Detection arrangement of optical scanning microscope and optical scanning microscope

Abstract

A detection arrangement for an optical scanning microscope includes a beam splitting element configured to receive reversely scanned detection light, split the detection light into two portions, direct a first portion of the detection light into a first light path and direct a second portion of the detection light into a second light path. The first and second optical paths each include a dispersive element configured to spectrally separate the detection light in a spectrally separating direction and an array detector for receiving the spectrally separated detection light. A beam splitting element, first and second dispersive elements are configured such that a first reference direction corresponding to a first spectral separation direction in which reverse imaging is performed via a first optical path toward a plane arranged before a beam splitting element and a second spectral separation direction in which reverse imaging is performed via a second optical path toward a plane arranged before a beam splitting element correspond The two reference directions are different from each other.

Description

Technical Field

[0001] This invention relates to a detection arrangement for an optical scanning microscope. The invention also relates to an optical scanning microscope. Background Technology

[0002] Image scanning microscopy (ISM) is an advanced fluorescence microscopy technique that improves spatial resolution and signal-to-noise ratio, surpassing the capabilities of traditional confocal microscopy. In traditional confocal microscopy, a single point detector, such as a single photomultiplier tube, is used to detect fluorescence emitted from the sample. In ISM, the point detector is replaced by a multi-element photodetector, which comprises multiple photodetector elements (pixels) arranged in an array. Each photodetector element in the array outputs a detector signal upon receiving fluorescence. When the sample is scanned using a focused laser, each photodetector element detects a small image of the illuminated sample at each scan position. Appropriate algorithms are used to fuse the multiple scan images, thereby reconstructing a single, high-resolution image of the sample.

[0003] While information from different photodetector elements can be used to improve spatial image resolution and signal-to-noise ratio, one of the main limitations of current ISM technology is the limited applicability of these photodetectors in the quantitative analysis of the spectral composition of emitted fluorescence. For example, the existing solution described by F. Strasser et al. in Biomed. Opt. Expr, Vol. 10 (2019), p. 2513, is only applicable to a narrow band of the visible spectrum and suffers from optical loss due to the generation of higher-order diffraction and the use of only a single polarization state. Summary of the Invention

[0004] The purpose is to provide a detection setup for an optical scanning microscope and an optical scanning microscope that can achieve spectral imaging with high spatial resolution and high signal-to-noise ratio.

[0005] The above-mentioned objectives are achieved by the subject matter of the independent claims. Preferred embodiments are defined in the dependent claims and the description below.

[0006] The proposed detection arrangement for an optical scanning microscope includes a beam-splitting element configured to receive descanned detection light, split the detection light into two parts, and guide a first part of the detection light into a first optical path and a second part into a second optical path. The first optical path includes a first dispersive element configured to spectrally separate the detection light along a first spectral separation direction; and a first array detector configured to receive the spectrally separated detection light. The second optical path includes a second dispersive element configured to spectrally separate the detection light along a second spectral separation direction; and a second array detector configured to receive the spectrally separated detection light. The beam-splitting element, the first dispersive element, and the second dispersive element are configured such that a first reference direction corresponding to the first spectral separation direction of descanned imaging in a plane arranged before the beam-splitting element via the first optical path and a second reference direction corresponding to the second spectral separation direction of descanned imaging in a plane arranged before the beam-splitting element via the second optical path are different from each other.

[0007] This detection arrangement is configured to receive detection light from a descanned arrangement and can therefore be used as a detection arrangement in, for example, existing confocal laser scanning microscopes or image scanning microscopes. The descanned detection light typically corresponds to an image of a small particle that emits detection light (e.g., fluorescence) as it is scanned by a focused illumination beam (e.g., a laser focus used to excite a fluorophore). This particle can be considered a point source of the detection light. The descanned detection light received by the detection arrangement is split into a first part and a second part by a beam-splitting element. This generates two copies of the detection light. Each copy is guided through one of the dispersive elements before being detected by one of the array detectors. If the detection light includes a wide wavelength range, the dispersive element fans the detection light spectrally out along one of the spectral separation directions. However, if the detection light is monochromatic or consists of a narrow wavelength range (common in fluorescence microscopy), the dispersive element deflects the detection beam along the spectral separation direction instead of fanning it out. Based on the deflection produced by the dispersive element, the spectral composition of the detection light can be inferred. The deflection of the detection light is detected by an array detector, which can be a two-dimensional array of photodetector elements. By comparing successive images captured by the array detector, changes in the deflection of the detection light cause apparent movement of the image of the detection light on each array detector. However, it is necessary to distinguish between the detection light deflection caused by the dispersive elements and the deflection caused by the scanning itself. For example, the movement of the illumination focus relative to the point source of the detection light can be caused by the convolution of the point spread function (PSF) of the scanning illumination with the point source. Since the detection light emitted from the point source is detected by the array detector, and the array detector provides spatial information in addition to intensity information, the image of the emitting particles will still appear to move on the array detector even if the detection light received by the detection arrangement has been scanned in reverse. Therefore, the beam-splitting elements, the first dispersive element, and the second dispersive element of the proposed detection arrangement are arranged such that the first spectral separation direction relative to the detection light image on the first array detector is oriented differently from the second spectral separation direction relative to the detection light image on the second array detector. There is no preferred direction for the point image movement caused by the scanning, and it exhibits a similar appearance on each of the two array detectors. However, the motion caused by the dispersive elements (e.g., due to (hypothetical) color changes in the particles, such as changes in emitted light wavelength, especially changes in the fluorescence wavelength emitted by the particles) always occurs along their respective spectral separation directions, and these directions are different for each array detector. Therefore, the proposed detection arrangement ensures that the deflection of the detection light caused by the dispersive elements can be distinguished from the deflection caused by the scanning itself by comparing two images captured by the two array detectors. Thus, the proposed detection arrangement enables image scanning microscopy with high spatial resolution and high signal-to-noise ratio by using two array detectors to detect the detection light.The proposed detection arrangement can also reconstruct the spectral information of the detection light without causing significant loss of the detection light and resulting in a deterioration of the signal-to-noise ratio.

