Confocal microscopy with photon reallocation

Abstract

The present invention relates to a scanning confocal photon relocation microscope configured to operate in a coherent (reflection or transmission) imaging mode and to have a confocal pinhole whose diameter is between 2 and 4 airy units, and to the use of such a microscope for observing virus particles in suspension.

Description

[Technical field]

[0001] The present invention relates to the field of optical microscopy. [Background technology]

[0002] Optical microscopes play a vital role in biology because they allow the rapid observation of living specimens, unlike, for example, electron microscopes, which require complex preparation procedures incompatible with the preservation of life. However, their resolving power is inevitably limited by the diffraction of light. According to Abbe theory, for a conventional optical microscope, the maximum resolution d is given by:

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[0003] So-called "super-resolution" techniques make it possible to surpass the Abbe resolution limit by using fluorescent markers and / or non-linear effects. These techniques are complex to implement.

[0004] Furthermore, since biological objects are three-dimensional in nature, it is necessary to obtain high spatial resolution in the axial direction (the Abbe limit concerns the lateral resolution in the plane perpendicular to the optical axis). Confocal microscopy allows obtaining images with a very shallow depth of field (on the order of a few hundred nanometers) and thus "cutting" the sample to access its three-dimensional structure. This technique is most often used in conjunction with fluorescent markers, but it is also possible to utilize it in reflection mode without the use of markers.

[0005] Confocal microscopes use a point illumination source, the image of which is projected by the objective onto the sample to be observed. Light from the sample (backscattered light in the case of a reflecting confocal microscope, or fluorescent emission if a fluorescent marker is used) is focused onto a pinhole optically conjugate to the point source and detected, for example, by a photomultiplier tube. The function of the pinhole is to suppress radiation outside the focal plane of the objective, thus realizing optical sectioning. The image of the sample is acquired point by point by scanning. More precisely, by a two-dimensional scan in two directions perpendicular to the optical axis, an image of a slice of the sample centered in the focal plane of the objective is obtained. By adding an axial scan of this focal plane, a three-dimensional image is obtained.

[0006] As the pinhole diameter decreases, the slice thickness decreases and thus the axial resolution improves, but the improvement is small for reductions of more than 1 Airy Unit (AU). An Airy Unit is the diameter of the Airy Spot on the microscope,

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[0007] In confocal microscopy, the use of pinholes with diameters less than 1 AU can provide an improvement in lateral resolution with respect to the Abbe limit, which could theoretically even be 30%, but this comes at the expense of a worse signal-to-noise ratio.

[0008] The photon relocation method, first proposed in (Sheppard 1988), could in principle improve the lateral resolution of a confocal microscope by a factor of two. The idea behind this method is to use, instead of photomultiplier tubes or, more generally, point emission detectors, a matrix detector, which allows to acquire one elementary image for each acquisition point. The image is then resized (ideally scaled down by a factor of two) by digital or optical means, before moving to the next scanning point. The final image is obtained by integrating the various scan images acquired in succession, each time shifted by one scan step with respect to the previous image.

[0009] Purely optical implementations of the photon relocation scheme are described in (York 2013), (De Luca 2013), (Curd 2015), and (Roth 2017). It consists of applying a first angle scan to the illumination beam, applying an inverse angle scan to the beam from the sample, and then applying a second angle scan to the same beam from the sample, synchronized with the first scan. The second angle scan has an amplitude normalized with respect to the cross-sectional area of ​​the beam, ideally a factor of 2 larger than the first scan. "Normalized with respect to the cross-sectional area of ​​the beam" means that the magnitude α / M must be greater than 1 and ideally equal to 2, where α is the ratio of the amplitude of the second angle scan to the amplitude of the first angle scan and M is the ratio of the cross-sectional area of ​​the beam from the sample to the cross-sectional area of ​​the illumination beam.

[0010] As with conventional confocal microscopy, imaging can be parallelized using a matrix array of pinholes and a matrix array of microlenses, see for example the aforementioned article (York 2013).

[0011] Photon remapping has been applied in particular to confocal fluorescence microscopy, see for example the aforementioned articles (York 2013), (De Luca 2013) and (Curd 2015), where an improvement in lateral resolution of a factor of 1.5 was observed. To the inventor's knowledge, photon remapping has only been described so far in the application of reflection imaging (DuBose 2019). However, in this article, this is not a problem of a microscope, but of an ophthalmoscope, where the objective lens is replaced by the crystalline lens of the patient's eye, therefore by a lens with a low numerical aperture, resulting in a lateral resolution of a few tens of micrometers. [Prior art documents] [Non-patent literature]

[0012] [Non-Patent Document 1] Sheppard 1988 [Non-Patent Document 2] York 2013 [Non-Patent Document 3] De Luca 2013 [Non-Patent Document 4] Curd 2015 [Non-Patent Document 5] Roth 2017 [Non-Patent Document 6] DuBose 2019 Summary of the Invention [Problem to be solved by the invention]

