Photon redistribution confocal microscope

Abstract

This invention relates to a scanning confocal photon redistribution microscope configured to operate in coherent (reflection or transmission) imaging mode and having a confocal pinhole with a diameter between 2 and 4 Airy units. The invention also relates to using such a microscope to observe suspended viral particles.

Description

Technical Field

[0001] This invention relates to the field of optical microscopy methods. Background Technology

[0002] Optical microscopy plays a crucial role in biology because it enables high-rate observation of living samples, unlike methods such as electron microscopy, which require complex preparation procedures and are incompatible with preserving life. However, its resolving power is inevitably limited by light diffraction. According to Abbe's theory, for a conventional optical microscope, the maximum resolution d is given by the following equation.

[0003]

[0004] Where λ is the wavelength of the light used (between 380 nm and 780 nm for visible light), and NA is the numerical aperture, which should not easily exceed 1.4 for biological samples. The resolution under visible light cannot exceed 135 nm, as 135 nm is not suitable for observing very small structures such as viral particles.

[0005] So-called "super-resolution" techniques make it possible to exceed the Abbe resolution limit by utilizing fluorescent labeling and / or nonlinear effects. These techniques are complex to implement.

[0006] Furthermore, since biological objects are inherently three-dimensional, high spatial resolution in the axial direction is also required (the Abbe limit refers to lateral resolution in a plane perpendicular to the optical axis). Confocal microscopy methods make it possible to obtain images with very shallow depth of field (on the order of hundreds of nanometers), and thus "section" the sample from a perspective that accesses its three-dimensional structure. This technique is often associated with the use of fluorescent labeling, but it can also be used for reflectivity without labeling.

[0007] Confocal microscopy uses a point illumination source, the image of which is projected onto the sample being observed through the objective lens. Light from the sample (backscattered light in the case of reflective confocal microscopy; fluorescence emission when using fluorescent labeling) is focused onto a pinhole optically conjugate to the point source and then detected, for example, by a photomultiplier tube. The pinhole serves to suppress radiation not originating from the focal plane of the objective lens, thus achieving optical sectioning. Images of the sample are obtained point-by-point by scanning. More precisely, two-dimensional scanning in two directions perpendicular to the optical axis allows for obtaining images of a slice of the sample centered on the focal plane of the objective lens. By adding an axial scan of this focal plane, a three-dimensional image is obtained.

[0008] As the pinhole diameter decreases, the slice thickness decreases, and therefore the axial resolution improves, but the gain drops when the decrease exceeds 1 Airy unit (AU). An Airy unit is the diameter of the Airy disk in a microscope and is equal to...

[0009] Using a pinhole with a diameter of less than 1 AU, confocal microscopy methods theoretically allow for a gain of up to 30% in lateral resolution relative to the Abbe limit. However, this is achieved at the cost of a reduced signal-to-noise ratio.

[0010] The photon redistribution technique, first proposed by Sheppard in 1988, can, in principle, increase the lateral resolution of confocal microscopy by up to two times. The idea behind this technique is to replace photomultiplier tubes, or more generally, point radiation detectors, with matrix detectors that allow for the acquisition of a primitive image for each acquisition point. The image size is then adjusted digitally or optically (ideally reduced to half its original size) before moving to the next scan point. The final image is obtained by integrating the successively acquired scans, each scan having moved one scan step relative to the previous one.

[0011] The purely optical implementation of photon redistribution techniques has been described in (York 2013), (De Luca 2013), (Curd 2015), and (Roth 2017). It involves applying a first angular scan to the illumination beam, performing a reverse angular scan to the beam from the sample, and then applying a second angular scan to the same beam from the sample synchronously with the first scan. The magnitude of the second angular scan, normalized relative to the beam cross-sectional area, is larger than that of the first scan, ideally twice as large. "Normalized relative to the beam cross-sectional area" means that if α is the ratio between the magnitude of the second angular scan and the magnitude of the first angular scan, and M is the ratio between the cross-sectional area of ​​the beam from the sample and the cross-sectional area of ​​the illumination beam, then the value of α / M must be greater than 1, and ideally equal to 2.

[0012] Similar to conventional confocal microscopy, acquisition can be performed using a matrix array of pinholes and a matrix array of microlenses in parallelization—for example, see the aforementioned article (York 2013).

[0013] Photon redistribution has been specifically applied to confocal fluorescence microscopy methods, as seen in the aforementioned articles (York 2013), (De Luca 2013), and (Curd 2015), where a 1.5-fold gain in lateral resolution was observed. To the inventors' knowledge, the only application of photon redistribution in reflection to date is described in (DuBose 2019). However, in this paper, this is not a problem of microscopy, but of ophthalmoscopy, in which the objective lens is replaced by a crystalline lens from the patient's eye, and therefore by a lens with a low numerical aperture, and thus the lateral resolution is on the order of tens of micrometers. Summary of the Invention

[0014] This invention aims to provide a scanning confocal microscope with improved resolution in three dimensions (lateral and axial). According to the invention, this is achieved by using a confocal pinhole in coherent imaging mode with a diameter (or more generally, the maximum lateral dimension) between 2 and 4 Airy units, ideally equal to 3 Airy units. The inventors have recognized that in coherent imaging mode, for a diameter of 3 Airy units, the lateral resolution increases with increasing pinhole diameter, reaching twice the Abbe limit. Conversely, in fluorescence operating mode, the lateral resolution is independent of the pinhole diameter. Coherent imaging means that the detected photons are illumination photons that have undergone elastic scattering only by the optical system of the sample and the microscope; for example, this could be a problem of reflection imaging (considered in detail below) or transmission imaging. In contrast, fluorescence imaging or Raman scattering imaging is considered incoherent because the illumination photons undergo inelastic scattering.