[0008] In this embodiment, the first reference direction and the second reference direction are opposite to or perpendicular to each other. With this arrangement, the apparent motion of the detection light image caused by color changes on the array detector will be in opposite or perpendicular directions, respectively. By selecting the reference directions, it is easier to separate the motion caused by color changes from the motion caused by the scanning itself, thereby enabling more reliable extraction of spectral information.

[0009] In another embodiment, the beam-splitting element is configured to mirror or reflect the second portion of the detection light guided into the second detection optical path. The beam-splitting element can be a neutral beam splitter, preferably configured to uniformly split the received reverse-scan detection light with a reflectance / transmittance ratio equal to or close to 1, for example, reflecting the second portion of the detection light and allowing the first portion of the detection light to pass through. Thus, the image captured by one of the array detectors will be mirrored relative to the other array detectors. Motion caused by scanning can be easily identified by finding mirror symmetry. This allows for the isolation of motion caused by color changes and enables more reliable extraction of spectral information.

[0010] In another embodiment, the beam-splitting element is configured to rotate a second portion of the detection light guided into the second detection optical path. In this embodiment, the image imaged along the first optical path is rotated relative to the image imaged along the second optical path. For example, the beam-splitting element may include an Abbe-König prism for rotating the second portion of the detection light. Similar to mirroring the second portion of the detection light, by rotating the second portion of the detection light, motion caused by scanning can be easily identified by finding rotational symmetry corresponding to the rotation of the second portion of the detection light.

[0011] In another embodiment, the beam-splitting element is configured to guide a first polarization component of the detection light as a first portion into a first optical path, and guide a second polarization component of the detection light as a second portion into a second optical path. In this embodiment, the detection light is split into first and second portions based on polarization. Assuming unpolarized light, such as fluorescence, this means the detection light is split almost uniformly. This means that each of the two array detectors can detect the maximum amount of detection light, thereby reducing optical loss and improving the signal-to-noise ratio. Alternatively, this uniform beam splitting can be achieved using a neutral beam splitter with a reflectance / transmittance ratio equal to or close to 1.

[0012] In another embodiment, the beam-splitting element includes an Abbe-König prism with a polarizing beam-splitting coating, configured to transmit a first polarization component of the detection light and reflect a second polarization component of the detection light. The Abbe-König prism rotates the second polarization component of the detection light. In this embodiment, the detection light is split into a first part and a second part based on polarization. Furthermore, the part including the second polarization component is rotated. Therefore, the Abbe-König prism not only ensures that the first part and the second part are split into nearly equal parts, but also rotates the image captured by the second array detector relative to the image captured by the first array detector. The advantages of these two features have been described above.

[0013] In another embodiment, the beam-splitting element includes a Wollaston prism. The Wollaston prism uses the optical property of birefringence to separate the detection light based on polarization. Unlike polarizing beam splitters, the Wollaston prism does not reflect the portion of the detection light, so both the first and second portions of the detection light retain their original orientation. This can be used to capture the same image using an array detector, making it easier to isolate motion caused by color changes.

[0014] In another embodiment, at least one of the first and second dispersive elements includes at least one dispersive prism. The dispersive prism has a wide operating wavelength range, including the visible spectrum and portions of the infrared and ultraviolet spectra. Furthermore, the dispersive prism does not produce higher-order diffraction that might occur with a diffraction grating. Higher-order diffraction might not be captured by the array detector. Therefore, using a dispersive prism prevents light loss and improves the signal-to-noise ratio.

[0015] In another embodiment, the first dispersive element includes a first dispersive prism, and the second dispersive element includes a second dispersive prism. The orientation of the first dispersive prism and the orientation of the second dispersive prism are associated by a single rotation about a rotation axis. For example, the rotation axis may be parallel to the side of the first dispersive prism. This is, for example, when the first and second optical paths are perpendicular to each other and do not include further reflecting elements. Combined with the beam-splitting element that reflects the second portion of the detection light, this results in the first and second reference directions being opposite to each other, thus having the advantages described above. Furthermore, in this embodiment, the optical arrangement of the first and second optical paths is particularly simple.

[0016] In another embodiment, the first dispersive element includes a first dispersive prism, and the second dispersive element includes a second dispersive prism. The second dispersive prism rotates about the optical axis relative to the first dispersive prism. For example, the second dispersive prism rotates about the optical axis in front of the beam-splitting element or at the second prism, wherein a second portion of the detection light enters the second prism. The rotation can be, in particular, 90° or 180°. This results in the first reference direction and the second reference direction being perpendicular to each other or opposite to each other, thereby having the advantages described above.

[0017] In another embodiment, the first and second dispersive elements are implemented via different surfaces of a dispersive prism. For example, the first and second dispersive elements can be implemented via two different sides of a single dispersive prism. Such a configuration can be advantageously used in conjunction with a beam-splitting element (e.g., a Wollaston prism) that splits the detection light into two beams forming an acute angle (especially less than 45°).

[0018] In another embodiment, the first and second dispersive elements are implemented using the same surface of a dispersive prism. For example, the first and second optical paths are made parallel before being guided into the dispersive prism. Using a single dispersive prism ensures consistent alignment and comparable dispersion of the detected light in both optical paths. Furthermore, in this embodiment, the optical configuration of the detection arrangement can be designed to be particularly compact.

[0019] In another embodiment, at least one of the first and second dispersive elements includes at least one of a planar grating, preferably a blazed grating, a prism grating, and a diffractive optical element. In particular, the blazed grating has high diffraction efficiency at a predetermined diffraction order, which can reduce light loss and improve the signal-to-noise ratio. The prism grating combines a dispersive prism with a grating, resulting in extremely low chromatic aberration. The diffractive optical element enables more complex light manipulation, thereby controlling the beam shape and precisely controlling the phase and amplitude of the detection light.