[0013] The object of the present invention is to provide a scanning confocal microscope with improved resolution in three dimensions (lateral and axial). According to the present invention, this object is achieved through the use of a confocal pinhole whose diameter (or, more generally, its maximum lateral dimension) is between 2 and 4 airy units, ideally equal to 3 airy units, in coherent imaging mode. The inventors have noticed that in coherent imaging mode, the lateral resolution improves with the opening of the pinhole at a diameter of 3 airy units up to twice the Abbe limit. In contrast, in the fluorescence mode of operation, the lateral resolution is independent of the pinhole diameter. Coherent imaging refers to a scheme in which the detected photons are the illuminating photons that have undergone only elastic scattering in the sample and in the microscope optics, which can be, for example, a matter of reflection imaging (a case considered in detail below) or transmission imaging. In contrast, fluorescence imaging or Raman scattering imaging are considered incoherent, since the illuminating photons undergo inelastic scattering.

[0014] By using a pinhole with a diameter of 3 Airy units, background subtraction is further optimized (however, the background is reduced by about 30% compared to a confocal microscope without photon relocation and with a pinhole of 1 AU). The idea of ​​"background subtraction" is clearly defined in (Sandison 1995).

[0015] Furthermore, the inventors have found that under these conditions the axial resolution is improved by a factor of 1.5 compared to that of conventional confocal microscopy.

[0016] In the end, by using illumination with a wavelength of 445 nm and an immersion objective lens with NA=1.3, the measurement was (86×86×248) nm 3 It is possible to obtain a spatial resolution of 100 nm. Moreover, it does not require fluorescent labeling, which makes the application of this technique easier and more general. [Means for solving the problem]

[0017] Thus, one subject of the present invention is a light source configured to generate at least one spatially coherent illumination light beam at an illumination wavelength; a first optical system configured to apply an angular scan to the illumination light beam; at least one microscope objective configured to receive as input the illumination light beam emerging from the first optical system, to focus it onto the sample, and to collect and collimate a light beam elastically scattered by said sample, referred to as a signal beam; a second optical system, which may coincide in whole or in part with the first optical system, and which is configured to receive as an input the signal beam collimated by the microscope objective lens, apply thereto an angular scan opposite to that applied to the illumination light beam, and focus it onto the first focal plane; a pinhole disposed in the first focal plane; a matrix image sensor disposed in the second focal plane; photon redistribution means which interact with the matrix image sensor to reconstruct an image of the sample; a scanning confocal photon relocation microscope comprising: a microscope objective lens and a second optical system configured to focus the signal beam at the illumination wavelength onto a matrix image sensor; The pinhole must have a diameter or maximum lateral dimension of 2 to 4 air units. It is characterized by:

[0018] According to a particular embodiment of such a scanning confocal microscope: At least the second optical system may include a beam splitter for splitting the signal beam from the illumination light beam. The light source may be configured to emit a blue, violet, or near-ultraviolet illumination light beam. The light source may be a laser. The microscope objective may be an immersion objective having a numerical aperture of 1 or greater. The photon repositioning means may include a third optical system configured to collect the signal beam that has passed through the pinhole, collimate it and apply to it an angular scan synchronized with that applied to the illumination light beam, the product of its amplitude and the cross-sectional area of ​​the collimated light beam in the third optical system being greater than the product of the amplitude of the scan applied by the second optical system and the cross-sectional area of ​​the collimated light beam in the second optical system, and to focus it in a second focal plane, and an assembly consisting of the microscope objective lens, the second optical system and the third optical system is configured to focus said signal beam of the illumination wavelength onto a matrix image sensor. The third optical system may be configured to apply an angular scan to the signal beam such that the amplitude of said scan multiplied by the cross-sectional area of ​​the beam is between 1.8 and 2.2 times the product of the amplitude of the angular scan applied to the illumination light beam by the first optical system and the cross-sectional area of ​​said beam.

[0019] The scanning confocal microscope may be configured to operate in reflection mode, the first optical system may comprise a first lens for converging an illumination light beam, a pinhole arranged in the focal plane of the first lens for spatial filtering of the illumination light beam, a beam splitter for reflecting a portion of said beam, a second lens for collimating said portion of the illumination beam, a first oscillating mirror or a system of oscillating mirrors to which said angular scan is applied, and an afocal system including a third and a fourth lens, the second optical system may include the afocal system, the first oscillating mirror or system of oscillating mirrors, the second lens, and the beam splitter configured to transmit a portion of the signal beam that is backscattered by the sample, The third optical system may include a fifth lens that collimates the signal beam passed through the pinhole, a second oscillating mirror or a system of oscillating mirrors that applies to it said angular scan synchronous with that applied to the illumination light beam, and a sixth lens that focuses it onto the second focal plane. - the light source may be configured to generate a plurality of said illumination light beams in parallel, which propagate through a first optical system, and a microscope objective lens to collect a plurality of respective signal beams, which then propagate along said second optical system, and the microscope further includes a matrix array of pinholes, one for each of said backscattered light beams, arranged in said second focal plane.