[0015] Using a pinhole with a diameter of 3 Airy units can further optimize background suppression (however, the background is reduced by about 30% compared to a confocal microscope with no photon redistribution and a pinhole of 1 AU). The concept of “background suppression” is precisely defined in (Sandison 1995).

[0016] Furthermore, the inventors discovered that under these conditions, the axial resolution increased by 1.5 times compared to that of a conventional confocal microscope.

[0017] Finally, using illumination at a wavelength of 445 nm and an immersion objective with NA = 1.3, (86 x 86 x 248) nm can be obtained. 3 The spatial resolution is excellent. Furthermore, it eliminates the need for fluorescent labeling, making the technology simpler and more widely applicable.

[0018] Therefore, one subject of the present invention is a scanning confocal photon redistribution microscope, comprising:

[0019] The light source is configured to generate at least one spatially coherent illumination beam at the illumination wavelength;

[0020] A first optical system is configured to perform an angular scan on the illumination beam;

[0021] At least one microscope objective is configured to: receive the illumination beam output from the first optical system as input and focus it onto the sample; and collect and collimate a beam referred to as a signal beam that is elastically scattered by the sample;

[0022] A second optical system, which is wholly or partially consistent with the first optical system, is configured to receive the signal beam collimated by the microscope objective as input, apply an angular scan to the signal beam opposite to the angular scan applied to the illumination beam, and focus the signal beam in a first focal plane.

[0023] The pinhole is located in the first focal plane;

[0024] A matrix image sensor is arranged in the second focal plane; and

[0025] A photon redistribution component interacts with the matrix image sensor to reconstruct an image of the sample;

[0026] The feature is that: the microscope objective and the second optical system are configured to focus the signal beam at the illumination wavelength onto the matrix image sensor; and

[0027] The characteristic feature is that the diameter or maximum lateral dimension of the pinhole is between 2 and 4 air units.

[0028] According to a specific embodiment of this scanning confocal microscope:

[0029] - At least the second optical system may include: a beam splitter for separating the signal beam from the illumination beam.

[0030] - The light source can be configured to emit blue, violet, or near-ultraviolet illumination beams.

[0031] - The light source can be a laser.

[0032] - The microscope objective may be an immersion objective with a numerical aperture greater than or equal to 1.

[0033] The photon redistribution component may include a third optical system configured to collect the signal beam passing through the pinhole, collimate the signal beam, apply an angle scan to the signal beam synchronized with the angle scan applied to the illumination beam, and such that the product of the amplitude of the signal beam and the cross-sectional area of ​​the beam collimated by the third optical system is greater than the product of the amplitude of the scan applied by the second optical system and the cross-sectional area of ​​the beam collimated by the second optical system, and focus the signal beam in a second focal plane; the assembly including the microscope objective, the second optical system, and the third optical system is configured to focus the signal beam at the illumination wavelength onto the matrix image sensor.

[0034] - The third optical system can be configured to apply an angular scan to the signal beam such that the product of the scan amplitude and 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 beam by the first optical system and the cross-sectional area of ​​the beam.

[0035] The scanning confocal microscope can be configured to operate in reflection mode, and:

[0036] - The first optical system may include a first lens for focusing the illumination beam, a pinhole disposed in the focal plane of the first lens for performing spatial filtering of the illumination beam, a beam splitter for reflecting a portion of the beam, a second lens for collimating the portion of the illumination beam, a first oscillating mirror or oscillating mirror system for applying the angular scan to the illumination beam, and a focusless system including a third lens and a fourth lens.

[0037] - The second optical system may include the afocal system, the first oscillating mirror or oscillating mirror system, the second lens, and the beam splitter, the beam splitter being configured to transmit a portion of the signal beam backscattered by the sample; and

[0038] The third optical system may include a fifth lens for collimating the signal beam passing through the pinhole, a second oscillating mirror or oscillating mirror system for applying an angle scan to the signal beam that is synchronized with the angle scan applied to the illumination beam, and a sixth lens for focusing the signal beam in the second focal plane.

[0039] The light source can be configured to generate multiple illumination beams in parallel, which propagate through the first optical system, thereby the microscope objective collects multiple corresponding signal beams, which then propagate along the second optical system, and the scanning confocal microscope further includes a matrix array of pinholes arranged in the second focal plane, with each backscattered beam using one pinhole.