[0020] In another embodiment, the first optical path does not include a first reflecting element or includes at least one first reflecting element, and the second optical path does not include a second reflecting element or includes at least one second reflecting element. The number of both the first and second reflecting elements is either even or odd. In other words, the number of reflecting elements in both the first and second optical paths is either even or odd, where zero is considered even. This ensures that the relative parity of the image generated by the beam-splitting element does not change due to the reflective elements arranged in the first and second optical paths. For example, if the beam-splitting element mirrors the second portion of the detection light, the images captured by the first and second array detectors will be mirrored. Thus, even if the first and second dispersive elements are implemented through the same surface of a dispersive prism, they can have different reference orientations.

[0021] A further advantage is that the optical path length from the focal plane of the optical scanning microscope to the first array detector is equal to the optical path length from the focal plane of the optical scanning microscope to the second array detector. This maintains consistency between the images captured by the first and second array detectors and simplifies the optical design of the detection arrangement.

[0022] In another embodiment, the detection arrangement includes a pinhole. Preferably, the pinhole is positioned upstream of the beam-splitting element, i.e., before the beam-splitting element. The pinhole excludes defocused light, thereby improving image quality.

[0023] The present invention also relates to an optical scanning microscope. The optical scanning microscope includes an excitation source configured to generate excitation light, an objective lens facing a sample space and configured to guide the excitation light into the sample space and receive detection light from the sample space, and a scanning unit arranged along the optical path between the excitation source and the objective lens and configured to selectively guide the excitation light into different regions of the sample space via the objective lens (e.g., in a serpentine scanning manner). The optical scanning microscope also includes the detection arrangement described above and a main beam splitter configured to guide the excitation light into the objective lens via the scanning unit and guide the detection light into the detection arrangement.

[0024] Optical scanning microscopes offer the same advantages as the detection setups described above. In particular, optical scanning microscopes can be equipped with the features related to the detection setups described herein. Furthermore, the detection setups described above can be equipped with the features related to optical scanning microscopes described herein.

[0025] In embodiments, the optical scanning microscope further includes an acousto-optic device disposed downstream of or as part of the main beam splitter. The acousto-optic device is configured to receive detection light during reverse scanning and selectively guide a portion of the detection light into the detection arrangement, while guiding another portion of the detection light away from the detection arrangement. Excitation light can be reflected in the sample space and leak through the main beam splitter into the detection arrangement. The acousto-optic device can be controlled to selectively deflect certain wavelengths or bands. This property is used to deflect the leaked excitation light away from the detection arrangement. WO 99 / 42884 A1 discloses an exemplary main beam splitter including an acousto-optic device.

[0026] In another embodiment, the excitation source is a pulsed laser source, and at least one of the array detectors in the detection arrangement is used to record the arrival time of a single photon relative to the laser pulse generated by the excitation source. By recording the arrival time of a single photon relative to a reference signal, fluorescence lifetime characteristics can be determined using optical scanning microscopy. This allows optical scanning microscopy to be used for fluorescence microscopy applications, such as fluorescence lifetime imaging microscopy (FLIM). Attached Figure Description

[0027] The specific embodiments will be described below with reference to the accompanying drawings, wherein: Figure 1 This is a schematic diagram of an optical scanning microscope including a detection arrangement according to an embodiment; Figure 2 This is a schematic diagram of a detection arrangement according to an embodiment including two dispersive elements, the orientation of which is associated by rotation about a single axis. Figure 3 This is a schematic diagram of a detection arrangement according to an embodiment including two dispersive elements, the orientation of which is associated by mirror symmetry; Figure 4 This is a schematic diagram of a detection arrangement according to an embodiment including two dispersive elements, the orientation of which is associated by rotation about the optical axis; Figure 5 This is a schematic diagram of a detection arrangement according to another embodiment, wherein two dispersive elements are formed from the same surface of a dispersive prism; Figure 6 This is a schematic diagram of a detection arrangement according to another embodiment, which includes a Wollaston prism; Figure 7 This is a schematic diagram of a detection arrangement according to another embodiment, which includes a Wollaston prism and an Abbe-König prism; Figure 8 A schematic diagram of an Abbe-König prism with a polarizing beam splitter coating; and Figure 9 This is a schematic diagram of an optical scanning microscope according to another embodiment, which includes an acousto-optic device. Detailed Implementation

[0028] Figure 1 This is a schematic diagram of an optical scanning microscope 100 according to an embodiment, which includes a detection arrangement 102. The optical scanning microscope 100 exemplarily includes a single objective lens 104 facing a sample 106 arranged in a sample space 108. The optical scanning microscope 100 also includes an excitation light source 110, a scanning unit 112, and a main beam splitter 114.

[0029] Excitation source 110 is configured to generate excitation light 116, particularly excitation light 116 having a single wavelength or a narrow band. Excitation source 110 may include one or more lasers to generate laser light as excitation light 116. In particular, excitation source 110 may include a continuous spectrum laser and an arrangement of replaceable filters or a tunable laser to selectively generate excitation light 116 with different wavelengths. Excitation source 110 may include other optical elements, such as lenses and apertures, for forming the excitation light 116 into a beam. The excitation light 116 generated by excitation source 110 is guided to scanning unit 112 by main beam splitter 114. Scanning unit 112 is configured to deflect excitation light 116 to selectively guide excitation light 116 into different regions of sample space 108 via objective lens 104. This allows the sample 106 to be scanned using the excitation light 116 focused by objective lens 104. To deflect excitation light 116, scanning unit 112 may include, for example, one or more galvanometers or acousto-optic deflectors. The optical path of the excitation light is as follows: Figure 1As shown, the line is marked with a dashed line starting from the excitation source 110 and ending at the sample 106.

