Detection device for optical scanning microscope and optical scanning microscope

Abstract

To provide a detection device for an optical scanning microscope.SOLUTION: A detection device includes a beam splitting element configured to receive descanned detection light, split the detection light into two portions, direct a first portion of the detection light into a first beam path, and direct a second portion of the detection light into a second beam path. The first beam path and the second beam path include, respectively, a dispersive element configured to spectrally separate the detection light along a spectral separation direction and an array detector configured to receive the spectrally separated detection light. The beam splitting element, a first dispersive element, and a second dispersive element are configured such that a first reference direction corresponding to a first spectral separation direction imaged back to a plane arranged before the beam splitting element via the first beam path and a second reference direction corresponding to a second spectral separation direction imaged back to the plane arranged before the beam splitting element via the second beam path are different from each other.SELECTED DRAWING: Figure 1

Description

[Technical Field]

[0001] The present invention relates to a detection device for an optical scanning microscope.The present invention further 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 beyond the capabilities of conventional confocal microscopy. In conventional confocal microscopy, a single point detector, such as a single photomultiplier tube, is used to detect fluorescence emitted from a sample. In the ISM approach, the point detector is replaced by a multi-element photodetector, which includes multiple photodetector elements (pixels) arranged in a photodetector array. Each photodetector element in the array is configured to output a detector signal upon receiving fluorescence. As the sample is scanned with the laser focus, each photodetector element detects a small image of the illuminated sample at each scan position. Appropriate algorithms are then used to combine the multiple scan images to reconstruct a single high-resolution image of the sample.

[0003] While information from various photodetector elements can be used to improve spatial image resolution and signal-to-noise ratio, one major limitation of current ISM is the limited availability of photodetectors to quantify the emitted fluorescence in terms of its spectral content. Existing methods, such as those described in F. Strasser et al., Biomed. Opt. Expr 10(2019) 2513, are limited to a narrow band of the visible light spectrum and suffer from light losses due to the formation of higher diffraction orders and the use of a single polarization state. Summary of the Invention [Problem to be solved by the invention]

[0004] The object here is to provide a detection device for an optical scanning microscope and an optical scanning microscope that enable spectral imaging with high spatial resolution and a high signal-to-noise ratio. [Means for solving the problem]

[0005] The above-mentioned object is achieved by the subject matter of the respective independent claims. Advantageous embodiments are defined in the respective dependent claims and in the following description.

[0006] The proposed detection device for an optical scanning microscope includes a beam splitting element configured to receive descanned detection light, split the detection light into two portions, and direct the first portion of the detection light to a first beam path and the second portion of the detection light to a second beam path. The first beam 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 beam 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, which is imaged back via the first beam path onto a plane located in front of the beam splitting element, and a second reference direction corresponding to the second spectral separation direction, which is imaged back via the second beam path onto a plane located in front of the beam splitting element, are mutually different.