[0040] In this case, the scanning confocal microscope can be configured to operate in reflection mode, and:

[0041] - The light source may include a first array of microlenses for generating and focusing the plurality of illumination beams;

[0042] The scanning confocal microscope may include at least one oscillating mirror having a front reflective surface and a rear reflective surface, the front surface forming a portion of the first optical system and the second optical system, and the rear surface forming a portion of the third optical system; and

[0043] - The third optical system may include a second array of microlenses for increasing the cross-sectional area of ​​the backscattered beam incident on the rear surface of the oscillating mirror by 1.8 to 2.2 times.

[0044] Another subject of this invention is the use of this scanning confocal microscope for observing suspended viral particles.

[0045] Another subject of the present invention is a method for observing a sample, comprising the following steps:

[0046] - Generate at least one spatially coherent and collimated illumination beam at the illumination wavelength;

[0047] - Apply an angle scan to the illumination beam;

[0048] - The illumination beam is focused onto the sample through a microscope objective;

[0049] - A beam of light, referred to as a signal beam, that is elastically scattered by the sample at the illumination wavelength is collected through the microscope objective or another microscope objective;

[0050] - Apply an angular scan to the signal beam that is opposite to the angular scan applied to the illumination beam, and focus the signal beam in the first focal plane;

[0051] - Spatial filtering of the signal beam is performed by means of pinholes arranged in the first focal plane, the diameter or maximum lateral dimension of which is between 2 and 4 Airy units;

[0052] - Collect the signal beam passing through the pinhole, collimate the signal beam, and apply an angle scan to the signal beam synchronized with the angle scan applied to the illumination beam, such that the product of the amplitude of the signal beam and the cross-sectional area of ​​the signal beam is greater than the product of the amplitude of the angle scan applied to the illumination beam and the diameter of the illumination beam, and focus the signal beam in the second focal plane; and

[0053] - The signal beam is detected by a matrix image sensor arranged in the second focal plane.

[0054] Another subject of the present invention is a method for observing a sample, comprising the following steps:

[0055] Generate at least one spatially coherent and collimated illumination beam (FE) at the illumination wavelength;

[0056] An angle scan is applied to the illumination beam;

[0057] The illumination beam is focused onto the sample using a microscope objective lens;

[0058] A beam of light, referred to as a signal beam, that is elastically scattered by the sample at the illumination wavelength is collected through the microscope objective or another microscope objective.

[0059] An angular scan opposite to the angular scan applied to the illumination beam is applied to the signal beam, and the signal beam is focused in the first focal plane;

[0060] Spatial filtering of the signal beam is performed by means of pinholes arranged in the first focal plane, wherein the diameter or maximum lateral dimension of the pinholes is between 2 and 4 Airy units.

[0061] The signal beam passing through the pinhole is detected by a matrix image sensor arranged in the second focal plane, the acquisition rate of the matrix image sensor being synchronized with the angular scanning of the illumination beam; and

[0062] Digital photon redistribution processing is applied to the images acquired by the matrix image sensor. Attached Figure Description

[0063] Other features, details, and advantages of the invention will become apparent upon reading the description given with reference to the accompanying drawings, which are illustrated by way of example and show, respectively:

[0064] [ Figure 1 [Image of a confocal photon redistribution microscope according to a first embodiment of the present invention;]

[0065] [ Figure 2 ], showing [ Figure 1 The graph shows how the performance of a confocal microscope depends on the diameter of the confocal pinhole.

[0066] [ Figure 3 ],

[0067] [ Figure 4 ]and

[0068] [ Figure 5 Experimental results demonstrating the technical effectiveness of the present invention;

[0069] [ Figure 6 ]and[ Figure 7 The results of numerical simulations demonstrate another technical effect of the present invention;

[0070] [ Figure 8 A diagram of a confocal photon redistribution microscope according to a second embodiment of the present invention; and

[0071] [ Figure 9 [Image of a confocal photon redistribution microscope according to a third embodiment of the present invention.] Detailed Implementation

[0072] As described above, photon redistribution technology involves recording an image of the sample through a pinhole when the sample is illuminated by a highly focused beam of light. The image captured through the pinhole is positioned at a specific location on the acquisition camera, where the interval between two points scanned on the sample is s and the de-magnification is M. The signal recorded by the camera... The following is given, where Represents the coordinates within the sample plane and Represents the coordinates within the detector plane:

[0073]

[0074] Among them, H e For the illumination (or excitation) extension function, H d The detection spread function is given, and U is the field scattered by the sample.