[0030] The sample 106 is illuminated using excitation light 116, generating detection light 118. Detection light 118 is collected by objective lens 104 and guided back to main beam splitter 114 via scanning unit 112. Since scanning unit 112 is positioned between main beam splitter 114 and objective lens 104, the deflection of excitation light 116 is opposite to that of detection light 118. Regardless of the deflection angle of scanning unit 112, detection light 118 is guided to a single point. In other words, detection light 118 is scanned in reverse. The reverse-scanned detection light 118 is then guided by main beam splitter 114 into detection arrangement 102. The optical path of detection light 118 is as follows: Figure 1 As shown, the dotted line starting from sample 106 is used for marking.

[0031] The detection arrangement 102 includes a beam splitter 120, a first dispersive element 122a, a second dispersive element 122b, a first array detector 124a, and a second array detector 124b. As an example only, the detection arrangement 102 also includes a pinhole 126 disposed on the conjugate image plane.

[0032] The detection light 118 is received by the detection arrangement 102 through a pinhole 126, which limits the beam diameter of the detection light 118 entering the detection arrangement 102 and / or suppresses light originating from the defocused region of the sample space 108, allowing only the detection light 118 originating from the focused region of the objective lens 104 to enter the detection arrangement 102. After passing through the pinhole 126, the detection light 118 reaches the beam splitter 120. In this embodiment, the beam splitter 120 is a neutral beam splitter that divides the incident light into two nearly identical portions. The first portion 128a of the detection light 118 is guided into a first optical path 130a, which includes a first dispersive element 122a and a first array detector 124a. The second portion 128b of the detection light 118 is guided into a second optical path 130b, which includes a second dispersive element 122b and a second array detector 124b. The dispersive elements 122a and 122b are exemplarily shown as dispersive prisms. However, the dispersive elements 122a and 122b may include one or more of the following: a dispersive prism, a plane grating (preferably a blazed grating), a prism grating, and a diffractive optical element.

[0033] In each of the two optical paths 130a and 130b, the detection light 118 first passes through one of the dispersive elements 122a and 122b. Each of the dispersive elements 122a and 122b deflects the detection light 118 based on its wavelength to perform spectral separation along spectral separation directions 132a and 132b. For example, the dispersive elements 122a and 122b can deflect shorter wavelengths more than longer wavelengths. The spectral separation directions 132a and 132b may coincide, but are in principle independent of each other. The first dispersive element 122a performs spectral separation of the detection light 118 along the first spectral separation direction 132a, and the second dispersive element 122b performs spectral separation of the detection light 118 along the second spectral separation direction 132b.

[0034] Next, the spectrally separated detection light 118 is received by array detectors 124a and 124b. Each of the array detectors 124a and 124b comprises an array of photodetector elements, preferably a two-dimensional array of photodetector elements, such as photodiodes (e.g., single-photon avalanche diodes (SPADs)) or photomultiplier tubes (PMTs) (e.g., gallium arsenide phosphide (GaAsP) type PMTs). Each photodetector element, acting as a single-pixel detector, captures a portion of the spectrally separated detection light 118 at a different position in the array. Therefore, the array detectors 124a and 124b enable the detection of the spatial distribution of the intensity of the spectrally separated detection light 118. When the sample 106 is scanned with excitation light 116, each of the array detectors 124a and 124b detects at least one spatial distribution at each scan position. By collecting the spatial distribution, a single high-resolution image of the sample 106 can be reconstructed. This imaging technique, called image scanning microscopy (ISM), offers higher spatial resolution and signal-to-noise ratio compared to conventional confocal laser scanning microscopy (CLSM).

[0035] During the scanning of sample 106, a single particle (e.g., a single fluorophore) can be imaged multiple times in successive steps. Therefore, the motion of the excitation light 116 causes motion in the image of the particle on array detectors 124a, 124b. Similarly, color changes in the particle cause the dispersive elements 122a, 122b to deflect it at different angles, thus resulting in apparent motion in the image. By distinguishing between these two types of motion, spectral information can be extracted, thereby expanding the capabilities of ISM technology.

[0036] The beam-splitting element 120, the first dispersive element 122a, and the second dispersive element 122b are arranged and configured to facilitate the computational demixing of scanning effects and spectral separation effects. When the first spectral separation direction 132a is back-imaged onto the sample 106, a first reference direction 134a perpendicular to the optical axis of the objective lens 104 is defined. Similarly, when the second spectral separation direction 132b is back-imaged onto the sample 106, a second reference direction 134b, also perpendicular to the optical axis of the objective lens 104, is defined. The aforementioned elements are arranged and configured such that the first reference direction 134a and the second reference direction 134b are different from each other, for example, perpendicular to each other. In other words, the first spectral separation direction 132a is oriented differently relative to the image of the detection light 118 on the first array detector 124a than the second spectral separation direction 132b is oriented differently relative to the image of the detection light 118 on the second array detector 124b. The following description, in conjunction with... Figures 2 to 8 The description includes various embodiments of the detection arrangement 102 that implement this configuration.

[0037] To resolve scanning and spectral separation effects, multi-view deconvolution algorithms can be used, for example, by minimizing the difference between the detected spatial distribution and the reconstructed signal to solve for the reconstructed image. A signal formation model can then be used to calculate the reconstructed signal from the estimated reconstructed image. Multi-view deconvolution can also be combined with pixel redistribution methods. For example, pixel redistribution can be performed in directions perpendicular to the spectral separation directions 132a and 132b, while multi-view deconvolution can be performed along these directions.

[0038] The detection light 118 may include fluorescence generated by a fluorophore excited by the excitation light 116. However, the detection light 118 may also include, for example, a small amount of the excitation light 116 reflected in the sample space 108. Such excitation light 116 may leak into the detection arrangement 102 through the main beam splitter 114; in other words, it may leak into the detection arrangement 102. The following will combine... Figure 9 An embodiment for resolving excitation light leakage 116 is described.

[0039] Figure 2 This is a schematic diagram of the detection arrangement 200 according to an embodiment.