[0007] The detection device is configured to receive the detection light from the descanner and can therefore be used, for example, as the detection device for an existing confocal laser scanning microscope or image scanning microscope. The descanned detection light typically corresponds to an image of a small particle, which emits detection light, e.g., fluorescence, as a result of being scanned by a focused illumination light beam, e.g., a laser focus used to excite a fluorescent material. The particle can be considered a point source of the detection light. The descanned detection light received by the detection device is split into a first portion and a second portion by a beam-splitting element, thereby generating two copies of the detection light. Each copy is then directed by one of the dispersive elements before being detected by one of the array detectors. When the detection light has a broadband of wavelengths, the dispersive element spectrally fanns out the detection light along one of the spectrally separated directions. However, when the detection light is monochromatic or consists of a narrow wavelength band, as is often the case in fluorescence microscopy, the dispersive element does not fan out the detection light but rather deflects the beam of detection light in the spectrally separated directions. Therefore, conclusions about the spectral composition of the detected light can be drawn based on the deflection by the dispersive element. The deflection of the detected light is detected by an array detector, which can be a two-dimensional array of photodetector elements. By comparing multiple consecutive images acquired by the array detector, changes in the deflection of the detected light form an apparent shift of the image of the detected light on each array detector. However, the deflection of the detected light caused by the dispersive element must be distinguished from the deflection caused by the scanning itself. For example, such shift can result from a shift of the illumination focus relative to the point source of the detected light, due to the convolution of the scanned illumination point spread function (PSF) with the point source. Because the detection of the detected light emitted from the point source is performed by the array detector, which provides spatial information in addition to intensity information, the image of the emitted particle appears to shift on the array detector even when the detected light received by the detection device is descanned.Therefore, the beam-splitting element, first dispersive element, and second dispersive element of the proposed detection device are arranged so that the first spectral separation direction for the image of the detected light at the first array detector is oriented differently from the second spectral separation direction for the image of the detected light at the second array detector. The movement of the point-like image due to scanning has no preferred direction and appears similar on each of the two array detectors. However, the movement due to, for example, a (virtual) color change of the particle caused by the dispersive element, e.g., a change in wavelength of the emitted light, particularly the wavelength change of the fluorescence emitted by the particle, always exists in different spectral separation directions for each of the array detectors. Therefore, the proposed detection device ensures that by comparing two images acquired by the two array detectors, the deflection of the detected light caused by the dispersive element can be distinguished from the deflection caused by the scanning itself. This not only enables image scanning microscopy with high spatial resolution and a high signal-to-noise ratio by detecting the detected light using two array detectors, but also allows the reconstruction of spectral information about the detected light without the risk of a significant loss of detected light compromising the signal-to-noise ratio.

[0008] In one embodiment, the first and second reference directions are opposite or perpendicular to each other. In such an arrangement, the apparent shift of the detected light image at the array detector due to color shifts is opposite or perpendicular, respectively. By selecting the reference directions in this way, the task of separating shifts due to color shifts from shifts due to the scanning itself is easier to perform, which means that spectral information can be extracted more reliably.

[0009] In another embodiment, a beam splitting element is configured to mirror or reflect the second portion of the detection light directed into the second detection beam path. The beam splitting element may be a neutral beam splitter configured to uniformly split the received and descanned detection light, preferably with a reflection-to-transmission ratio of 1 or close to 1, e.g., reflecting the second portion of the detection light and passing the first portion of the detection light. In this case, the image acquired by one of the multiple array detectors is mirrored compared to the other array detectors. This allows for easy identification of shifts due to scanning by finding mirror symmetry. This allows for separation of shifts due to color changes, allowing for more reliable extraction of spectral information.

[0010] In another embodiment, the beam splitting element is configured to rotate the second portion of the detection light directed into the second detection beam path. According to this embodiment, the image formed along the first beam path is rotated relative to the image formed along the second beam path. For example, the beam splitting element can include an Abbe-König prism that rotates the second portion of the detection light. Similar to mirroring the second portion of the detection light, by finding a rotational symmetry corresponding to the rotation of the second portion of the detection light, the movement due to scanning can be easily distinguished from the rotation of the second portion of the detection light.

[0011] In another embodiment, the beam splitting element is configured to direct a first polarization component of the detected light as a first portion into a first beam path and a second polarization component of the detected light as a second portion into a second beam path. In this embodiment, the detected light is split into a first portion and a second portion based on polarization. Assuming unpolarized light, such as fluorescent light, this means that the detected light is split approximately evenly. This means that the maximum possible amount of detected light is detected by each of the two array detectors, thereby reducing light loss and improving the signal-to-noise ratio. Alternatively, such an even split can be achieved by using a neutral beam splitter with a reflection / transmission ratio of 1 or close to 1.