[0075] Equation (1) assumes that the sample is moved to achieve scanning, while it is more practical to keep the sample stationary and move the illumination beam. Mathematically, this corresponds to coordinates. The changes give:

[0076]

[0077] If the detector is large enough relative to the pinhole size, the latter's weighting factor can be ignored. If a reduction factor M is further introduced, it can be written... And (2) becomes:

[0078]

[0079] The system's optical transfer function It can be written as, where, Spatial frequency coordinates:

[0080]

[0081] Ce and Cd are the illumination and detection optical transfer functions, respectively. Ideally, if but Otherwise, it is 0. It should be noted that the support... It has a radius proportional to 1 / (M-1). It has been shown in Sheppard 1988 and DuBose 2019 that for M = 1 / 2, the maximum cutoff frequency of the optical transfer function is its maximum value k. max=2k0, which corresponds to a magnification of 2x, resulting in double the lateral resolution compared to conventional confocal microscopy methods. This magnification can be achieved by rescanning the amplitude, which is equal to twice the amplitude required to compensate for the illumination point scanning the sample. As a variant, the rescanning amplitude can be equal to the amplitude required to compensate for the illumination point scanning the sample, with the magnification provided by the lens system. See below for reference. Figure 2 Intermediate solutions are also possible, as discussed; more generally, it is important that the amplitude of the rescan divided by the product of the amplitude of the illumination scan and the optical magnification is approximately equal to 2.

[0082] Generally, a confocal photon redistribution microscope according to the present invention comprises: a light source; a first optical system interacting with said light source; an objective lens for scanning the surface of a sample with a focused beam; a second optical system for collecting and focusing light backscattered or transmitted by the sample (to compensate for angular deflection introduced for performing the scan); a pinhole in the focal plane of the second optical system for performing confocal filtering; and a third optical system for applying a “re-scan” to the beam from the pinhole and focusing it onto a camera. The various optical systems may be partially identical, which makes it possible to limit the number of optical components.

[0083] [ Figure 1 The image shows an optical diagram of a confocal photon redistribution microscope according to a first embodiment of the present invention.

[0084] The light source SL is a laser that emits a beam FE (illumination beam) with a wavelength of λ = 445 nm. The beam FE is focused by a first converging lens L1 (focal length 200 mm) and is spatially purified by a first pinhole P1 with a diameter of 50 μm located in the focal plane of the lens.

[0085] The spatially filtered illumination beam FE is reflected by a beam splitter LS (50% transmission - 50% reflection) and collimated by a second converging lens L2 (focal length 200 mm). A first system of two oscillating mirrors (only one is shown for simplicity, MO1), such as galvanometer-type mirrors, imparts a time-varying deflection to the beam FE to perform a two-dimensional scan. This deflected beam FE is focused onto the sample E by a microscope objective OM (an apochromatic objective immersed in silicone oil, magnification 60x, numerical aperture NA = 1.3), causing a diffraction-limited focal spot to scan the sample surface. If the sample is transparent or translucent, the focal spot can be located below the surface at a depth that can be changed by axially moving the objective or the sample, allowing for the acquisition of a three-dimensional image via tomography.

[0086] The afocal system consisting of converging lenses L3 and L4 ensures an optical conjugate relationship between the pupil of the objective lens and the midpoint of the two oscillating mirrors (or a single oscillating mirror MO1); typically, the distance between the two oscillating mirrors is negligible relative to the focal length of L3. All optical components included between lens L1 and objective lens OM—including the non-essential mirror M1 that deflects the beam FE upstream of pinhole P1 to make the device more compact—form the first optical system or illumination optical system SO1.

[0087] The backscattered light from the sample is collected by the objective lens OM, forming a signal beam FR. The signal beam FR propagates in the opposite direction to the illumination beam along the same optical path as the beam splitter LS. This optical path includes the pair of oscillating mirrors MO1, which compensate for the time-varying deflection of the illumination beam as it scans the sample. The component of FR reflected by the beam splitter LS is lost, while the component passing through the beam splitter LS is detected and reflected 90° by mirror M2 (mirror M2 is not essential and is only used to make the setup more compact). Components L4, L3, MO1, L2, LS, and M2 form a second optical system or collecting optical system SO2. Notably, SO2 partially coincides with SO1 because the microscope operates on a reflective basis.

[0088] Lens L2 has a dual function: collimating the illumination beam focused by L1 and spatially filtered by P1 before diverging, and focusing the signal beam FR collimated from the objective lens OM and the afocal systems L3 and L4. A second pinhole P2 is placed in the focal plane PF1 of lens L2, where the signal beam FR is focused. Unlike pinhole P1, P2 is an essential feature of this invention, and its diameter (or more generally, its maximum lateral dimension if it is not circular) significantly affects the microscope's performance. Reference will be made below to […]. Figure 2 This will be discussed in detail.

[0089] The key point to emphasize is that Figure 1 It is not scaled proportionally. In fact, the distance between the oscillating mirror system MO1 and L3 must be equal to the distance between L3 and the focal plane PF1. Furthermore, lenses L2 and L5 are formed in […]. Figure 1 The embodiment of the second afocal system has a magnification of 1 (unit).

[0090] Since the beam FR diverges as it passes through pinhole P2, it is collimated by converging lens L5 (focal length 200mm) and guided toward a second system of two oscillating mirrors (only one MO2 is shown for simplicity), such as a galvanometer type. This second system of oscillating mirrors applies a time-varying deflection to the beam FR to perform a two-dimensional scan as the starting point for photon redistribution. This deflection is synchronized with the deflection applied to the beam FR, and its amplitude α2 is greater than the amplitude α1 of the deflection applied by MO1.