[0020] In this case, the scanning confocal microscope may be configured to operate in reflection mode, the light source may include a first array of microlenses for generating and focusing said plurality of illumination light beams; The scanning confocal microscope may include at least one oscillating mirror having a reflective front surface and a reflective rear surface, the front surface forming part of the first and second optical systems and the rear surface forming part of a third optical system; The third optical system may include a second array of microlenses that increases the cross-sectional area of ​​the backscattered light beam incident on the rear face of the oscillating mirror by a factor of 1.8 to 2.2.

[0021] Another subject of the invention is the use of these scanning confocal microscopes for observing virus particles in suspension.

[0022] Another subject of the invention is a method for observing a sample, comprising the steps of: - generating at least one illumination light beam of a spatially coherent and collimated illumination wavelength; - applying an angle scan thereto; - focusing it on the sample by a microscope objective; - collecting, by means of said or another microscope objective, a beam of light at said illumination wavelength, called the signal beam, elastically scattered by the sample; - applying to the signal beam an angular scan opposite to that applied to the illumination light beam, focusing it onto a first focal plane; - performing spatial filtering of the signal beam by means of a pinhole arranged in said first focal plane, the pinhole having a diameter or maximum lateral dimension of between 2 and 4 airy units; - collecting the signal beam that has passed through the pinhole, collimating it and applying to it an angular scan synchronous with that applied to the illumination light beam, such that the product of its amplitude and the cross-sectional area of ​​the signal beam is greater than the product of the amplitude and the diameter of the angular scan applied to the illumination light beam, and focusing it on a second focal plane; - detecting the signal beam by a matrix image sensor arranged in a second focal plane; Includes.

[0023] Another subject of the invention is a method for observing a sample, comprising the steps of: generating at least one spatially coherent and collimated illumination light beam (FE) at an illumination wavelength; applying an angle scan thereto; focusing it onto the sample by a microscope objective; collecting, by said or another microscope objective, a beam of light at said illumination wavelength, referred to as a signal beam, that has been elastically scattered by the sample; applying an angular scan to the signal beam in an opposite direction to that applied to the illumination light beam, focusing it onto a first focal plane; performing spatial filtering of the signal beam by a pinhole located in the first focal plane, the pinhole having a diameter or maximum lateral dimension of 2 to 4 airy units; detecting the signal beam passing through the pinhole by a matrix image sensor disposed in a second focal plane, the imaging rate of which is synchronized with the angular scanning of the illumination light beam; applying a digital light remapping process to an image captured by the matrix image sensor; Includes.

[0024] Other characteristics, details and advantages of the invention will become apparent from reading the description given with reference to the attached drawings, given by way of example and respectively showing: [Brief description of the drawings]

[0025] [Figure 1] 1 is a schematic diagram of a confocal photon relocation microscope according to a first embodiment of the present invention. [Diagram 2] FIG. 2 is a graph showing how the performance of the confocal microscope of FIG. 1 depends on the diameter of the confocal pinhole. [Diagram 3] [Figure 4] and [Diagram 5] 1 is an experimental result demonstrating the technical effect of the present invention. [Figure 6] 11 is a numerical simulation result demonstrating another technical effect of the present invention. [Figure 7] 11 is a numerical simulation result demonstrating another technical effect of the present invention. [Figure 8] 2 is a schematic diagram of a confocal photon relocation microscope according to a second embodiment of the present invention. [Figure 9] 4 is a schematic diagram of a confocal photon relocation microscope according to a third embodiment of the present invention. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0026] As already explained, the photon relocation method consists in recording an image of a sample through a pinhole when it is illuminated by a very tightly focused light beam. This image obtained through the pinhole is located at a specific position on the recording camera, with s being the distance between the two points of the sample scan and M being the demagnification. The signal recorded by the camera is

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[0027] Equation (1) assumes that the sample is moved and scanned, but it is more practical to keep the sample stationary and move the illumination beam. Mathematically, this is expressed as the coordinates

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[0028] If the detector is large enough compared to the pinhole size, the pinhole weighting factor can be neglected. Furthermore, when a reduction factor M is introduced,

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[0029] The optical transfer function of this system is

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[0030] In general, a confocal photon relocation microscope according to the invention includes a light source, a first optical system that interacts with the light source, an objective lens that scans the surface of the sample with a focused light beam, a second optical system that collects the light backscattered or transmitted by the sample and focuses it to compensate for the angular deviation introduced to perform the scan, a pinhole in the focal plane of the second optical system that performs confocal filtering, and a third optical system that applies a "rescan" to the light beam from the pinhole and focuses it onto a camera. The various optical systems may be partially coincident, making it possible to limit the number of optical components.