[0040] Figure 2 The illustrated detection arrangement 200 includes a beam-splitting element 120, exemplaryly configured as a neutral beam splitter with a reflectance / transmittance ratio of 1. This type of beam splitter is also referred to as a 50:50 beam splitter. The beam-splitting element 120 reflects a second portion 128b of the detection light 118, thereby mirroring it. Figure 2In the diagram, the first arrow A1 indicates the motion of the imaging particle (such as a single fluorophore) relative to the optical axis. The other arrows A2a and A2b show the direction of motion of the particle image relative to the optical axis in the first and second optical paths 130a and 130b, respectively. It can be seen that because the second portion 128b of the detection light 118 is reflected by the beam-splitting element 120, the motion in the second optical path 130b is mirrored relative to the motion in the first optical path 130a.

[0041] The detection arrangement 200 also includes two dispersive elements 202a and 202b, exemplary configured as dispersive prisms with triangular bases. The triangular bases of the two dispersive prisms are parallel. The orientation of the dispersive elements 202a and 202b is associated only by rotation about an axis R. Figure 2 In this diagram, axis R is perpendicular to the plane of the attached drawing and intersects the plane at the position where the detection light 118 is divided into first and second optical paths 130a and 130b. In other words, the orientation of the dispersive elements 202a and 202b is associated with rotation and lateral displacement about the side of the dispersive prism. The arrangement of the dispersive elements 202a and 202b, combined with the mirror image of the detection light 118 in the second optical path 130b, causes the first spectral separation direction 132a to be oriented differently relative to the image of the detection light 118 on the first array detector 124a than the second spectral separation direction 132b is oriented differently relative to the image of the detection light 118 on the second array detector 124b. Figure 2 The embodiment shown implements a detection arrangement 200 in which the first reference direction 134a and the second reference direction 134b are opposite to each other.

[0042] Figure 3 This is a schematic diagram of a detection arrangement 300 according to another embodiment. Figure 2 Compared to the detection setup 200 shown, Figure 3 The difference in the detection arrangement 300 shown is that the dispersive elements 202a and 202b are oriented differently, and an additional reflective element 302 is added to the second optical path 130b.

[0043] The reflective element 302 is arranged in the second optical path 130b such that the first and second optical paths 130a and 130b remain parallel before reaching the first and second dispersive elements 202a and 202b, respectively. The second reflection in the second optical path 130 restores the parity of the image of the detection light 118 in the two optical paths 130a and 130b. This means, as described above... Figure 2 Unlike the embodiment shown, the images on the first and second array detectors 124a and 124b are not mirrored. Figure 2 similar, Figure 3In the image, the first arrow A1 indicates the motion of the imaged particle relative to the optical axis, while the other arrows A2a and A2b show the direction of motion of the image of the particle relative to the optical axis in the first and second optical paths 130a and 130b, respectively.

[0044] Related to the above Figure 2 The illustrated embodiment is similar, with the bases of the two dispersive prisms forming dispersive elements 202a and 202b parallel and exemplaryly formed as triangular bases. However, unlike the above... Figure 2 In the illustrated embodiment, the orientations of the dispersive elements 202a and 202b are correlated through mirror symmetry. This results in the first spectral separation direction 132a being... Figure 3 Downward, while the second spectral separation direction 132b is in Figure 3 In the upward direction, that is, the first and second spectral separation directions 132a and 132b are far apart from each other. This fact, combined with the equal parity of the images on the first and second array detectors 124a and 124b, results in the first reference direction 134a and the second reference direction 134b being opposite to each other.

[0045] Figure 4 This is a schematic diagram of a detection arrangement 400 according to another embodiment. Figure 2 Compared to the detection setup 200 shown, Figure 4 The difference in the detection arrangement 400 shown is that the orientation of the dispersive elements 202a and 202b is related by rotation about the optical axis.

[0046] The second dispersive element 202b is rotated about 90° about the optical axis relative to the first dispersive element 202a, wherein the optical axis upstream of the beam splitter 120 serves as a reference. This arrangement, combined with the mirror symmetry between the images on the first and second array detectors 124a, 124b, results in the first reference direction 134a being perpendicular to the second reference direction 134b.

[0047] Figure 5 This is a schematic diagram of a detection arrangement 500 according to another embodiment. In such... Figure 5 In the illustrated embodiment, the two dispersive elements 502a and 502b are formed from the same surfaces 504a and 504b of a single dispersive prism 506, which exemplarily has a triangular base.

[0048] Figure 5The detection arrangement 500 shown includes a beam splitter element 120, which may be a neutral 50:50 beam splitter. A first portion 128a of the detection light 118 is formed by the detection light 118 entering the first optical path 130a through the beam splitter element 120. A second portion 128b of the detection light 118 is formed by the detection light 118 reflected by the beam splitter element 120 into the second optical path 130b. This means that the second portion 128b of the detection light 118 is immediately mirrored relative to the first portion 128a of the detection light 118 after passing through the beam splitter element 120. The first optical path 130a includes three reflective elements 508a that guide the first portion 128a of the detection light 118 onto the first surface 504a of the dispersive prism 506. After passing through the dispersive prism 506, the first portion 128a of the detection light 118 exits the dispersive prism 506 via the second surface 504b. The dispersion effect (i.e., the refractive index varies with wavelength) causes the detection light 118 of different wavelengths to refract at different angles. Therefore, the first and second surfaces 504a and 504b of the dispersive prism 506 form a first dispersive element 502a. The second optical path 130b includes only one reflective element 508b, which guides the second portion 128b of the detection light into the first surface 504a of the dispersive prism 506. Similar to the first portion 128a, the second portion 128b of the detection light 118 passes through the dispersive prism 506 and exits from the dispersive prism 506 via the second surface 504b. Therefore, the first and second surfaces 504a and 504b of the dispersive prism 506 also form a second dispersive element 502b. This results in the first spectral separation direction 132a being the same as the second spectral separation direction 132b.