[0091] More precisely, the deflection exerted by the second system of oscillating mirror MO2 is ideally twice as large as the deflection exerted by the first system of oscillating mirror MO1 (or more generally between 1.8 and 2.2).

[0092] More generally, the magnification of the afocal system L2–L5 can have a value G other than 1. In this case, the cross-sectional area of ​​beam FR when it strikes MO2 is larger than that of the same beam when it strikes MO1, and by a factor of M. In this case, the product Mα2 must be greater than α1, and ideally twice α1.

[0093] The beam deflected by the system of the oscillating mirror MO2 is focused by the converging lens L6 (focal length 200mm) in the second focal plane PF2.

[0094] Components L5, MO2, and L6 form a third optical system or a photon redistribution optical system SO3.

[0095] The matrix image sensor CMI is positioned in the focal plane PF2 of lens L6. Its integration time is greater than or equal to half the scanning cycle of the system of oscillating mirror MO2, which makes it possible to calculate the integral of equation (3) similarly. As a variant, the integration time can be shorter and the image acquisition rate can be higher, but in this case, digital integration needs to be performed after acquisition.

[0096] [ Figure 2 ] showed [ Figure 1 The dependence of certain performance standards of the microscope (but the validity of the results is more general) on the diameter of the pinhole P2 is described, and this allows for comparison with those obtained through other confocal microscopy methods and techniques. The diameter of P2 is expressed in Airy units (AU). As mentioned above, an Airy unit is the diameter of the Airy disk in a microscope and is equal to... NA is the numerical aperture of the objective lens OM, and λ is the wavelength under discussion.

[0097] The curves CRR, CFR, and CF were plotted respectively. Figure 1 The lateral resolution of [the microscope], incoherent confocal photon redistribution microscopy (e.g., fluorescence-based microscopy), and conventional confocal microscopy is compared. More precisely, the curves show how the parameter "x" depends on the diameter of the pinhole, where "x" is the value that makes the lateral resolution [variable from] [the pinhole diameter]. Provided. Note:

[0098] - In the case of conventional confocal microscopy (curve CF), x increases as the diameter of the pinhole decreases; therefore, a pinhole with a diameter of approximately 1 AU is usually selected.

[0099] - In the case of incoherent confocal photon redistribution microscopy (curve CFR), the lateral resolution is independent of the pinhole diameter and is better than that of conventional confocal microscopy, approximately 1.5 times the resolution of conventional confocal microscopy.

[0100] - In the case of coherent confocal photon redistribution microscopy according to the invention (curve CRR), the lateral resolution increases with increasing pinhole diameter before stabilizing (leveling off) after the diameter reaches 3 AU. Starting from approximately 2 AU, the lateral resolution is significantly better than that in coherent microscopy methods. Under optimal conditions (3 AU), the lateral resolution is twice that of conventional confocal microscopy methods.

[0101] The RF curve illustrates the variation of background suppression with the diameter of the pinhole (the variation is the same in the three techniques considered above). Background suppression decreases between 1 and 2 AU, then stabilizes between 2 and 4 AU at a value approximately 30% lower than the value obtained with a diameter of 1 AU, and then drops sharply above 4 AU (not shown). The slight increase between 3 and 4 AU is likely an artifact.

[0102] In summary, for pinholes with diameters between 2 and 4 AU, this invention enables a doubling of lateral resolution compared to a confocal microscope with a pinhole of 1 AU size and no photon redistribution, at the cost of degradation included in background suppression.

[0103] Experiments have demonstrated the improved lateral resolution achieved using this invention.

[0104] [ Figure 3 ] shows the use of [ Figure 1 The images show photographs (panels) of a microscope (labeled b) and d), where d) is a magnified view of the area defined by the dashed line in b), and images of the USAF resolution test pattern (group 11 element) acquired using a confocal microscope (photographs a) and c) with the same light source and objectives but without photon redistribution. The former is much sharper. Photograph e) illustrates the pattern of the test pattern. Images were obtained without deconvolution.

[0105] [ Figure 4 The image shows silver nanorods (diameter: 90 nm ± 5 nm; length: tens of micrometers) immersed in refractive index-matched oil. This image was created using […]. Figure 1Photographs of the microscope (labeled b) and d) in the figure, where d) is a magnification of the area defined by the dashed line in b), and those acquired using a confocal microscope (a) and c) with the same light source and objectives but without photon redistribution. The former is much sharper. Photograph e) shows the contours extracted along the dashed line from photographs c) and d): the curve CRR corresponds to photograph d) and allows differentiation of two nanorods that are not resolved in the curve CR corresponding to photograph c). The GAUSS curves are Gaussian fits to the CRR and allow estimation of the nanorod diameters: the full widths at the half-maxima of the two Gaussian distributions are 92.6 nm and 91.2 nm, consistent with the expected values.

[0106] The microscope according to the present invention can be used to detect viruses, etc. Figure 5 The image shows silica particles with a diameter of approximately 100 nm (thus comparable to virus particles in diameter and refractive index) freely diffusing in an aqueous solution. [By [] Figure 1 Images were acquired using a microscope of the [type], but illuminated at a wavelength of 400 nm. Various images correspond to consecutive time intervals of 4 s. The diameter of the particles measured in the focused images is 105 nm.