[0031] FIG. 1 shows an optical schematic diagram of a confocal photon relocation microscope according to a first embodiment of the present invention.

[0032] The light source SL is a laser emitting a light beam FE (illumination beam) with a wavelength of λ=445 nm. The beam FE is focused by a first focusing lens L1 (focal length 200 mm) and spatially purified by a first pinhole P1 with a diameter of 50 μm, located in the focal plane of the lens.

[0033] The spatially filtered illumination beam FE is reflected by a beam splitter LS (transmission 50% - reflection 50%) and collimated by a second focusing lens L2 (focal length 200 mm). A first system of two oscillating mirrors, for example of the galvanometer type (only one MO1 is shown for simplicity), imparts a time-varying deflection to the beam FE, resulting in a two-dimensional scan. The beam FE thus deflected is focused by a microscope objective OM (apochromatic objective with magnification 60x and numerical aperture NA=1.3, immersed in silicone oil) onto the sample E, whereby the surface of the sample is scanned with a focal spot whose diameter is limited by diffraction. If the sample is transparent or translucent, the focal point can be located below the surface at a depth that can be varied by axially moving the objective or the sample, whereby a three-dimensional image is obtained in tomography.

[0034] The afocal system consisting of the converging lenses L3, L4 ensures an optical conjugate relationship between the pupil of the objective and the midpoint of the two oscillating mirrors (or one oscillating mirror MO1), typically the distance between the two oscillating mirrors being negligible with respect to the focal length of L3. All optical components included between the lens L1 and the objective OM form the first optical system, or illumination optical system SO1, including the non-essential mirror M1 for deflecting the beam FE upstream of the pinhole P1 to make the device more compact.

[0035] The light backscattered by the sample is collected by the objective lens OM, which forms a signal beam FR, which propagates in the opposite direction along the same optical path as the illumination beam to the beam splitter LS. This optical path includes a pair of oscillating mirrors MO1, which compensate for the time-varying deflection imparted to the illumination beam to scan the sample. The component of FR reflected by the splitter LS is lost, while the component that passes through it, intended to be detected, is reflected at 90° by the mirror M2 (this is not essential, only to make the device more compact). The assembly of L4, L3, MO1, L2, LS, M2 forms a second optical system, namely the collection optical system SO2. It will be seen that SO2 partially coincides with SO1, since the microscope is operated in reflection mode.

[0036] Lens L2 has two functions: to collimate the illumination beam, which diverges after being converged by L1 and spatially filtered by P1, and to converge the signal beam FR emerging collimated from the objective OM and the afocal system L3, L4. A second pinhole P2 is provided in the focal plane PF1 of lens L2, where the signal beam FR is focused. Unlike pinhole P1, P2 is an important feature of the invention, and its size in diameter (or, more generally, in its maximum lateral dimension if it is not circular) has a significant impact on the performance of the microscope. This is explained in more detail below with reference to [Fig. 2].

[0037] It is important to emphasize that Fig. 1 is not to scale. In reality, the distance between the system of oscillating mirrors MO1 and L3 must be equal to the distance between L3 and the focal plane PF1. Furthermore, lenses L2 and L5 form a second afocal system, which in the embodiment of Fig. 1 has unit magnification.

[0038] The beam FR diverges after passing through the pinhole P2, so it is collimated by a converging lens L5 (focal length 200 mm) and directed towards a second system of two oscillating mirrors, for example of the galvanometer type (for simplicity only one MO2 is shown). This second system of oscillating mirrors imparts a time-varying deflection to the beam FR, thereby performing a two-dimensional scan which is the origin of the photon relocation. This deflection is synchronous with the deflection imparted to the beam FR and its amplitude is α 1 times the amplitude of the deflection imparted by MO1. 1 Larger amplitude α 2 has.

[0039] More precisely, the deflection imparted by the second system of oscillating mirrors MO2 is greater than that imparted by the first system of oscillating mirrors MO1, ideally by a factor equal to 2 (more generally between 1.8 and 2.2).

[0040] More generally, the magnification of the afocal systems L2-L5 may have a value G other than 1, in which case the cross-sectional area of ​​the beam FR when it strikes MO2 is larger by a factor M than the cross-sectional area of ​​the same beam when it strikes MO1. In this case, it is the product Mα 2 This means that α 1 It must be larger, ideally twice as large.

[0041] The beam deflected by the system of oscillating mirrors MO2 is focused in a second focal plane PF2 by a converging lens L6 (focal length 200 mm).

[0042] The assembly L5, MO2 and L6 form a third optical system, namely the photon rearrangement optical system SO3.