[0012] In another embodiment, the beam splitting element includes an Abbe-König prism having a polarizing beam splitter coating configured to transmit a first polarization component of the detected light and reflect a second polarization component of the detected light. The Abbe-König prism rotates the second polarization component of the detected light. In this embodiment, the detected light is split into a first portion and a second portion based on polarization. Furthermore, the portion having the second polarization component is rotated. Thus, the Abbe-König prism not only ensures that the first and second portions are split approximately evenly, but also rotates the image acquired by the second array detector relative to the image acquired by the first array detector. The advantages of both features are described above.

[0013] In another embodiment, the beam splitting element includes a Wollaston prism. The Wollaston prism utilizes the optical property of birefringence to separate the detected light based on polarization. Unlike a polarizing beam splitter, the Wollaston prism does not reflect portions of the detected light, so both the first and second portions of the detected light maintain their original orientations. This can be exploited to capture the same image using an array detector, making it easier to separate shifts due to color changes.

[0014] In another embodiment, at least one of the first dispersive element and the second dispersive element includes at least one dispersive prism. The dispersive prism operates with a wide wavelength range of light, including the visible spectrum and portions of the infrared and ultraviolet spectrum. Furthermore, the dispersive prism does not produce higher diffraction orders that may be produced by a diffraction grating. Higher orders cannot be recorded by the array detector. Therefore, the use of 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 orientations of the first dispersive prism and the second dispersive prism are related by a single rotation about a rotation axis. For example, the rotation axis may be parallel to a side edge of the first dispersive prism. This corresponds to the case where the first and second beam paths are, for example, perpendicular to each other and do not include any additional reflecting elements. In combination with a beam-splitting element that reflects the second portion of the detection light, the first and second reference directions are opposite to each other, which has the advantages described above. Furthermore, this embodiment provides a particularly simple optical arrangement of the first and second beam paths.

[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 is rotated about its optical axis relative to the first dispersive prism. For example, the second dispersive prism is rotated about its optical axis directly in front of the beam splitting element or at the point of the second prism where the second portion of the detection light enters the second prism. The rotation here can be, in particular, 90° or 180°. This allows the first and second reference directions to be perpendicular or opposite to each other, respectively, with the advantages described above.

[0017] In another embodiment, the first and second dispersive elements are realized by different surfaces of a single dispersive prism. For example, the first and second dispersive elements can be realized by two different sides of a single dispersive prism. Such a configuration can be advantageously used in combination with a beam splitting element, such as a Wollaston prism, that splits the detected light into two beams that form an acute angle, particularly an angle of less than 45°.

[0018] In another embodiment, the first and second dispersive elements are realized by the same surface of a single dispersive prism. For example, the first and second beam paths are parallel to each other before being directed into the dispersive prism. The use of a single dispersive prism ensures consistent alignment and uniform dispersion of the detection light in the two beam paths. Furthermore, this embodiment allows for a particularly compact optical configuration of the detection device.

[0019] In another embodiment, at least one of the first dispersive element and the second dispersive element comprises at least one of a planar grating, preferably a blazed grating, a grism, and a diffractive optical element. In particular, a blazed grating provides high diffraction efficiency at a predetermined diffraction order, thereby reducing light loss and improving signal-to-noise ratio. A grism combines a dispersive prism and a diffraction grating and exhibits very low chromatic aberration. A diffractive optical element enables more complex light manipulation, allows control of the beam shape, and provides precise control of the phase and amplitude of the detected light.

[0020] In another embodiment, the first beam path does not include a first reflective element or includes at least one first reflective element, and the second beam path does not include a second reflective element or includes at least one second reflective element. The number of first reflective elements and the number of second reflective elements are both even or both odd. In other words, the number of reflective elements in the first beam path and the number of reflective elements in the second beam path are both even or both odd, with zero being treated as an even number. This ensures that the relative parity of the images formed by the beam splitting element is not changed by the reflective elements disposed in the first and second beam paths. For example, if the beam splitting element mirrors the second portion of the detected light, the images acquired by the first and second array detectors are mirrored. This allows the first and second dispersive elements to have different reference directions even though they are realized on the same surface of a dispersive prism.