[0049] The number of reflective elements 508a in the first optical path 130a and the number of reflective elements 508b in the second optical path 130b are both odd numbers—three and one. This means that the mirror symmetry of the first and second portions 128a, 128b of the detection light 118 is preserved. Alternatively, the number of reflective elements 508a in the first optical path 130a and the number of reflective elements 508b in the second optical path 130b can both be even numbers, for example, four and two, respectively. This would restore the parity of the images captured by the first and second array detectors 124a, 124b.

[0050] One of the reflecting elements 508a in the first optical path 130a is arranged such that the first portion 128a of the detection light 118 is not only mirrored but also rotated. Figure 2 Similarly, the first arrow A1 indicates the motion of the imaged particle relative to the optical axis, while the other arrows A2a and A2b show the direction of motion of the particle image relative to the optical axis in the first and second optical paths 130a and 130b, respectively. Figure 5As can be seen, the images on the first and second array detectors 124a and 124b are not only mirror images of each other, but also rotated by 90° relative to each other. Because the first and second spectral separation directions 132a and 132b are the same, this results in the first reference direction 134a and the second reference direction 134b being perpendicular to each other.

[0051] Figure 6 This is a schematic diagram of a detection arrangement 600 according to another embodiment. In this embodiment, the beam splitting element 120 includes a Wollaston prism 602, which splits the detection light 118 according to polarization.

[0052] The Wollaston prism 602 splits the incident detection light 118 into two orthogonally polarized beams that diverge at a specific angle, thereby generating first and second portions 128a and 128b of the detection light 118. The first portion 128a of the detection light 118 is then guided into the first surface 606a of the dispersive prism 604. The second portion 128b of the detection light 118 is guided into the second surface 606b of the same dispersive prism 604. The first and second surfaces 606a and 606b exemplarily form an included angle of 90°. Both the first and second portions 128a and 128b of the detection light 118 exit from the dispersive prism 604 via the third surface 606c and are subsequently guided into the first and second array detectors 124a and 124b, respectively. Therefore, in Figure 6 In the illustrated embodiment, the first surface 606a and the third surface 606c of the dispersive prism 604 form a first dispersive element 608a, and the second surface 606b and the third surface 606c of the dispersive prism 604 form a second dispersive element 608b. In this embodiment, the first and second dispersive elements 608a and 608b can also be considered as two dispersive prisms with quadrilateral bases, wherein... Figure 6 The fourth side, indicated by the dashed line, is shared. Due to the arrangement of the first and second surfaces 606a and 606b, the first and second spectral separation directions 132a and 132b are opposite to each other. Figure 6 In this embodiment, the first spectral separation direction 132a is downward and the second spectral separation direction 132b is upward. This results in the first reference direction 134a and the second reference direction 134b being opposite to each other.

[0053] exist Figure 6In the illustrated embodiment, the dispersive prism 604 forming the dispersive element is exemplarily composed of two parts 610a and 610b, which together form a cube. Each part 610a and 610b is made of a different material. The refractive indices of the different materials are chosen such that the dispersive prism 604 effectively functions as a parallel plate at a predetermined center wavelength. This minimizes astigmatism, thereby improving image quality. In any embodiment using a dispersive prism as a first dispersive element 122a, 202a, 502a, 608a, 710a and / or a second dispersive element 122b, 202b, 502b, 608b, 710b, a similar dispersive prism combining two different materials can be used to achieve effective parallel plate function at a predetermined center wavelength.

[0054] Generally, the spectral dispersion achieved by the dispersive elements 122a, 122b, 202b, 202b, 502a, 502b, 608a, 608b, 710a, and 710b relative to the diffraction limit (i.e., the size of the point spread function (PSF)) can be adjusted by selecting the material and cutting angle of the dispersive prism; and by adjusting the overall optical design of the detection arrangements 102, 200, 300, 400, 500, 600, and 700 (e.g., by selecting the focal length and pupil diameter accordingly). The selection of spectral dispersion depends on the required balance between spectral resolution and signal-to-noise ratio for the detection arrangements 102, 200, 300, 400, 500, 600, and 700, and also on economic considerations regarding the number of array detectors 124a and 124b (which necessarily also include readout electronics).

[0055] Figure 7 This is a schematic diagram of a detection arrangement 700 according to another embodiment. Figure 6 Compared to the detection setup shown in 600, Figure 7 The difference in the detection arrangement 700 is that the beam splitting element 702 additionally includes an Abbe-König prism 704 with a polarizing beam splitter coating.

[0056] The Abbe-König prism 704 rotates one polarization component 118b of the detection light 118, while the other polarization component 118a passes through the Abbe-König prism 704 without rotation. Which polarization is rotated depends on the polarization beamsplitter coating. The detection light 118 then strikes the Wollaston prism 602, which generates the first and second portions 128a, 128b of the detection light 118 by deflecting the two polarization components 118a, 118b at different angles. The working principle of the Abbe-König prism 704 will be discussed below in conjunction with... Figure 8 To provide a more detailed explanation.

[0057] For example only, Figure 7The detection arrangement 700 shown includes a dispersive prism 706 having two surfaces 708a and 708b, forming first and second dispersive elements 710a and 710b, respectively. (Refer to the above text) Figure 5 The described embodiments are similar, resulting in the first and second spectral separation directions 132a, 132b being identical. Further, the Wollaston prism 602 is arranged such that the first and second optical paths 130a, 130b lie in a plane parallel to the side of the dispersive prism 706 (or in other words, the third surface 708c of the triangular dispersive prism 706). This results in the first optical path 130a and the second optical path 130b intersecting the first surface 706a at the same height above the third surface 708c.

[0058] With Figure 2 As in the image, the first arrow A1 indicates the motion of the imaged particle relative to the optical axis, while the other arrows A2a and A2b show the direction of motion of the particle's image relative to the optical axes of the two polarization components 118a and 118b, respectively. For example, in... Figure 7 As can be seen, because the first and second spectral separation directions 132a and 132 are the same, the rotation of one of the polarization components 118a and 118b causes the first and second reference directions 134a and 134b to be opposite.

