), the resolution decreases. The Airy unit is the diameter of the microscope Airy disk, which is

[0009] Confocal microscopy using pinholes with diameters smaller than 1 AU can theoretically improve lateral resolution by up to 30% relative to the Abbe limit. However, this comes at the expense of reduced signal-to-noise ratio.

[0010] Photon redistribution, first proposed by Sheppard in 1988, can, in principle, increase the lateral resolution of confocal microscopy by a factor of two. The idea behind this technique is to replace photomultiplier tubes, or more generally point-radiation detectors, with a matrix detector that acquires a base image for each acquisition point. The image is then resized (ideally by a factor of two) by digital or optical means before moving on to the next scan point. The final image is obtained by combining consecutive scans, each offset by a scan step size relative to the previous one.

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

[0012] As with conventional confocal microscopy, pinhole arrays and microlens arrays can be used to parallelize acquisition, see for example the aforementioned article (York 2013).

[0013] Photon redistribution technology has been applied in particular to confocal fluorescence microscopy, as shown in the aforementioned articles (York 2013), (De Luca 2013), and (Curd 2015), where a lateral resolution improvement of 1.5 times was observed. To the best of the inventors' knowledge, the only application of photon redistribution in reflection is described in (DuBose 2019). However, this article does not mention microscopes, but rather ophthalmoscopy, in which the objective lens is replaced by the lens of the patient's eye and therefore exhibits a small numerical aperture, resulting in a resolution of approximately tens of micrometers.

[0014] Document EP 4012476 and the article (Aguilar 2020) describe a confocal microscopy device and method for photon redistribution by reflection or transmission, which optimizes spatial resolution through appropriate aperture size.

[0015] Confocal microscopy using photon redistribution is a point-by-point scanning imaging technique. Therefore, long acquisition times are required, which is disadvantageous or even impossible for certain applications, such as in situ characterization in non-destructive testing for rapid diagnostics in the biomedical, materials, and micro / nano / nanotechnology industries.

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

[0017] According to the present invention, this goal is achieved by dispersing a light beam in a first direction to form "spectral lines" that illuminate the sample. Consequently, multiple points on the sample aligned along the first direction are illuminated simultaneously with different wavelengths. Consequently, only a scan in a second direction (non-parallel to the first, preferably perpendicular) is required to inspect a portion of the sample surface.

[0018] The act of scanning in only a single direction, rather than two, significantly reduces image acquisition time. Furthermore, in some embodiments of the present invention, this allows the use of resonant mirrors that oscillate at very high frequencies (on the order of tens of kHz), further reducing acquisition time. The use of such resonant mirrors would be difficult to envision if, according to prior art, two simultaneous scans in the first and second directions were required.

[0019] Replacing scanning in a spatial direction with spectral lines has been proposed, for example, as described in (Tearney 1998) and (Hwang 2015). More specifically, (Tearney 1998) involves a fiber-optic confocal microscope designed for incorporation into an endoscope, while the microscope in (Hwang 2015) replaces scanning in a second direction with spatially variable frequency modulation. These systems are unable to exceed the Abbe limit and, in particular, are incompatible with the principle of photon redistribution.

[0020] Therefore, an object of the present invention is a photon redistribution imaging system comprising:

[0021] - a polychromatic light source configured to generate at least one spatially coherent illumination beam within an illumination wavelength range;

[0022] a first optical system configured to disperse the illumination beam with a first angular dispersion DA1 and to focus it on the sample to form a first spectral line oriented along a first direction;

[0023] a second optical system configured to collect the beam elastically scattered by the sample, referred to as the signal beam, to apply a second angular dispersion DA2 to the signal beam and to focus it in a focal plane so as to form a second spectral line oriented along a second direction;

[0024] - a matrix image sensor arranged in said image focal plane;

[0025] - means for causing a relative displacement between the first spectral line and the sample in a third direction perpendicular to the first direction; and

[0026] - means for forming an image of the sample by photon redistribution based on one or more images acquired by said matrix image sensor corresponding to a plurality of different positions adopted by the first spectral line on the sample;

[0027] wherein the first optical system comprises a first dispersive optical device for dispersing the illumination light beam with a first angular dispersion DA1, and the second optical system comprises a second dispersive optical device for applying the second angular dispersion DA2 to the illumination light beam;

[0028] The length of the second spectral line in the second direction is a factor A of the length of the first spectral line in the first direction G times, A G =M G DA2 / DA1>1, M G is the ratio of the signal beam size on the first dispersive optical device measured along the first direction to the signal beam size on the second dispersive optical device measured along the second direction.

[0029] According to a specific embodiment of the present invention:

[0030] The factor A G It can be 1.8 to 2.2.