[0021] Furthermore, it is advantageous if 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, which allows for maintaining consistency between the images acquired by the first array detector and the second array detector and simplifies the optical design of the detection device.

[0022] In another embodiment, the detector comprises a pinhole, preferably located upstream of the beam splitting element, i.e., in front of the beam splitting element, which eliminates out-of-focus light and thus improves imaging quality.

[0023] The present invention also relates to an optical scanning microscope, comprising: an excitation light source configured to generate excitation light; an objective lens directed toward a sample space and configured to direct the excitation light into the sample space and receive detection light from the sample space; and a scanning unit arranged along a beam path between the excitation light source and the objective lens and configured to selectively direct the excitation light via the objective lens to various regions of the sample space, e.g., in a meandering pattern. The optical scanning microscope further comprises a detection device as described above; and a main beam splitter configured to direct the excitation light via the scanning unit to the objective lens and to direct the detection light to the detection device.

[0024] The optical scanning microscope has the same advantages as the detection device described above. In particular, the optical scanning microscope can be complemented by the features described herein in relation to the detection device. Furthermore, the detection device described above can be complemented by the features described herein in relation to the optical scanning microscope.

[0025] In one embodiment, the optical scanning microscope further includes an acousto-optical device located downstream of or part of the main beam splitter. The acousto-optical device is configured to receive the descanned detection light and selectively direct a portion of the detection light to the detection device and not direct another portion of the detection light to the detection device. The excitation light can be reflected in the sample space by passing a leak of the main beam splitter to the detection device. The acousto-optical device can be controlled to selectively deflect predetermined wavelengths or predetermined wavelength bands. This property is used to deflect the leaked excitation light away from the detection device. An exemplary main beam splitter including an acousto-optical device is disclosed in WO 99 / 42884.

[0026] In another embodiment, the excitation light source is a pulsed laser light source, and at least one of the array detectors of the detection device is configured to record the arrival time of individual photons relative to the laser pulse generated by the excitation light source. By recording the arrival time of individual photons relative to a reference signal, the optical scanning microscope can be used to determine fluorescence lifetime characteristics. This allows the optical scanning microscope to be used in fluorescence microscopy applications, such as fluorescence lifetime imaging microscopy (FLIM).

[0027] In the following, specific embodiments will be described with reference to the drawings. [Brief explanation of the drawings]

[0028] [Figure 1] 1 is a schematic diagram illustrating an optical scanning microscope including a detection device according to one embodiment. [Figure 2] 1 is a schematic diagram illustrating a detection device according to an embodiment including two dispersive elements whose orientations are related by rotation about a single axis. [Figure 3] 1 is a schematic diagram illustrating a detection device according to an embodiment including two dispersive elements whose orientations are related by mirror symmetry; [Figure 4]1 is a schematic diagram illustrating a detection apparatus according to an embodiment including two dispersive elements related by rotation about an optical axis; [Figure 5] FIG. 10 is a schematic diagram showing a detection device according to another embodiment in which the two dispersive elements are formed by the same surface of a dispersive prism. [Figure 6] FIG. 10 is a schematic diagram illustrating a detection device according to another embodiment including a Wollaston prism. [Figure 7] FIG. 10 is a schematic diagram of a detection device according to another embodiment including a Wollaston prism and an Abbe-König prism. [Figure 8] FIG. 1 is a schematic diagram showing an Abbe-König prism with a polarizing beam splitter coating. [Figure 9] FIG. 1 is a schematic diagram illustrating an optical scanning microscope according to another embodiment including an acousto-optic device. DETAILED DESCRIPTION OF THE INVENTION

[0029] 1 is a schematic diagram of an optical scanning microscope 100 with a detection device 102 according to one embodiment. The optical scanning microscope 100 illustratively includes a single objective lens 104 directed toward a sample 106 disposed in a sample space 108. The optical scanning microscope 100 further includes an excitation light source 110, a scanning unit 112, and a main beam splitter 114.