[0059] Figures 2 to 7 The embodiments of detection arrangements 200, 300, 400, 500, 600, and 700 shown do not explicitly include pinhole 126. However, pinhole 126 may be arranged upstream of beam splitting elements 120 and 702, i.e., in the direction from which the detection light 118 originates (applicable to all embodiments).

[0060] Figure 8 This is a schematic diagram of the Abbe-König prism 704 with a polarizing beam splitter coating.

[0061] The Abbe-König prism 704 consists of two parts, 800a and 800b. The first part, 800a, is shaped like an isosceles triangle with a vertex angle of 125°, as shown below. Figure 8 The first part 800a is arranged at the bottom as shown. The second part 800b is cubic in shape with a notch 802 at the bottom. The notch 802 is sized to accommodate the first part 800a, ensuring there is no air gap between the first and second parts 800a and 800b. Therefore, the first and second parts 800a and 800b contact each other to form a first optical surface 804a and a second optical surface 804b with an included angle of 125°. Both optical surfaces 804a and 804b are coated with a polarizing beam splitter coating.

[0062] The first polarization component 118a of the detected light 118 is in Figure 8 The second polarization component 118b of the detection light 118 is indicated by a dashed line. Figure 8 The first polarization component 118a is not reflected by the polarizing beamsplitter coating and therefore passes through the first and second optical surfaces 804a and 804b without deflection. Conversely, the second polarization component 118a is reflected by the polarizing beamsplitter coating. Therefore, the second polarization component 118b is reflected from the first optical surface 804a towards the top of the second portion 800b. At the top of the second portion 800b, the second polarization component undergoes total internal reflection and is reflected downwards. Then, the second polarization component is reflected by the second optical surface 804b and thus becomes parallel to the first polarization component 118a again. Both the first and second polarization components 118a and 118b exit parallel from the Abbe-König prism 704, but the second polarization component 118b has a longer optical path length through the Abbe-König prism 704.

[0063] Figure 9 This is a schematic diagram of an optical scanning microscope 900 according to another embodiment. Figure 1 Compared to the optical scanning microscope 100 shown, Figure 9 The difference in the optical scanning microscope 900 shown is that it includes an acousto-optic device 902.

[0064] An acousto-optic device 902 is exemplarily arranged downstream of the main beam splitter 114. However, the acousto-optic device 902 can also be part of the main beam splitter 114, which can be formed as an acousto-optic main beam splitter. The acousto-optic device 902 includes an acousto-optic medium 904 and a transducer 906. A radio frequency signal applied to the transducer 906 generates acoustic waves in the acousto-optic medium 904, which locally affect the refractive index of the acousto-optic medium 904. This anisotropy of the refractive index forms a refractive grating in the acousto-optic medium 904, the characteristics of which are determined by the radio frequency signal applied to the transducer 906. This refractive grating causes the light 116, 118 approaching the acousto-optic device 902 to undergo multi-level diffraction based on the acoustic wave frequency and amplitude (and therefore the radio frequency signal applied to the transducer 906).

[0065] In the optical scanning microscope 900 of this embodiment, an acousto-optic device 902 is used to selectively deflect a portion of the detection light 118 away from the detection arrangement 102, for example, by guiding a portion of the detection light 118 into a beam collector or away from the opening of the pinhole 126. For example, the wavelength of the excitation light 116 can be deflected away from the detection arrangement 102, thereby preventing the excitation wavelength from leaking into the detection arrangement 102. Similarly, the acousto-optic device 902 can be operated to allow only detection light 118 with a predetermined wavelength to enter the detection arrangement 102, for example, the emission wavelength of one or more specific fluorophores arranged in the sample 106.

[0066] In all the accompanying drawings, identical or functionally similar elements are designated by the same reference numerals. The term "and / or" (which may be abbreviated as " / ") as used herein includes any and all combinations of one or more of the listed related items.

[0067] Although some aspects are described within the contents of the device, these aspects clearly also represent descriptions of the corresponding methods, where modules or devices correspond to method steps or features of method steps. Similarly, aspects described within the contents of method steps also represent descriptions of corresponding modules, components, or features in the corresponding device.

[0068] Figure Labels 100 Optical Scanning Microscope 102 Inspection Setup 104 Objective Lenses 106 samples 108 Sample Space 110 Excitation Light Source 112 scanning units 114 Main beam splitter 116 Excitation Light 118 Detection Light 118a and 118b polarization components 120 beam splitter 122a, 122b dispersive elements 124a and 124b array detectors 126 pinholes 128a, 128b Detection of light portion 130a and 130b optical paths 132a, 132b spectral separation directions 134a, 134b Reference directions 200 Inspection Setup 202a, 202b dispersive elements 300 Inspection Setup 302 Reflective element 400 Inspection Setup 500 Inspection Setup 502a, 502b dispersive elements 504a and 504b surfaces 506 Dispersion Prism 508a and 508b reflective elements 600 Inspection Setup 602 Wollaston Prism 604 Dispersion Prism 606a, 606b, 606c surfaces 608a and 608b dispersive elements Parts 610a and 610b 700 Inspection Setup 702 Beam Splitter 704 Abbe-König Prism 706 Dispersion Prism 708a, 708b, 708c surfaces 710a and 710b dispersive elements 800a and 800b sections 802 Gap 804a and 804b optical surfaces 900 Optical Scanning Microscope 902 Acoustic and visual device 904 Acousto-optic medium 906 transducer Arrows A1, A2a, A2b R Rotation axis