[0031] The first optical system may include:

[0032] - a first collimation system configured to collimate the illumination light beam;

[0033] - said first dispersive optical device;

[0034] - a focusing optical system configured to receive as input the illumination beam dispersed by said first dispersive optical device and to focus it on a sample.

[0035] The second optical system may include:

[0036] - the focusing system or another focusing system for collecting the signal beam, collimating the signal beam and directing it to the first dispersive optical device or another dispersive optical device, so that the signal beam is spectrally recombined;

[0037] - the first dispersive optical device or the further dispersive optical device, configured to perform spectral recombination on the signal light beam;

[0038] The second dispersive optical device has a second angular dispersion DA2 for spectrally dispersing the signal light beam in the second direction; and

[0039] - at least one optical element configured to focus the signal light beam spectrally dispersed by the second dispersive optical device in a focal plane called the image focal plane.

[0040] In this case, the second optical system may include a pinhole arranged in a plane conjugate to a focal plane of a focusing system of the second optical system in order to perform confocal filtering.

[0041] The device for forming an image of the sample by photon redistribution based on the multiple images of the second spectral line acquired by the matrix image sensor may include a digital image processor. In this case, the device for causing a relative displacement between the first spectral line and the sample may include a sample translation stage, or may also include a variable orientation mirror shared by the first and second optical systems, the variable orientation mirror being configured to apply an angular scan along a third direction to the illumination beam and an opposite angular scan to the spectrally recombined signal beam.

[0042] According to other embodiments

[0043] - the means for causing 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, the first variable orientation mirror being configured to apply an angular scan along a third direction to the illumination beam and an opposite angular scan to the spectrally recombined signal beam; and

[0044] - the means for forming an image of the sample by photon redistribution comprises a second variable orientation mirror configured to apply an angular scan along a fourth direction perpendicular to the second direction to the signal light beam spectrally dispersed by the second dispersive optical device;

[0045] The angular scan applied by the second variable orientation mirror is synchronized with the angular scan applied by the first mirror, and the amplitude of the angular scan applied by the second variable orientation mirror is A times the amplitude of the angular scan applied by the first mirror. S / M S times, where A S A factor of 1.8 to 2.2, M S is the ratio of the size of the illumination beam on the second variable orientation mirror measured along the fourth direction to the size 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 can be structurally independent of the second variable orientation mirror, and the first dispersive optical device is separate from the second dispersive optical device.

[0046] The first dispersive optical device may be a diffraction system having the angular dispersion DA1 in a first diffraction order and the angular dispersion DA2 = 2DA1 in a second diffraction order, and the second optical system is configured to collect light scattered by the diffraction system in the second diffraction order.

[0047] In other embodiments, the first and second variable orientation mirrors can be formed by two separate reflective regions of the same variable orientation support. For example, the orientation support. The first dispersive optical device can be separate from the second dispersive optical device.

[0048] The light source may include a first group of multiple microlenses aligned in a third direction to generate a plurality of the illumination light beams in parallel, so that the first optical system forms a plurality of first spectral lines oriented along a first direction and arranged in the third direction on the sample; and wherein the second optical system may include a second group of multiple microlenses aligned in the third direction to form a plurality of first spectral lines oriented along the first direction and arranged in a fourth direction perpendicular to the second direction on the matrix image sensor.

[0049] It is worth noting that, contrary to what is taught, for example, in (Tearney 1998) and (Hwang 2015), the imaging system according to the present invention performs a second spatial dispersion operation to form a second spectral line on the image sensor that is longer (ideally twice) than the first spectral line projected onto the sample. This is necessary to enable photon redistribution.

[0050] Other features, details and advantages of the invention will become apparent from a reading of the description given with reference to the accompanying drawings, which, by way of example, show:

[0051] Figure 1 shows a schematic diagram of a confocal microscope using photon redistribution according to a first embodiment of the present invention;

[0052] Figure 2 shows a schematic diagram of a confocal microscope using photon redistribution according to a second embodiment of the present invention;

[0053] Figure 3 shows a schematic diagram of a confocal microscope using photon redistribution according to a third embodiment of the present invention;

[0054] Figure 4 shows a schematic diagram of a confocal microscope using photon redistribution according to a fourth embodiment of the present invention;

[0055] Figure 5 shows a schematic diagram of a confocal microscope using photon redistribution according to a fifth embodiment of the present invention;

[0056] Figure 6 A schematic diagram showing a confocal microscope using photon redistribution according to a sixth embodiment of the present invention; and

[0057] Figure 7shows a schematic diagram of a confocal microscope using photon redistribution according to a seventh embodiment of the present invention;

[0058] Figure 8 A schematic diagram of a confocal microscope using photon redistribution according to an eighth embodiment of the present invention is shown.