[0107] The improvement in axial resolution was verified through numerical simulation. Figure 6 Photographs a) and b) show two images of two overlapping spheres of submicron size. While the images of the two spheres are not resolved in conventional confocal imaging (photograph a), the method of this invention allows them to be distinguished using a pinhole with a diameter equal to 3 AU (illumination wavelength: 455 nm; objective numerical aperture 1.3). Figure 6 Photo c) is a graph of the intensity measured along the z-axis passing through the centers of the two spheres (for the conventional confocal microscopy method, the curve CF). ax And the CRR curve for the method of the present invention ax This confirms that what is seen in photograph b) is indeed an improvement in resolution, and not an aliasing effect. Photographs d) and e) show images of axially oriented periodic structures; the periodicity is visible in the image of photograph e) obtained by the method of the present invention, but not in the image of photograph d) corresponding to the conventional confocal microscopy method.

[0108] [ Figure 7 The diagram illustrates the dependence of axial resolution on pinhole diameter for conventional confocal microscopy methods (faded line "confocal") and for the method according to the invention (darker line "re-scanning"). It can be seen that, in the case of the invention, the dependence of axial resolution on pinhole diameter is weak, although there is a slight improvement in axial resolution for diameters greater than 2 AU. Furthermore, for pinholes with a diameter greater than or equal to 1 AU, the invention enables significantly better axial resolution than conventional confocal microscopy methods.

[0109] [ Figure 1 The field of view of a microscope is limited—or requires a long acquisition time—because it uses a single focused beam. Figure 8 An alternative embodiment is shown that enables overcoming this limitation through massive parallelization. This setup uses a matrix array of microlenses to create a series of source points. This makes it possible to parallelize measurements within the sample plane to increase speed while maintaining a large field of view (scanning only a very small area between two measurement points). The image of each source point is filtered through a matrix array of filter apertures. To improve the lateral resolution by a factor of 2, each point is rescanned through a second matrix array of microlenses, with the rescan size reduced to half. This approach makes it possible to achieve imaging approaching kHz in a field of view of at least 50x50 μm² by parallelizing 50x50 measurement points in the field of view (one confocal measurement point per 1 μm in the sample), and also makes it possible to achieve a lateral resolution better than 100 nm while remaining within the imaging range of visible or near-ultraviolet wavelengths.

[0110] More precisely, Figure 8 The apparatus includes a light source SL' (e.g., a laser, but not necessarily a light source) equipped with a first matrix array RML1 of microlenses, which generates multiple focused illumination beams FE1, FE2, FE3 (only three are shown, but typically a two-dimensional matrix array of hundreds of beams is involved). These beams pass through a beam splitter LS' and then diverge, being refocused by a converging lens L10 onto the front FAV of an oscillating mirror MOD that reflects on both sides; for example, in the case of virus detection, this could be a problem with the resonating mirror in order to increase the scanning speed to “freeze” the movement of suspended particles. A scan in another direction is performed at a lower speed, causing the focus to follow a “serpentine” path; thus, it can be obtained by a current mirror (not shown) that also reflects on both sides.

[0111] The light beam reflected by the oscillating mirror is focused by the microscope objective OM (an apochromatic objective immersed in silicone oil, with a magnification of 60x and a numerical aperture NA = 1.3) onto the sample E' (a drop of aqueous solution containing suspended virus particles PV deposited on a microscope slide), so that the focal point (whose diameter is limited by diffraction) scans the surface of the sample. The afocal system consisting of converging lenses L20 and L30 ensures the optical conjugate relationship between the objective pupil and the oscillating mirror.

[0112] The backscattered beams FR1, FR2, and FR3 from sample E' pass through the afocal system L20 and L30 in opposite directions, are reflected by the frontal FAV of mirror MOD to compensate for the scanning of the illumination beam, are refocused by lens L10, and are reflected by beam splitter LS'. They then undergo filtering through a matrix array of pinholes MP, the pinholes having a function and size similar to […]. Figure 1 The function and size of the pinhole P2. Two mirrors M10, M20 (optional) and two converging lenses L60, L50 forming the afocal system allow the beam from the matrix array MP of the pinhole to be guided onto the second array RML2 of converging microlenses, which focuses the beam, thereby reducing the size of each focal point to half. After passing through another converging lens L40, the beams FR1–FR3 ​​are reflected by the back face FAR of the resonant mirror MOD and the back face of the current mirror (not shown) for photon redistribution. The combined effect of the afocal system L60, L50, lens L40, and the array RML2 of microlenses is to double the cross-sectional area of ​​each signal beam impacting the resonant mirror MOD. This is necessary because the amplitude of the rescanning must be equal to the amplitude of the scan of the illumination beam.

[0113] The final converging lens L70 focuses the signal beam onto the matrix image sensor CMI. The microlens array RML2 serves to halve the cross-sectional area of ​​the focal point of the signal beam.