Claims

1. A detection arrangement (102, 200, 300, 400, 500, 600, 700) for an optical scanning microscope (100, 900), said detection arrangement (102, 200, 300, 400, 500, 600, 700) comprising: Beam splitting elements (120, 702) are configured to receive the detection light (118) in reverse scanning, split the detection light (118) into two parts (128a, 128b), guide the first part (128a) of the detection light (118) into the first optical path (130a), and guide the second part (128b) of the detection light (118) into the second optical path (130b). The first optical path (130a) includes: a first dispersive element (122a, 202a, 502a, 608a, 710a) configured to perform spectral separation on the detection light (118) along a first spectral separation direction (132a); and a first array detector (124a) configured to receive the spectrally separated detection light (118); and The second optical path (130b) includes: a second dispersive element (122b, 202b, 502b, 608b, 710b) configured to perform spectral separation of the detection light (118) along a second spectral separation direction (132b); and a second array detector (124b) configured to receive the spectrally separated detection light (118). The beam splitting elements (120, 702), the first dispersive elements (122a, 202a, 502a, 608a, 710a), and the second dispersive elements (122b, 202b, 502b, 608b, 710b) are configured such that a first reference direction (134a) corresponding to the first spectral separation direction (132a) of planar reverse imaging arranged in front of the beam splitting elements (120, 702) via the first optical path (130a) and a second reference direction (134b) corresponding to the second spectral separation direction (132b) of planar reverse imaging arranged in front of the beam splitting elements (120, 702) via the second optical path (130b) are different from each other.

2. The detection arrangement (102, 200, 300, 400, 500, 600, 700) according to claim 1, wherein, The first reference direction (134a) and the second reference direction (134b) are opposite to each other or perpendicular to each other.

3. The detection arrangement (102, 200, 300, 400, 500) according to claim 1 or 2, wherein, The beam splitter (120) is configured to mirror the second portion (128b) of the detection light (118) that is guided into the second detection optical path.

4. The detection arrangement (700) according to any one of the preceding claims, wherein, The beam splitter (702) is configured to rotate the second portion (128b) of the detection light (118) that is guided into the second detection optical path.

5. The detection arrangement (600, 700) according to any one of the preceding claims, wherein, The beam splitting elements (120, 702) are configured to guide the first polarization component of the detection light (118) as the first part (128a) into the first optical path (130a), and guide the second polarization component of the detection light (118) as the second part (128b) into the second optical path (130b).

6. The detection arrangement (700) according to claim 5, wherein, The beam splitting element (702) includes an Abbe-König prism (704) with a polarizing beam splitting coating, which is configured to transmit the first polarization component (118a) of the detection light (118) and reflect the second polarization component (118b) of the detection light (118).

7. The detection arrangement (700) according to claim 5 or 6, wherein, The beam splitter (702) includes a Wollaston prism (602).

8. The detection arrangement (102, 200, 300, 400, 500, 600, 700) according to any one of the preceding claims, wherein, At least one of the first dispersive element (122a, 202a, 502a, 608a, 710a) and the second dispersive element (122b, 202b, 502b, 608b, 710b) includes at least one dispersive prism (506, 604, 706).

9. The detection arrangement (102, 200, 400) according to claim 8, wherein, The first dispersive element (122a, 202a) includes a first dispersive prism, and the second dispersive element (122b, 202b) includes a second dispersive prism, wherein the orientation of the first dispersive prism and the orientation of the second dispersive prism are associated by a single rotation about a rotation axis (R).

10. The detection arrangement (400) according to claim 8 or 9, wherein, The first dispersive element (202a) includes a first dispersive prism, and the second dispersive element (202b) includes a second dispersive prism, wherein the second dispersive prism rotates about the optical axis relative to the first dispersive prism.

11. The detection arrangement (600) according to any one of claims 8 to 10, wherein, The first dispersive element (608a) and the second dispersive element (608b) are implemented through different surfaces (606a, 606b, 606c) of a dispersive prism (604).

12. The detection arrangement (500) according to any one of claims 5 to 10, wherein, The first dispersive element (502a) and the second dispersive element (502b) are implemented through the same surfaces (504a, 504b) of a dispersive prism (506).

13. The detection arrangement (102) according to any one of claims 1 to 8, wherein, At least one of the first dispersive element (122a) and the second dispersive element (122b) includes at least one of a planar grating, preferably a blazed grating, a prism grating, and a diffractive optical element.

14. The detection arrangement (102, 200, 400, 500, 600, 700) according to any one of the preceding claims, wherein, The first optical path (130a) does not include a first reflective element (508a) or includes at least one first reflective element (508a), and the second optical path (130b) does not include a second reflective element (508a) or includes at least one second reflective element (508a), wherein the number of first reflective elements (508a, 508b) and the number of second reflective elements (508a, 508b) are both even or both odd.

15. The detection arrangement (102) according to any one of the preceding claims, wherein, The detection arrangement (102) includes a pinhole (126).

16. An optical scanning microscope (100, 900), comprising: An excitation light source (110) is configured to generate excitation light (116). An objective lens (104) is oriented toward the sample space (108) and configured to guide the excitation light (116) into the sample space (108) and receive the detection light (118) from the sample space (108). A scanning unit (112) is arranged along the optical path between the excitation light source (110) and the objective lens (104), and is configured to selectively guide the excitation light (116) into different regions of the sample space (108) through the objective lens (104); The detection arrangement according to any one of the preceding claims (102, 200, 300, 400, 500, 600, 700); and The main beam splitter (114) is configured to guide the excitation light (116) into the objective lens (104) via the scanning unit (112) and guide the detection light (118) into the detection arrangement (102, 200, 300, 400, 500, 600, 700).

17. The optical scanning microscope (900) according to claim 16 further includes an acousto-optic device (902) disposed downstream of or as part of the main beam splitter (114), the acousto-optic device (902) being configured to receive a reverse-scanning detection light (118) and selectively guide a portion of the detection light (118) into the detection arrangement (102, 200, 300, 400, 500, 600, 700) and guide another portion of the detection light (118) not into the detection arrangement (102, 200, 300, 400, 500, 600, 700).

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