[0059] In the drawings, like reference numerals designate corresponding elements.

[0060] exist Figure 1 In the illustrated apparatus, a polychromatic light source SL generates a spatially coherent, but typically temporally incoherent, light beam FE. Source SL comprises, for example, a broad-spectrum source SLS, such as an arc lamp, a light-emitting diode, or a pulsed laser. A spectral filter FSC selects a portion of the spectrum of source SLS. The wavelength range selected by filter FSC, together with the angular dispersion DA1, determines the range of the first spectral line scanned across the sample. Its size is a compromise—a wide spectral range reduces the constraints imposed by the angular dispersion DA1 and allows for the utilization of a larger fraction of the total light intensity emitted by source SLS, but makes compensating for chromatic aberrations (axial and longitudinal) in the imaging system more difficult, especially when using f-theta scan lenses, which typically tolerate a spectral width Δλ / λ of approximately 10%. With a large spectral range, the resolution of the field of view also becomes variable (optical resolution is proportional to the local wavelength).

[0061] Converging lens L 110 And the pinhole P1 arranged in its focal plane performs spectral filtering to ensure the spatial coherence of the light beam FE.

[0062] The light beam FE emitted from the light source SL is reflected by the beam splitter BS, which directs said light beam to a first optical system SO1 intended to illuminate the sample E. In addition to the beam splitter BS, said optical system SO1 comprises a first converging lens L for collimating the light beam FE. 105 ; Plane mirror M 102 (optional), two additional converging lenses L forming an afocal system 104 , L 103 , a second pinhole P2 is arranged in its common focal plane, so the pinhole P2 is optically conjugate with the first pinhole P1 at the output of the light source SL. The light beam leaves the collimating lens L 103 , and is directed to a diffraction grating G1 having an angular dispersion DA1 in a first direction x. The light beam diffracted by the grating G1 passes through two other converging lenses L 101 , L 102 The afocal system is composed of a plane mirror M before being focused on the sample E by the microscope objective MO. 102The spectral dispersion of the grating G1 and the focusing of the objective lens MO form a spectral line LS1 on the sample, ie an elongated focal spot oriented in the x direction, where the wavelength of the light varies monotonically with the position in said direction x.

[0063] By lens L 101 and L 102 The main function of the resulting afocal system 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 microscope. In addition, the afocal system can also be used to adjust the size of the light beam.

[0064] The sample E is mounted on a translation stage PT allowing it to be moved in a direction y perpendicular to x. In this way, the spectral line LS1 scans one surface of the sample.

[0065] The second optical system SO2 collects the beam FS (“signal beam”) elastically scattered by the sample, ie scattered without a change in wavelength, to form a second spectral line LS2 on the matrix image sensor CMI. Figure 1 The imaging system shown is operated in reflection mode, which allows sharing of optical elements between the first and second optical systems. More specifically, all elements of the first optical system SO1 also belong to the second optical system, which also includes additional components. In addition, the light backscattered by the sample E is collected and collimated by the microscope objective MO and spectrally reconstructed by the same diffraction grating G1 that disperses it on the outward path. Similar to the confocal microscope according to the prior art, the light is collected by the lens L 103 and L 104 The resulting afocal system allows performing confocal filtering, as well as modifying the size of the light beam FS.

[0066] The light beam FS passes through the beam splitter BS, which reflects the light beam FE from the light source SL. The converging lenses L on both sides of the beam splitter are 105 and L 106 An afocal system is formed, which allows the second diffraction grating G2 to be placed in a plane conjugate to the pupil of the objective lens MO. This second diffraction grating again disperses the light in the direction x. Lens L 107 A second spectral line LS2 oriented along the x-direction is formed on the matrix image sensor CMI.

[0067] The matrix image sensor, which can be manufactured using CMOS or CCD technology, has a large number of pixels in the x-direction, determined by the spatial range of spectral lines available to the optical system SO2, typically several hundred pixels. The number of pixels in the y-direction may be smaller, on the order of ten or a few dozen, as these pixels are used solely for implementing the photon redistribution algorithm implemented by the digital image processor PNI. To implement this algorithm, the digital image processor PNI must be synchronized with the translation stage. The digital image processor PNI can be, for example, a suitably programmed microprocessor or a dedicated digital circuit.