[0043] The matrix image sensor CMI is located in the focal plane PF2 of the lens L6. Its integration time is greater than or equal to half the scan time of the system of oscillating mirrors MO2, which makes it possible to calculate the integral of equation (3) by analogy. In variants, the integration time can be shorter and the image acquisition speed faster, but in this case a digital integration must be performed after acquisition.

[0044] [Figure 2] shows the dependence of the specific performance criteria of the microscope of [Figure 1] (although the validity of the results is more general) on the diameter of the pinhole P2, so that these can be compared with those obtained by other confocal microscopy techniques. The diameter of P2 is expressed in Airy units AU. The Airy unit is the diameter of the Airy spot of the microscope,

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[0045] The curves CRR, CFR, and CF plot the lateral resolution of the microscope of [Figure 1], an incoherent confocal photon relocation microscope (e.g., of the fluorescent type), and a conventional confocal microscope, respectively. More precisely, these curves show how the parameter "x" depends on the pinhole diameter, where "x" is the lateral resolution.

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[0046] Curve RF shows the variation of background rejection as a function of pinhole diameter (this variation is the same for all three methods described above). Between 1 and 2 AU, the background rejection decreases, then between 2 and 4 AU it stabilizes at a value about 30% lower than that obtained for a diameter of 1 AU, and drops off sharply above 4 AU (not shown). The slight increase between 3 and 4 AU may be an artifact.

[0047] In conclusion, for pinhole diameters of 2-4 AU, the present invention makes it possible to obtain twice the lateral resolution compared to a confocal microscope with a pinhole of size 1 AU without photon reallocation, at the expense of a significantly reduced background rejection.

[0048] The improvement in lateral resolution provided by the present invention has been demonstrated experimentally.

[0049] [Figure 3] shows images of the USAF Resolution Test Chart (11th group of elements) from the microscope of [Figure 1] (panels b) and d), where panel d) is an enlargement of the area enclosed by the dashed line in panel b)) and from a confocal microscope without photon relocation (panels a) and c)) using the same light source and the same objective. The former is much sharper. Panel e) shows the pattern of the test chart. The image was obtained without deconvolution.

[0050] [Figure 4] shows the images of silver nanorods (diameter 90 nm + / - 5 nm, length tens of micrometers) immersed in refractive index matching oil, acquired with the microscope of [Figure 1] (panels indicated as b) and d), where panel d) shows an enlargement of the area enclosed by the dashed line in panel b) and images acquired with a confocal microscope without photon relocation (panels a) and c) using the same light source and the same objective. The former is much sharper. Panel e) shows the profile along the dashed line extracted from panels c) and d), where the curve CRR corresponds to panel d) and allows to distinguish two nanorods, which are not resolved by the curve CR corresponding to panel c). The curve GAUSS is a Gaussian fitting of the CRR, which allows to estimate the diameter of the nanorods, the full width at half maximum of the two Gaussian distributions are 92.6 nm and 9.12 nm, which is in agreement with the expected values.

[0051] The microscope according to the invention can be used, inter alia, for the detection of viruses. [Figure 5] shows an image of a silica particle, freely diffused in an aqueous solution, with a diameter of about 100 nm and therefore comparable to a virus particle in terms of both diameter and refractive index. The image was acquired by a microscope of the type of [Figure 1], but with illumination of a wavelength of 400 nm. The various images correspond to successive times separated by 4 s. The diameter of the particle, measured in the focused image, was 105 nm.

[0052] The improvement of the axial resolution was demonstrated by numerical simulations. Panels a) and b) of FIG. 6 show two images of two overlapping balls with submicron dimensions. In the case of conventional confocal imaging (panel a), the images of the two balls are not resolved, whereas the method of the present invention allows them to be distinguished with a pinhole diameter of 3 AU (illumination wavelength: 455 nm, objective numerical aperture 1.3). Panel c) of FIG. 6 shows a graph of the intensity measured along the z-axis passing through the centers of the two balls (curve CF ax is the curve CRR for conventional confocal microscopes ax(c) relates to the method of the invention. This confirms that what is seen in panel b) is indeed an improvement in resolution and not an aliasing effect. Panels d) and e) show images of axially oriented periodic structures, where the periodicity is visible in the image of panel e) obtained by the method of the invention but not in the image of panel d) corresponding to conventional confocal microscopy.

[0053] [Figure 7] shows the dependence of the axial resolution on the pinhole diameter for a conventional confocal microscope (light line "confocal") and for the method according to the invention (dark line "rescan"). As can be seen, for the present invention the axial resolution is not so dependent on the pinhole diameter, but is slightly improved for diameters larger than 2 AU. Moreover, for pinhole diameters larger than or equal to 1 AU, the present invention makes it possible to achieve an axial resolution significantly higher than that of a conventional confocal microscope.