[0030] The excitation light source 110 is configured to generate excitation light 116, particularly excitation light 116 having a single wavelength or a narrow wavelength band. The excitation light source 110 can include one or more lasers that generate laser light as the excitation light 116. In particular, the excitation light source 110 can include a continuous wave laser, an exchangeable filter device, or a tunable laser to selectively generate excitation light 116 having different wavelengths. The excitation light source 110 can also include additional optical elements, such as lenses and apertures, for shaping the beam from the excitation light 116. The excitation light 116 generated by the excitation light source 110 is directed toward the scanning unit 112 by the main beam splitter 114. The scanning unit 112 is configured to deflect and selectively direct the excitation light 116 to various regions of the sample space 108 via the objective lens 104. This allows the sample 106 to be scanned using the excitation light 116 focused by the objective lens 104. To deflect the excitation light 116, the scanning unit 112 may include, for example, one or more galvanometric mirrors or acousto-optical deflectors. The beam path of the excitation light is shown in Figure 1 using a dashed line starting from the excitation light source 110 and terminating at the sample 106.

[0031] The excitation light 116 is used to illuminate the sample 106, generating the detection light 118. The detection light 118 is collected by the objective lens 104 and directed back toward the main beam splitter 114 via the scanning unit 112. Because the scanning unit 112 is positioned between the main beam splitter 114 and the objective lens 104, the polarization state of the excitation light 116 is reversed with respect to the detection light 118. Here, regardless of the deflection angle of the scanning unit 112, the detection light 118 is directed toward a single point. The detection light 118 is, in effect, descanned. The descanned detection light 118 is then directed toward the detection device 102 by the main beam splitter 114. The beam path of the detection light 118 is shown in FIG. 1 using a dotted line starting from the sample 106.

[0032] The detector 102 includes a beam splitting element 120, a first dispersive element 122a, a second dispersive element 122b, a first array detector 124a, and a second array detector 124b. By way of example only, the detector 102 further includes a pinhole 126 located at a conjugate image plane.

[0033] The detection light 118 is received by the detection device 102 through a pinhole 126 that limits the beam diameter of the detection light 118 entering the detection device 102 and / or suppresses light coming from the focal region of the sample volume 108, so that only the detection light 118 coming from the focal region of the objective lens 104 enters the detection device 102. Following the pinhole 126, the detection light 118 reaches the beam-splitting element 120. In this embodiment, the beam-splitting element 120 is a neutral beam splitter that splits the incident light into two substantially identical portions. A first portion 128a of the detection light 118 is directed to a first beam path 130a that includes a first dispersive element 122a and a first array detector 124a. A second portion 128b of the detection light 118 is directed to a second beam path 130b that includes a second dispersive element 122b and a second array detector 124b. The dispersive elements 122a, 122b are illustratively shown as dispersive prisms, however, the dispersive elements 122a, 122b may include one or more of a dispersive prism, a planar grating, preferably a blazed diffraction grating, a grism, and a diffractive optical element.

[0034] In each of the two beam paths 130a, 130b, the detection light 118 first passes through a respective dispersive element 122a, 122b. Each of the dispersive elements 122a, 122b spectrally separates the detection light 118 along a spectral separation direction 132a, 132b by deflecting the detection light 118 based on its wavelength. For example, the dispersive elements 122a, 122b may deflect shorter wavelengths more than longer wavelengths. The spectral separation directions 132a, 132b may coincide with each other but are essentially independent of each other. The first dispersive element 122a spectrally separates the detection light 118 along the first spectral separation direction 132a, and the second dispersive element 122b spectrally separates the detection light 118 along the second spectral separation direction 132b.

[0035] The spectrally separated detected light 118 is then received by array detectors 124a and 124b. Each of the array detectors 124a and 124b includes 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) PMTs. Each detector element functions as a single pixel detector that captures a portion of the spectrally separated detected light 118 at a different position in the array. Thus, the array detectors 124a and 124b can detect a spatial distribution of the intensity of the spectrally separated detected light 118. As the sample 106 is scanned with the excitation light 116, at least one spatial distribution is detected by each of the array detectors 124a and 124b at each scan position. From the collection of spatial distributions, a single high-resolution image of the sample 106 can be reconstructed. This imaging technique is known as image scanning microscopy (ISM) and offers improved spatial resolution and signal-to-noise ratio compared to conventional confocal laser scanning microscopy (CLSM).