[0114] [ Figure 1 ]and[ Figure 8 The embodiment of the microscope achieves photon redistribution by means of a purely optical means, through scanning (i.e., "re-scanning") of a signal beam with an appropriate amplitude synchronized with the scanning of the illumination beam. However, photon redistribution can also be achieved through digital processing. In this case, the camera CMI, whose acquisition rate is synchronized with the oscillating mirror MO1, is typically arranged flush with the pinhole P2, which can be integrated into the camera itself. Digital processing—as described, for example (Mueller 2010)—is implemented by a processor PNI, which receives the image acquired by the camera CMI as input. The third optical system SO3 can be omitted. The microscope according to this embodiment [ Figure 9 The photon redistribution is also shown in []. Figure 8 It is implemented digitally in a parallel architecture.

[0115] The invention has been described with reference to two specific embodiments, but is not limited thereto.

[0116] For example, it can be applied to the case of transmission confocal microscopy. In this case, two microscope objectives are required—one for illuminating the sample and the other for collecting the transmitted light. Furthermore, the first and second optical systems must be completely separated, which requires additional components.

[0117] Typically, the scanning of the sample by the focused beam and the photon redistribution scanning are two-dimensional. However, in some cases, single-dimensional scanning may be sufficient, which allows for a reduction in the number of oscillating mirrors.

[0118] If the illumination has sufficient spatial coherence (Strehl ratio > 80%), optical filtering through lens L1 and pinhole P1 or through a matrix array of microlenses RML1 and a matrix array of pinholes MP is not strictly required; in situations such as [ Figure 8 In the case of parallelized systems, coherence must be understood as being at the scale of each elementary beam; global coherence is not required. Similarly, optical relays L3-L4 and L20-L30 are not strictly necessary, but their presence is generally required to ensure that the oscillating mirror MO1 or MOD is optically conjugate with the objective's pupil. Otherwise, at large deflection angles, the objective's pupil will not be fully illuminated, which reduces the numerical aperture and thus spatial resolution. Likewise, using a laser as the illumination source is not necessary.

[0119] At least some lenses may not converge, but diverge and / or be replaced by other focusing or defocusing devices (such as concave or convex mirrors).

[0120] In principle, a beam splitter LS can be replaced by a beam splitter cube, but this is disadvantageous because the latter component introduces stray reflections.

[0121] More generally, besides [ Figure 1 ]and[ Figure 8 Optical setups other than those shown in the figures can be used to produce various constituent optical systems of the microscope according to the invention. Furthermore, in the description of the figures, the dimensions of the optical elements are given only by way of example.

[0122] The illumination wavelength can be any wavelength, but it is preferred to use light in the green (495-570 nm), blue (450-495 nm), or ultraviolet (380-450 nm) or even near-ultraviolet (300-380 nm) range, as this allows for lateral spatial resolution of 100 nm or less, while avoiding the technical difficulties associated with using shorter wavelengths. Similarly, selecting an immersion microscope objective with a high numerical aperture (greater than or equal to 1) can maximize spatial resolution, but it is not essential.

[0123] Finally, the detection and identification of viral particles is merely an application of […]. Figure 8 An example of a parallelized microscope of the type shown.

[0124] References

[0125] (Sheppard 1988): CJR Sheppard, “Super-resolution in Confocal Imaging”, Optik 80, No. 2, pp. 53-54.

[0126] (York 2013): A.York et al., “Instant super-resolution imaging in livecells and embryos via analog image processing”, Nat.Methods, November 2013, 10(11), pp. 1122-1126.

[0127] (De Luca 2013): GMRDe Luca “Re-scan confocal microscopy: scanning twice for better resolution”, Biomedical Optics Express, Vol. 4, No. 11, November 2013.

[0128] (Curd 2015): A. Curd et al., "Construction of an instant structuredillumination microscope", Methods 88 (2015) pp. 37-47.

[0129] (Roth 2017): S. Roth, “Development of a new microscopy method: Optical Photon Reassignment Microscopy”, PhD dissertation, Friedrich-Schiller University, Jena (DE), March 29, 2017.

[0130] (DuBose 2019): TBDuBose et al., “Super-resolution retinal imaging using optically reassigned scanning laser ophthalmology”, Nature Photonics, Vol. 13, April 2019, pp. 257-262.

[0131] (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, July 1, 1995, pp. 3576-3588.

[0132] (Mueller 2010): CB Mueller, J. Enderlein "Image Scanning Microscopy", PRL104, 198101 (2010).