[0054] The field of view of the microscope of FIG. 1 is limited or requires long acquisition times because it uses one focused beam. FIG. 8 shows an alternative embodiment that makes it possible to overcome this limitation through large-scale parallelization. In this configuration, a matrix array of microlenses is used to create a series of source points. This allows parallelizing the measurements in the plane of the sample, making it possible to maintain a large field of view while still increasing the speed (only a very small area between two measurement points is scanned). The image of each of the source points is filtered by a matrix array of filter holes. To increase the lateral resolution by a factor of 2, each of the points is rescanned by a second matrix array of microlenses with a size reduced by a factor of 2. This scheme allows a resolution of at least 50×50 μm by parallelizing 50×50 measurement points in the field of view (one confocal measurement point for every 1 μm in the sample). 2 It is possible to achieve imaging approaching kHz over a field of view of 100 nm with lateral resolution better than 100 nm while remaining in the visible or near-ultraviolet wavelength imaging range.

[0055] More precisely, the device of [Fig. 8] comprises a light source SL' (for example, but not limited to, a laser) equipped with a first matrix array of microlenses RML1 generating a number of focused illumination beams FE1, FE2, FE3 (only three are shown, but typically it is a matter of a two-dimensional matrix array of several hundred beams). These beams pass through a beam splitter LS', then diverge and are refocused by a focusing lens L10 on the front face FAV of a double-reflective oscillating mirror MOD, which in the case of an application to the detection of viruses can be for example a matter of a resonant mirror, increasing the scanning speed to "freeze" the movement of the particles in the suspension. Scanning in the other direction is performed slower, making the focal spot follow a "serpentine" path, which can then be acquired by a galvanometer mirror (not shown), also double-reflective.

[0056] The beam reflected by the oscillating mirror is focused by the microscope objective OM (apochromatic objective with magnification 60x and numerical aperture NA=1.3, immersed in silicone oil) onto the specimen E' (a drop of aqueous solution containing the virus particles PV in suspension, deposited on a microscope slide), so that a focal spot, the diameter of which is limited by diffraction, scans the surface of the specimen. An afocal system consisting of focusing lenses L20, L30 ensures an optical conjugate relationship between the pupil of the objective and the oscillating mirror.

[0057] The beams FR1, FR2, FR3 backscattered by the sample E' pass through the afocal system L20, L30 in the opposite direction, are reflected by the front face FAV of the mirror MOD to compensate the scanning of the illumination beam, are refocused by the lens L10 and are reflected by the beam splitter LS'. They are then filtered by the matrix array of pinholes MP, whose function and size are similar to those of the pinhole P2 in [Fig. 1]. Two converging lenses L60, L50 and two mirrors M10, M20 (optional), which form the afocal system, allow the beams from the matrix array of pinholes MP to be directed to a second array of converging microlenses RML2, which converges the beams, thereby reducing the size of each focal spot by a factor of 2. After passing through another converging lens L40, the beams FR1 to FR3 are reflected by the rear face FAR of the resonator mirror MOD for the purpose of photon relocation, and by the rear face of a galvanometer mirror (not shown). The combined effect of the afocal system L60, L50, the lens L40, and the array of microlenses RML2 is to double the cross-sectional area of ​​each signal beam FR impinging on the resonant mirror MOD. This is necessary because the amplitude of the rescan is necessarily equal to the amplitude of the scan of the illumination beam.

[0058] A final focusing lens L70 focuses the signal beam onto the matrix image sensor CMI. The function of the array of microlenses RML2 is to halve the cross-sectional area of ​​the focal point of the signal beam.

[0059] The embodiments of [Fig. 1] and [Fig. 8] realize photon relocation through purely optical means by scanning (i.e. "rescanning") the signal beam with an appropriate amplitude in synchronization with the scanning of the illumination light. However, it is also possible to realize photon relocation by digital processing. In this case, a camera CMI with an imaging rate synchronized with the oscillating mirror MO1 is typically placed at the same level as the pinhole P2, which may be integrated in the camera itself. The digital processing is carried out by a processor PNI, described for example in (Mueller 2010), which receives as input the images acquired by the camera CMI. The third optical system SO3 may be omitted. A microscope according to this embodiment is shown in [Fig. 9]. Photon relocation can also be realized digitally in the parallel architecture of [Fig. 8].

[0060] The present invention has been described with respect to two specific embodiments, but is not limited thereto.

[0061] For example, this can be adapted for a transmitted light confocal microscope, where two microscope objectives are needed: one to illuminate a point on the sample and the other to collect the transmitted light. Furthermore, the first and second optical systems must be completely separate, which requires more components.

[0062] Typically, scanning of the sample with a focused light beam and scanning for photon relocation are two-dimensional, however in some cases one-dimensional scanning may be sufficient, thereby reducing the number of oscillating mirrors.