[0036] During scanning of the sample 106, a single particle, e.g., a single fluorophore, can be imaged multiple times in successive steps. Therefore, movement of the excitation light 116 can also cause a movement of the particle's image on the array detectors 124a, 124b. Similarly, a change in particle color can result in different deflection angles by the dispersive elements 122a, 122b, thus causing an apparent movement of the image. By distinguishing between these two types of movement, spectral information can be extracted, extending the capabilities of ISM technology.

[0037] The beam splitting element 120, the first dispersive element 122a, and the second dispersive element 122b are positioned and configured to allow for a calculated separation of the scanning and spectral separation effects. The first spectral separation direction 132a defines a first reference direction 134a that is perpendicular to the optical axis of the objective lens 104 when imaged back onto the sample 106. Similarly, the second spectral separation direction 132b defines a second reference direction 134b that is perpendicular to the optical axis of the objective lens 104 when imaged back onto the sample 106. The above-mentioned elements are positioned and configured such that the first reference direction 134a and the second reference direction 134b are different, e.g., perpendicular to each other. In other words, the first spectral separation direction 132a for the image of detected light 118 at the first array detector 124a is oriented differently from the second spectral separation direction 132b for the image of detected light 118 at the second array detector 124b. Various embodiments of detection device 102 embodying such configurations are described below with reference to Figures 2-8.

[0038] To separate the effects of scanning and spectral separation, a multi-view deconvolution algorithm can be used, which finds a reconstruction by, for example, minimizing the difference between the detected spatial distribution and the reconstructed signal. The reconstructed signal can be calculated from the estimated reconstruction using a signal formation model. Multi-view deconvolution can also be combined with pixel reassignment techniques. For example, pixel reassignment can be performed in a direction perpendicular to the spectral separation directions 132a and 132b, and multi-view deconvolution can be performed along the spectral separation directions 132a and 132b.

[0039] The detected light 118 may include fluorescence produced by fluorophores excited by the excitation light 116. However, the detected light 118 may also include a small amount of excitation light 116 that has been reflected, for example, in the sample space 108. This excitation light 116 may also leak through the main beam splitter 114 to, or in other words, into, the detection device 102. An embodiment that addresses this leakage of excitation light 116 is described below with reference to FIG.

[0040] FIG. 2 is a schematic diagram of a detection device 200 according to one embodiment.

[0041] The detection device 200 according to FIG. 2 includes a beam splitting element 120, exemplarily formed as a neutral beam splitter with a reflection / transmission ratio of 1. Such a beam splitter is also referred to as a 50:50 beam splitter. The beam splitting element 120 reflects the second portion 128b of the detection light 118, thereby mirroring the second portion 128b. In FIG. 2, a first arrow A1 indicates the movement of an imaged particle, e.g., a single fluorophore, relative to the optical axis. Furthermore, arrows A2a and A2b indicate the movement directions of the particle image relative to the optical axis in the first beam path 130a and the second beam path 130b, respectively. As can be seen, the movement in the second beam path 130b is mirrored compared to the movement in the first beam path 130a due to the second portion 128b of the detection light 118 being reflected by the beam splitting element 120.