Claims

1. A scanning confocal photon redistribution microscope, comprising: The light source (SL) is configured to generate at least one spatially coherent illumination beam (FE) at the illumination wavelength. A first optical system (SO1) is configured to perform an angular scan on the illumination beam; At least one microscope objective (OM) is configured to: receive the illumination beam output from the first optical system as input and focus it onto the sample (E); and collect and collimate a beam (FR) elastically scattered by the sample, referred to as a signal beam. A second optical system (SO2) is fully or partially consistent with the first optical system and is configured to receive the signal beam collimated by the microscope objective as input, apply an angular scan to the signal beam opposite to the angular scan applied to the illumination beam, and focus the signal beam in the first focal plane (PF1). A pinhole (P2) is positioned in the first focal plane; A third optical system (SO3) is configured to collect the signal beam that has passed through the pinhole, collimate the signal beam, apply an angle scan to the signal beam that is synchronized with the angle scan applied to the illumination beam, and make the product of the scan amplitude and the cross-sectional area of ​​the beam collimated by the third optical system greater than the product of the scan amplitude applied by the second optical system and the cross-sectional area of ​​the beam collimated by the second optical system, and focus the signal beam in the second focal plane; as well as A matrix image sensor (CMI) is arranged in the second focal plane (PF2); Its features are: The components including the microscope objective, the second optical system, and the third optical system are configured to focus the signal beam at the illumination wavelength onto the matrix image sensor; and The diameter or maximum lateral dimension of the pinhole is between 2 and 4 air units.

2. The scanning confocal photon redistribution microscope as described in claim 1, wherein, At least the second optical system includes a beam splitter (LS) for separating the signal beam from the illumination beam.

3. The scanning confocal photon redistribution microscope as described in any of the preceding claims, wherein, The light source (SL) is configured to emit blue, violet, or near-ultraviolet illumination beams.

4. The scanning confocal photon redistribution microscope as described in claim 1, wherein, The light source (SL) is a laser.

5. The scanning confocal photon redistribution microscope as described in claim 1, wherein, The microscope objective (OM) is an immersion objective with a numerical aperture greater than or equal to 1.

6. The scanning confocal photon redistribution microscope as described in claim 1, wherein, The third optical system is configured to apply an angular scan to the signal beam such that the product of the scan amplitude and 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 beam by the first optical system and the cross-sectional area of ​​the beam.

7. The scanning confocal photon redistribution microscope as described in claim 1, configured to operate in a reflective manner, wherein: - The first optical system (SO1) includes a first lens (L1) for focusing the illumination beam, a pinhole (P1) arranged in the focal plane of the first lens for performing spatial filtering of the illumination beam, a beam splitter (LS) for reflecting a portion of the beam, a second lens (L2) for collimating the portion of the illumination beam, a first oscillating mirror (MO1) or oscillating mirror system for applying the angular scan to the illumination beam, and a focusless system including a third lens (L3) and a fourth lens (L4); - The second optical system includes the afocal system, the first oscillating mirror (MO1) or oscillating mirror system, the second lens (L2), and the beam splitter (LS), the beam splitter (LS) being configured to transmit a portion of the signal beam backscattered by the sample; and The third optical system includes a fifth lens (L5) for collimating the signal beam that has passed through the pinhole, a second oscillating mirror (MO2) or oscillating mirror system for applying an angle scan to the signal beam that is synchronized with the angle scan applied to the illumination beam, and a sixth lens (L6) for focusing the signal beam in the second focal plane.

8. The scanning confocal photon redistribution microscope as described in claim 1, wherein, The light source is configured to generate multiple illumination beams (FE1, FE2, FE3) in parallel, which propagate through the first optical system, thereby the microscope objective collects multiple corresponding signal beams (FR1, FR2, FR3), which then propagate along the second optical system, and the scanning confocal photon redistribution microscope includes a matrix array (MP) of pinholes arranged in the second focal plane, with one pinhole for each backscattered beam.

9. The scanning confocal photon redistribution microscope as described in claim 8, configured to operate in a reflective manner, wherein: The light source includes a first array of microlenses (RML1) for generating and focusing the plurality of illumination beams; The scanning confocal photon redistribution microscope includes at least one oscillating mirror (MOD) having a front reflective surface (FAV) and a rear reflective surface (FAR), the front surface forming part of the first and second optical systems, and the rear surface forming part of the third optical system; and The third optical system includes a second array of microlenses (RML2) for increasing the cross-sectional area of ​​the backscattered beam incident on the rear surface of the oscillating mirror by 1.8 to 2.2 times.

10. The use of the scanning confocal photon redistribution microscope as described in claim 1, for observing suspended viral particles (PV).

11. A method for observing a sample (E), comprising the following steps: Generate at least one spatially coherent and collimated illumination beam (FE) at the illumination wavelength. An angle scan is applied to the illumination beam; The illumination beam is focused onto the sample (E) through a microscope objective (OM); A beam of light, referred to as the signal beam (FR), which is elastically scattered by the sample at the illumination wavelength, is collected through the microscope objective or another microscope objective. An angle scan opposite to that applied to the illumination beam is applied to the signal beam, and the signal beam is focused in the first focal plane (PF1); Spatial filtering of the signal beam is performed by a pinhole (P2) arranged in the first focal plane, the diameter or maximum lateral dimension of which is between 2 and 4 Airy units. The signal beam that has passed through the pinhole is collected, the signal beam is collimated and an angle scan is applied to the signal beam in sync with the angle scan applied to the illumination beam, such that the product of the amplitude of the signal beam and the cross-sectional area of ​​the signal beam is greater than the product of the amplitude of the angle scan applied to the illumination beam and the diameter of the illumination beam, and the signal beam is focused in the second focal plane (PF2). as well as The signal beam is detected by a matrix image sensor arranged in the second focal plane.

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