[0063] Optical filtering by lens L1 and pinhole P1 or by matrix array of microlenses RML1 and matrix array of pinholes MP is not strictly necessary if the illumination has sufficient spatial coherence (Strehl ratio > 80%); in the case of parallelized systems, such as that of [Fig. 8], it must be understood that the coherence is on the scale of each elementary beam, and no overall coherence is necessary. Similarly, the optical relays L3-L4 and L20-L30 are not essential in principle, their presence is generally necessary to ensure that the oscillating mirror MO1 or MOD is optically conjugate with the objective pupil, otherwise, at large deflection angles, the objective pupil will not be sufficiently illuminated, thereby reducing the numerical aperture and therefore the spatial resolution. Similarly, the use of a laser as an illumination source is not essential.

[0064] At least some of the lenses may be diverging rather than converging and / or may be replaced by other focusing or defocusing means, for example concave or convex mirrors.

[0065] The beam splitter LS could essentially be replaced by a splitter cube, but this would suffer from the spurious reflections introduced by that component.

[0066] More generally, optical configurations other than those shown in Figures 1 and 8 may be used to fabricate the various constituent optical systems of a microscope according to the invention. Furthermore, in the description of the figures, the dimensions of the optical elements are given by way of example only.

[0067] The illumination wavelength can be any wavelength, but the use of green (495-570 nm), blue (450-495 nm), or violet (380-450 nm), or even near-ultraviolet (300-380 nm) light is preferred, as it allows for lateral spatial resolution on the order of 100 nm or less while avoiding the technical problems associated with shorter wavelengths. Similarly, choosing an immersion microscope objective with a high numerical aperture (1 or greater) can maximize spatial resolution, but is not in itself essential.

[0068] Finally, detection and identification of virus particles is just one example of an application of parallelized microscopy of the type shown in [Figure 8].

[0069] References (Sheppard 1988): CJR Sheppard, “Super-resolution in Confocal Imaging”, Optik 80, No. 2, pages 53,54. (York 2013): AGYork et al. “Instant super-resolution imaging in live cells and embryos via analog image processing”, Nat. Methods 2013, November, 10(11), pages 1122-1126. (De Luca 2013):GMRDe Luca “Re-scan confocal microscopy:scanning twice for better resolution”Biomedical Optics Express,Vol.4,No.11,November 2013. (Curd 2015): A. Curd et al. “Construction of an instant structured illumination microscope”, Methods 88 (2015) pages 37-47. (Roth 2017):S.Roth“Development of a new microscopy method:Optical Photon Reassignment Microscopy”,doctoral thesis,Friedrich-Schiller University,Jena (DE),March 29,2017. (DuBose 2019):T.B.DuBose et al.“Super-resolution retinal imaging using optically reassigned scanning laser ophthalmology”,Nature Photonics,Vol.13,April 2019,pages 257-262. (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.19,1st July 1995,pages 3576-3588. (Mueller 2010):C.B.Mueller,J.Enderlein“Image Scanning Microscopy”,PRL 104,198101 (2010).

Claims

1. a light source (SL) configured to generate at least one spatially coherent illumination light beam (FE) of an illumination wavelength; a first optical system (SO1) configured to apply an angular scan to the illumination light beam; at least one microscope objective (OM) configured to receive as input the illumination light beam emerging from the first optical system, to focus it onto a sample (E), and to collect and collimate a light beam (FR) elastically scattered by the sample, called a signal beam; a second optical system (SO2), which may coincide in whole or in part with the first optical system, and which is configured to receive as input the signal beam collimated by the microscope objective lens, apply thereto an angular scan opposite to that applied to the illumination light beam, and focus it onto a first focal plane (PF1); A pinhole (P2) located in the first focal plane; a matrix image sensor (CMI) disposed in a second focal plane (PF2); a photon redistribution means (SO3, CNI) interacting with said matrix image sensor (CMI) to reconstruct an image of said sample; In a scanning confocal photon relocation microscope comprising: the microscope objective lens and the second optical system are configured to focus the signal beam at the illumination wavelength onto the matrix image sensor; The pinhole has a diameter or maximum lateral dimension of 2 to 4 air units. A scanning confocal photon relocation microscope comprising:

2. 2. The scanning confocal microscope of claim 1, wherein at least the second optical system includes a beam splitter (LS) for splitting the signal beam from the illumination light beam.

3. A scanning confocal microscope according to any one of claims 1 to 2, wherein the light source (SL) is configured to emit a blue, violet or near-ultraviolet illumination light beam.

4. The scanning confocal microscope according to any one of claims 1 to 3, wherein the light source (SL) is a laser.

5. A scanning confocal microscope according to any one of claims 1 to 4, wherein the microscope objective (OM) is an immersion objective having a numerical aperture greater than or equal to 1.