[0068] In order for the algorithm to achieve an improvement in spatial resolution, the second spectral line LS2 must be longer in the x-direction, ideally by a factor of 2 or close to 2 (e.g., 1.8 to 2.2). This is illustrated in the upper left portion of the figure; it can be clearly seen that the segment of the spectral line LS2 corresponding to the wavelength interval Δλ is longer than the segment of the spectral line LS1 corresponding to the wavelength interval Δλ. This elongation can be achieved by using a second diffraction grating G2 whose angular dispersion DA2 is greater than the angular dispersion DA1 of the first diffraction grating; using a lens L 103 , L 104 , L 105 and L 006 The magnification of the beam generated by the component is M G ; or a combination of the two. Generally speaking, if M G is the diameter d of the light beam incident on grating G2 G2 The diameter d of the light beam incident on the grating G1 G1 The ratio (M G =d G2 / d G1 ), get M G DA2 / DA1=length(LS2) / length(LS1)=A G , where factor A G is 1.8 to 2.2, and ideally 2. For example, if M G =1, then a second diffraction grating will be selected whose angular dispersion is (approximately) twice that of the first diffraction grating.

[0069] Figure 2 The embodiment shown in Figure 1 The embodiment shown in FIG. 1 differs only in that the mirror M of the first optical system 102 is replaced by an oscillating mirror, or more generally by S1 with a variable orientation. More precisely, the change in the orientation of the mirror S1 is controlled to cause a displacement of the first spectral line LS1 on the sample E in the y direction; for this reason, S1 is hereinafter referred to as a "scanning mirror". In this way, the spectral line LS1 scans the sample E without translating the sample E in the y direction. Figure 1In the embodiment shown, the photon redistribution is performed digitally by an image processor PNI synchronized with the moving mirror S1.

[0070] Figure 3 The embodiment shown in Figure 2 The embodiment shown in FIG. 1 differs in that the second optical system SO2 further comprises an oscillating scanning mirror, or more generally a mirror S2 with a variable orientation, which is arranged between the lens L and the lens L. 107 Before, it is used to focus the light beam FS on the matrix image sensor CMI. 308 and L 309 The resulting afocal system allows the mirror S2 to be conjugated with the pupil of the microscope objective MO.

[0071] By controlling the orientation change of the mirror S2, a displacement of the second spectral line LS2 on the matrix image sensor in the y direction is caused, thereby achieving a purely optical photon redistribution. In order to obtain a higher resolution, this scan must be synchronized with the scan performed on the sample E by the first spectral line LS1 and have a higher, A S / M S times the amplitude, where A S 1.8 to 2.2 times, ideally 2, M S is the diameter d of the beam on mirror S2 S2 The diameter d of the beam on mirror S1 s1 The ratio between. Figure 3 In the embodiment shown, M G It is made of lens L 103 , L 104 , L 105 , L 106 , L 308 and L 309 The total magnification of the components that make up it.

[0072] and Figure 1 and Figure 2 Unlike the case of the embodiment shown in , the number of pixels of the matrix image sensor CMI in the y direction is determined by the scanning amplitude performed by the second spectral line LS2 and is typically of the same order of magnitude as the number of pixels in the y direction.

[0073] Furthermore, if the scanning mirrors S1 and S2 perform scanning fast enough, the image sensor CMI does not necessarily need to acquire a separate image for each position of the spectral lines LS1 and LS2: a single image can correspond to multiple positions. In extreme cases, acquiring a single image may be sufficient, eliminating the need for synchronization between the scanning mirrors S1 and S2 and the matrix image sensor.

[0074] exist Figure 1 、 Figure 2 and Figure 3In the embodiment shown in FIG, the scanning rate is limited by the synchronization requirements: Figure 1 In the case of , the scanning rate is limited by the synchronization requirements between the translation stage PT and the matrix image sensor CMI; Figure 2 In the case of , the scanning rate is limited by the synchronization requirements between S1 and the matrix image sensor CMI; Figure 3 In this case, the scan rate is limited by the need for synchronization between at least S1 and S2. Figure 4 The embodiment shown is able to overcome this limitation, wherein two scanning mirrors S1 and S2 are created on two opposite sides of the same oscillating or more generally variable orientation support SPO. In the specific embodiment shown in the figures, this is achieved by using two redirecting mirrors M 402 and M 403 This eliminates any need for synchronization and therefore makes it easier to use higher scanning frequencies (kHz range), such as are achieved by means of resonant supports SPO or polygon scanners. Since the angular amplitudes of the scans performed by the mirrors S1 and S2 are structurally identical, the second optical system must provide M for the beam FS between the two scanning mirrors. S times magnification, M S is approximately 2 (in other words, d S2 / d S1 =M S =A S , where A S =2, or at least approximately 2). Figure 4 In the embodiment shown, this is achieved by the lens L 105 , L 106 , L 408 and L 409 The formed afocal system is realized.