6. said photon relocation means comprising a third optical system (SO3) configured to collect the signal beam passing through the pinhole, collimate it and apply to it an angular scan synchronized with that applied to the illumination light beam, the product of its amplitude and the cross-sectional area of ​​the collimated light beam in the third optical system being greater than the product of the amplitude of the scan applied by the second optical system and the cross-sectional area of ​​the collimated light beam in the second optical system, and to focus it in a second focal plane, A scanning confocal microscope as described in any one of claims 1 to 5, wherein an assembly consisting of the microscope objective lens, the second optical system, and the third optical system is configured to focus the signal beam of the illumination wavelength onto a matrix image sensor.

7. The scanning confocal microscope of claim 6, wherein the third optical system is configured to apply an angular scan to the signal beam such that the amplitude of the scan multiplied by the cross-sectional area of ​​the beam is 1.8 to 2.2 times the product of the amplitude of the angular scan applied to the illumination light beam by the first optical system and the cross-sectional area of ​​the beam.

8. configured to operate in a reflective mode; the first optical system (SO1) comprises a first lens (L1) for converging the illumination light beam, a pinhole (P1) arranged in the focal plane of the first lens for spatial filtering of the illumination light beam, a beam splitter (LS) for reflecting a part of the beam, a second lens (L2) for collimating the part of the illumination beam, a first oscillating mirror (MO1) or a system of oscillating mirrors to which the angular scan is applied, and an afocal system comprising a third lens (L3) and a fourth lens (L4), the second optical system comprises the afocal system (L3, L4), the first oscillating mirror (MO1) or a system of oscillating mirrors, the second lens (L2) and the beam splitter (LS) configured to transmit the part of the signal beam backscattered by the sample, said third optical system comprises a fifth lens (L5) collimating the signal beam passed through the pinhole, a second oscillating mirror (MO2) or a system of oscillating mirrors applying to it the angular scan synchronous with that applied to the illumination light beam, and a sixth lens (L6) focusing it on the second focal plane; A scanning confocal microscope according to any one of claims 6 and 7.

9. A scanning confocal microscope as described in any one of claims 1 to 8, wherein the light source (SL') is configured to generate a plurality of the illumination light beams (FE1, FE2, FE3) in parallel, which propagate through the first optical system, and the microscope objective lens collects a plurality of respective signal beams (FR1, FR2, FR3), which then propagate along the second optical system and further includes a matrix array (MP) of pinholes, one for each of the backscattered light beams, arranged in the second focal plane.

10. configured to operate in a reflective mode; the light source (SL') includes a first array (RML1) of microlenses for generating and focusing the plurality of illumination light beams; the scanning confocal microscope includes at least one oscillating mirror (MOD) having a reflective front surface (FAV) and a reflective back surface (FAR), the front surface forming part of the first and second optical systems and the back surface forming part of the third optical system; the third optical system includes a second array of microlenses (RML2) that increases a cross-sectional area of ​​the backscattered light beam incident on the rear surface of the oscillating mirror by a factor of 1.8 to 2.2; A scanning confocal microscope according to claim 9 when dependent on any one of claims 6 to 8.

11. Use of a scanning confocal microscope according to any one of claims 1 to 10 for observing virus particles (PV) in a suspension.

12. In the observation method of the sample (E), generating at least one spatially coherent and collimated illumination light beam (FE) at an illumination wavelength; applying an angle scan thereto; focusing it on said sample (E) by a microscope objective (OM); collecting, by said or another microscope objective, a beam of light (FR) elastically scattered by said sample at said illumination wavelength, called a signal beam; applying an opposite angular scan to the signal beam to that applied to the illumination light beam, and focusing it onto a first focal plane (PF1); performing spatial filtering of the signal beam by a pinhole (P2) arranged in the first focal plane, the pinhole having a diameter or maximum lateral dimension of 2 to 4 airy units; collecting the signal beam that has passed through the pinhole, collimating it and applying to it an angular scan synchronous with that applied to the illumination light beam such that the product of its amplitude and the cross-sectional area of ​​the signal beam is greater than the product of the amplitude and the diameter of the angular scan applied to the illumination light beam, and focusing it on a second focal plane (PF2); detecting the signal beam by a matrix image sensor disposed in the second focal plane; The method includes:

13. In the observation method of the sample (E), generating at least one spatially coherent and collimated illumination light beam (FE) at an illumination wavelength; applying an angle scan thereto; focusing it on said sample (E) by a microscope objective (OM); collecting, by said or another microscope objective, a beam of light (FR) elastically scattered by said sample at said illumination wavelength, called a signal beam; applying an opposite angular scan to the signal beam to that applied to the illumination light beam, and focusing it onto a first focal plane (PF1); performing spatial filtering of the signal beam by a pinhole (P2) arranged in the first focal plane, the pinhole having a diameter or maximum lateral dimension of 2 to 4 airy units; detecting the signal beam passing through the pinhole by a matrix image sensor (CMI) disposed in the second focal plane, the imaging rate of which is synchronized with the angular scanning of the illumination light beam; applying a digital light remapping process to the image captured by the matrix image sensor; The method includes:

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