[0075] Figure 5 The embodiment shown in Figure 4 The embodiment shown in FIG is similar except that the functions of the two diffraction gratings G1 and G2 are performed by two independent regions of a single grating G, which is achieved by using a redirecting mirror M. 502 、M 503 and M 504 and in Figure 5 In the example shown, the beam FE passes through the beam splitter BS, while the beam FS is reflected by the beam splitter BS. Since the two independent gratings G1 and G2 are replaced by two areas of the same grating, that is, DA1 = DA2, the magnification M G must be approximately 2, in other words, at least approximately d G2 =2d G1 .

[0076] Figure 4 and Figure 5 The embodiment shown requires two diffraction gratings, or a single grating large enough to present two separate areas that can be illuminated independently of each other. Figure 6 The illustrated embodiment comprises a single grating, the spatial extent of which is not necessarily larger than the spatial extent of the light beam FE.

[0077] Figure 6 The system shown includes the above reference Figure 1 The light source SL. The light beam FE from the light source is collimated by the light beam FE collimating lens L 105 collimated and then redirected by the first mirror M 601 The spectrally dispersed beam is then deflected (optionally) and reaches the diffraction grating G1, which spectrally disperses the beam in the first diffraction order. The spectrally dispersed beam is then focused by two lenses L located in the afocal system. 601 and L 602 The length of the slit FR in the spectral dispersion direction x depends on the angular dispersion introduced by the grating, while the width in the direction y is determined by the pinhole of the conventional confocal microscope. 602 and L 105 The main function of the formed afocal system is to make the slit FR and the pinhole P1 of the light source SL optically conjugated. Alternatively, the slit FR can also be omitted.

[0078] The spatially filtered light beam dispersed by the grating G1 is directed by a scanning mirror S1 formed by the reflecting surface of an oscillating or variably oriented support SPO to the microscope objective MO, thereby obtaining a spectral line LS1 of the scanned 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 again to compensate for the scanning effect introduced in the "outward path", and before reaching the diffraction grating G1 again by the component L 601 -FR-L 602 The originality of this embodiment is that, instead of collecting the beam spectrally recombined by the first diffraction order of the grating, the second-order diffracted light (beam FS' in the figure) is used, which propagates in the opposite direction along the beam path. Since the second-order angular dispersion is twice the first-order angular dispersion, the diffracted beam FS' is not spectrally recombined, but has an angular dispersion twice the amplitude of the first-order diffracted beam FE (in other words, the spectral dispersion introduced on the "outward path" is overcompensated). This beam FS' is filtered by the lens L 607 and L 608 The resulting afocal system is magnified by a factor of about 2, or M S =2, which is d S2 =2d S1 , was mirrored 602 、M 603 、M 604Redirected (optionally), reflected by scanning mirror S2 formed on the same variable orientation support SPO as S1, and finally by lens L 609 Focusing to form a spectrum line LS2 on the matrix image sensor CMI. By construction, in this embodiment, d G1 =d G2 .

[0079] It should be noted that in the embodiment described above, the first optical system SO1 for forming the first spectral line LS1 on the sample E largely overlaps with the second optical system, and the same optical components are passed through by the light on the outward path (forming the first spectral line and illuminating the sample) and the return path (collecting and processing the signal beam). More specifically, in Figures 1 to 5 In the embodiment shown in , the first optical system is a subset of the second optical system. Figure 6 In the illustrated embodiment, this is not the case: in fact, the path of the signal beam is separated from the path of the optical beam using second-order diffraction of the diffraction grating. In other embodiments, the first and second optical systems may be completely or substantially non-intersecting. This is particularly true in imaging systems operating in transmission mode.

[0080] Figure 7 Such a system is shown in FIG. Its working principle is similar to Figure 3 Similar, except it operates in transmissive mode rather than reflective mode. Figure 1 、 Figure 2 、 Figure 4 and Figure 5 The embodiment shown in can also be modified to operate in transmissive mode.

[0081] exist Figure 7 In the system shown in FIG, the light beam FE emitted by the light source SL is converged by the focusing lens L 702 collimated and oriented by the variable mirror S 10 After reflection, the variable orientation mirror scans the light beam in the direction y. 703 -L 704 (Note that there is no pinhole, which is not necessary in SO1) and is mirrored 701 After deflection (optional), the beam FE is diffracted by the diffraction grating G 10 The diffraction grating is spectrally dispersed in the direction x through the afocal system L 703 -L 704 and lens L 702 Located in the Fourier plane of the pinhole P1 of the light source SL. 705 -L 706After being deflected again by the mirror M702 (also optional), the beam is focused by the first microscope objective MO1, thereby forming a spectral line LS1 on the sample E. Afocal system L 706 -L 705 The function of the grating is to optically conjugate the rear pupil of the microscope objective with the grating G1. All these elements constitute the first SO1 optical system.

[0082] The light that passes through the sample (signal beam FS) is collected and collimated by the second microscope objective MO2 located opposite the first microscope objective, and then diffracted by another diffraction grating G 11 Spectral recombination (note that these two gratings perform the functions of dispersion and spectral recombination, which in the previous embodiment were performed by grating G0 alone). 10 It's G 10 The roles are scattered, and DA 11 It's G 11 The roles of the characters are scattered, so ideally M G10-G11 DA 11 =DA 10 , where M G10-G11 By L 705 , L 706 、MO1、MO2、L 707 and L 708 The magnification factor formed, that is, the magnification of the light beam at the grating G 11 The diameter d on G11 With the beam at the grating G 10 The diameter d on G10 The ratio between the two. Redirection mirror M 703 and afocal system L 707 -L 708 So that the grating G 11 is optically conjugate with the rear pupil of MO2. Equivalently, L 705 , L 706 、MO1、MO2、L 707 and L 708 The resulting system can be considered as an optically conjugated G 10 and G 11 , and in G 11 The dispersion DA of the imaging in the plane 10 Equal to DA 11 The spectrally recombined beam consists of components L 709 -P2-L 710 Perform spatial filtering and reach the S 10 Synchronously operated variable orientation mirror S 11 , and its oscillation amplitude is divided by S 10 and S 11 The total magnification between M S10-S11 (Incident to S 11The diameter d of the beam on S11 With the incident S 10 The diameter d of the beam on S10 ratio between them) to compensate for the 10 The introduction of angular scanning (with grating G 10 and G 11 The situation is similar, there are two variable orientation mirrors S 10 and S 11 , which replace the single orientation mirror S1 in the embodiment operating in reflection mode). Equivalently, S 10 and S 11 can be considered as optically conjugated and in S 11 The image S in the plane 10 The angular displacement must be equal to or approximately equal to S 11 Then, the beam FS is spectrally dispersed by another diffraction grating G2 with angular dispersion DA2, so that M G2-G11 DA2 / DA 11 =A G =2 (at least approximately equal), where M G2-G11 Yes, here is d G2 (the diameter of the beam incident on G2) and d 11 Then, the spectrally heavily dispersed beam is reflected by another scanning mirror S2, which is aligned with S 10 and S 11 synchronous, and has approximately equal to 2A S2 M S10-S2 The oscillation amplitude, where M S2 Is an afocal system L 711 , L 712 , L 713 , L 714 The magnification factor (in other words, the diameter d of the beam on mirror S2) S2 with d S11 The ratio between them), and by the lens L 715 Focusing, thereby forming a spectral line LS2 oriented in the direction x, and scanning the matrix image sensor CMI in the direction y. 711 -L 712 and L 713 -L 714 Ensure optical conjugation between the various optical elements of system SO2. Note that this system, operating in transmission mode, has more components than the above-described embodiment and requires synchronization of the three scanning mirrors. To alleviate these synchronization constraints, S 10 、S 11 and S 12 It can be replaced by a single scanning or oscillating system, such as a resonant mirror or polygon scanner.

[0083] As mentioned above, in order to shorten the image acquisition time, the angular velocity of one or more scanning mirrors can be increased, but this is technically difficult. Figure 8 Another alternative is shown in , consisting in replacing the single beam with multiple beams MFE arranged along the y-direction.

[0084] Figure 7 The embodiment shown is Figure 3 The embodiment shown in FIG is similar to the embodiment shown in FIG, and only the differences are described in detail below. First, the light source SL is replaced by a source SLMF comprising a set of microlenses MML1 aligned in the direction y, which decomposes the single light beam emitted by the broad spectrum source SLS into multiple light beams MFE. The spatial coherence of these light beams is ensured by multiple pinholes MP1 arranged in the focal plane of each microlens, which replace the single pinhole P1. Similarly, the pinhole P2 used for confocal filtering is replaced by multiple pinholes MP2 aligned in the direction y. Note that Figure 3 Lens L 105 The lens L is arranged at the output of the source SLMF 810 Instead of collimating multiple light beams MFE and converging them by lens L 805 Instead, a second plurality of microlenses MML2 aligned in the direction y is used to focus the multiple signal beams MFS after they pass through the beam splitter BS. In this system, N > 1 spectral lines PLS1 and PLS2 in the direction y (and oriented along the direction x) are generated on the sample E and the matrix image sensor, respectively. Thus, by performing an angular scan that is N times smaller than that for a single spectral line, the same region of the sample can be imaged using the scanning mirrors S1 and S2. Consequently, the time required to acquire an image of the region of the sample is also reduced by a factor of N.

[0085] The invention has been described with reference to several embodiments, but variations are possible. For example:

[0086] The cross section of the beam does not necessarily have to be circular. In this case, the concept of diameter must be replaced by the concept of lateral size. More specifically, the concept of beam diameter on the grating must be replaced by the concept of beam size in the dispersion direction (direction x in the above example), and the concept of beam diameter on the scanning mirror must be replaced by the concept of beam size in the scanning direction (direction y in the above example).

[0087] - The internal structure of the light source SL is given as an example only; other structures are possible.

[0088] - All or part of the converging lens can be replaced by other optical elements, such as concave mirrors or diffraction systems, such as holographic lenses, etc.

[0089] - The diffraction grating, or parts thereof, can be replaced by other dispersive systems, such as three-dimensional diffractive structures, or even by refractive systems, such as prisms (except Figure 6 In the case of the embodiment shown in , a diffraction system is required).

[0090] One or more microscope objectives can be replaced by other types of focusing systems, such as concave mirrors or f-theta lenses. In this case, the dispersive element does not necessarily have to be placed in a plane conjugate to the pupil of the focusing system, but more generally in a plane conjugate to the Fourier plane of the specimen.

[0091] - The directions x and y do not necessarily have to be perpendicular to each other, although this is advantageous. They must be non-parallel.

[0092] - In the embodiment where the two scanning mirrors S1 , S2 are carried by the same variable orientation structure, they do not have to be arranged on opposite faces of the structure.

[0093] - The concept of a "variably-orientable" mirror or support structure covers both resonantly oscillating structures and structures whose orientation varies continuously or discretely in a controlled manner.

[0094] References

[0095] (Sheppard 1988): CJR Sheppard, "Super-resolution in Confocal Imaging," Optik, vol. 80, no. 2, pp. 53-54.

[0096] (York 2013): A.G. York et al. "Instant super-resolution imaging in live cells and embryos via analog image processing." Nature Methods, Vol. 10, No. 11, November 2013, pp. 1122-1126.

[0097] (De Luca 2013): GMR De Luca, “Re-scan confocal microscopy: scanning twice for better resolution,” Biomedical Optics Express, Vol. 4, No. 11, November 2013.

[0098] (Curd 2015): A. Curd et al. "Construction of an instant structured illumination microscope." Methods, vol. 88, 2015, pp. 37–47.

[0099] (DuBose 2019): TB DuBose et al. "Super-resolution retinal imaging using optically reassigned scanning laser ophthalmology," Nature Photonics, Vol. 13, April 2019, pp. 257-262.

[0100] (Sandison 1995): DR 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, July 1, 1995, pp. 3576-3588.

[0101] (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).

[0102] (Tearney 1998) GJ Tearney et al. “Spectrally encoded confocal microscopy,” Optics Letters, vol. 23, no. 15, August 1998.

[0103] (Hwang 2015) Hwang J. et al. “Frequency and spectrally-encoded confocal microscopy,” Optics Express, vol. 23, no. 5, March 9, 2015.

Claims

1. A photon redistribution imaging system, comprising: - a polychromatic light source (SL) configured to generate at least one spatially coherent illumination beam (FE) in an illumination wavelength range; a first optical system (SO1) configured to disperse the illumination beam with a first angular dispersion DA1 and to focus it on the sample (E) to form first spectral lines (LS1) oriented along a first direction (x); a second optical system (SO2) configured to collect a light beam (FS) elastically scattered by the sample, said light beam (FS) being referred to as the signal beam, to apply a second angular dispersion DA2 to said signal beam and to focus it in a focal plane so as to form a second spectral line (LS2) oriented along a second direction (x); - a matrix image sensor (CMI) arranged in said image focal plane; - means (PT, S1) for causing a relative displacement between the first spectral line and the sample in a third direction (y) perpendicular to the first direction; and - means for forming an image of the sample by photon redistribution based on one or more images acquired by said matrix image sensor corresponding to a plurality of different positions adopted by the first spectral line on the sample; Wherein, the first optical system includes a first dispersive optical device (G1, G 10 ), for dispersing the illumination light beam with a first angular dispersion DA1; and the second optical system includes a second dispersive optical device (G2) for applying the second angular dispersion DA2 to the illumination light beam; The length of the second spectral line in the second direction is a factor A of the length of the first spectral line in the first direction G times, A G =M G DA2 / DA1>1, M G is the ratio of the signal beam size on the first dispersive optical device measured along the first direction to the signal beam size on the second dispersive optical device measured along the second direction.

2. The photon redistribution imaging system according to claim 1, wherein: The factor A G It is 1.8 to 2.

2.

3. The photon redistribution imaging system according to any one of the preceding claims, wherein: The first optical system (SO1) comprises: -The first collimation system (L 105 , L 205 , L 305 , L 405 , L 505 , L 605 ), configured to collimate the illumination beam (FE); - the first dispersive optical device (G1, G 10 ); - a focusing optical system (MO, MO1) configured to receive as input the illumination beam dispersed by said first dispersive device and to focus it on a sample (E).

4. The photon redistribution imaging system according to claim 3, wherein: The second optical system (SO2) comprises: - the focusing system or another focusing system (MO, MO2) for collecting the signal beam, collimating the signal beam and directing it to the first dispersive optical device or another dispersive optical device, so that the signal beam is spectrally recombined; - said first (G1) or said further dispersive optical device (G 11 ), configured to perform spectral recombination on the signal light beam; - the second dispersive optical device (G2) has a second angular dispersion DA2 for spectrally dispersing the signal light beam in the second direction (x); and - at least one optical element (L 107 , L 207 , L 309 , L 409 , L 509 , L 609 ), configured to focus the signal light beam spectrally dispersed by the second dispersive optical device (G2) in a focal plane called the image focal plane.

5. The photon redistribution imaging system according to claim 4, wherein: The second optical system comprises a pinhole (P2) arranged in a plane conjugate to the focal plane of the focusing system of the second optical system (MO) in order to perform confocal filtering.

6. The photon redistribution imaging system according to any one of claims 4 or 5, wherein: The device for forming an image of the sample by photon redistribution based on the plurality of images of the second spectral line acquired by the matrix image sensor includes a digital image processor (PNI).

7. The photon redistribution imaging system according to claim 6, wherein: The means for causing a relative displacement between the first spectral line and the sample comprises a sample translation stage (PT).

8. The photon redistribution imaging system according to claim 6, wherein: The device for causing a relative displacement between the first spectral line and the sample includes a variable orientation mirror (S1) shared by the first and second optical systems, wherein the variable orientation mirror is configured to apply an angular scan to the illumination beam along a third direction and to apply an opposite angular scan to the spectrally recombined signal beam.

9. The photon redistribution imaging system according to any one of claims 3 to 5, wherein: - the means for causing 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, the first variable orientation mirror being configured to apply an angular scan along a third direction to the illumination beam and an opposite angular scan to the spectrally recombined signal beam; as well as - the means for forming an image of the sample by photon redistribution comprises a second variable orientation mirror (S2) configured to apply an angular scan along a fourth direction perpendicular to the second direction to the signal light beam spectrally dispersed by the second dispersive optical device; The angular scan applied by the second variable orientation mirror is synchronized with the angular scan applied by the first mirror, and the amplitude of the angular scan applied by the second variable orientation mirror is A times the amplitude of the angular scan applied by the first mirror. S / M S times, where A S A factor of 1.8 to 2.2, M S is the ratio of the size of the illumination beam on the second variable orientability mirror measured along the fourth direction to the size of the spectrally dispersed signal beam on the first variable orientability mirror measured along the third direction.

10. The photon redistribution imaging system according to claim 9, wherein: The first variable orientation mirror (S1) is structurally independent from the second variable orientation mirror (S2), and the first dispersive optical device (G1) is separate from the second dispersive optical device (G2).

11. The photon redistribution imaging system according to any one of claims 1 to 3 or claim 9 when dependent only on claim 3, wherein: The first dispersive optical device (G1) is a diffraction system exhibiting the angular dispersion DA1 in the first diffraction order and the angular dispersion DA2=2DA1 in the second diffraction order, and the second optical system is configured to collect light scattered by the diffraction system in the second diffraction order.

12. The photon redistribution imaging system according to any one of claims 9 or 11, wherein: The first and second variable orientation mirrors are formed by two separate reflecting areas of the same variable orientation support (SPO).

13. The imaging system of claim 12, wherein: The variable orientation support (SPO) is selected from a resonant mirror support and a polygon scanner.

14. The imaging system according to one of claims 12 and 13, wherein: The first dispersive optical device (G1) is separate from the second dispersive optical device (G2).

15. An imaging system according to any one of the preceding claims, wherein: The light source (SLMF) includes a first group of multiple microlenses (MML1) aligned in a third direction to generate a plurality of the illumination light beams in parallel, so that the first optical system (SO1) forms a plurality of first spectral lines (LS1) oriented along a first direction (x) and arranged in the third direction (y) on the sample (E); and wherein the second optical system (SO2) includes a second group of multiple microlenses (MML2) aligned in the third direction (y) to form a plurality of first spectral lines (LS1) oriented along the first direction (x) and arranged in a fourth direction (y) perpendicular to the second direction (x) on the matrix image sensor (CMI).

Images