[0042] The detection device 200 further includes two dispersive elements 202a and 202b, illustratively formed as dispersive prisms with triangular bases. The triangular bases of the two dispersive prisms are parallel. The orientations of the dispersive elements 202a and 202b are related only by rotation about a single axis R. In FIG. 2, the axis R here is perpendicular to the plane of the drawing, i.e., it intersects the plane of the drawing where the detection light 118 is divided into the first beam path 130a and the second beam path 130b. In other words, the orientations of the dispersive elements 202a and 202b are related by rotation and lateral displacement with respect to the side edges of the dispersive prisms. This arrangement of dispersive elements 202a, 202b, combined with the mirrored image of detected light 118 in second beam path 130b, results in a first spectral separation direction 132a for the image of detected light 118 at first array detector 124a that is oriented differently than a second spectral separation direction 132b for the image of detected light 118 at second array detector 124b. The embodiment shown in Figure 2 provides a detection apparatus 200 in which first and second reference directions 134a, 134b are opposite to each other.

[0043] Figure 3 is a schematic diagram of a detection arrangement 300 according to another embodiment. The detection arrangement 300 according to Figure 3 differs from the detection arrangement 200 according to Figure 2 in the orientation of the dispersive elements 202a, 202b and in that it includes an additional reflective element 302 in the second beam path 130b.

[0044] The reflecting element 302 is positioned in the second beam path 130b so that the first beam path 130a and the second beam path 130b are parallel before hitting the first dispersive element 202a and the second dispersive element 202b, respectively. A second reflection in the second beam path 130b restores the parity of the images of the detected light 118 in the two beam paths 130a and 130b, which means that, unlike the embodiment described above with reference to FIG. 2, the images on the first array detector 124a and the second array detector 124b are not mirrored. As in FIG. 2, in FIG. 3, a first arrow A1 indicates the movement of an imaged particle relative to the optical axis, and further arrows A2a and A2b indicate the movement directions of the particle images relative to the optical axis in the first beam path 130a and the second beam path 130b, respectively.

[0045] Similar to the embodiment described above with reference to FIG. 2, the bases of the two dispersing prisms forming the dispersive elements 202a and 202b, illustratively shaped as triangular bases, are parallel. However, unlike the embodiment described above with reference to FIG. 2, the orientations of the dispersive elements 202a and 202b are related by mirror symmetry, such that the first spectral splitting direction 132a faces downward in FIG. 3 and the second spectral splitting direction 132b faces upward in FIG. 3, i.e., the first spectral splitting direction 132a and the second spectral splitting direction 132b face away from each other. This relates to the fact that the parity of the image on the first array detector 124a and the parity of the image on the second array detector 124b are equal, and therefore the first reference direction 134a and the second reference direction 134b are opposite to each other.

[0046] Figure 4 is a schematic diagram of a detection arrangement 400 according to another embodiment. The detection arrangement 400 according to Figure 4 differs from the detection arrangement 200 according to Figure 2 in that the orientations of the dispersive elements 202a, 202b are related by a rotation about the optical axis.

[0047] The second dispersive element 202b is rotated about 90° about its optical axis compared to the first dispersive element 202a when the optical axis upstream of the beam splitting element 120 is used as a reference. This arrangement, combined with the mirror symmetry between the images of the first array detector 124a and the second array detector 124b, results in the first reference direction 134a and the second reference direction 134b being perpendicular to each other.

[0048] Figure 5 is a schematic diagram of a detection device 500 according to another embodiment. In the embodiment shown in Figure 5, two dispersive elements 502a, 502b are formed by identical surfaces 504a, 504b of a single dispersive prism 506, illustratively having a triangular base.

[0049] 5 includes a beam splitting element 120, which may be a 50:50 neutral beam splitter. A first portion 128a of the detection light 118 is formed by the detection light 118 passing through the beam splitting element 120 into a first beam path 130a. A second portion 128b of the detection light 118 is formed by the detection light 118 being reflected by the beam splitting element 120 into a second beam path 130b. This means that the second portion 128b of the detection light 118 is mirror-symmetrical compared to the first portion 128a of the detection light 118 directly behind the beam splitting element 120. The first beam path 130a includes three reflecting elements 508a that direct the first portion 128a of the detection light 118 towards a first surface 504a of a dispersing prism 506. The first portion 128a of the detected light 118 passes through the dispersing prism 506 and exits the dispersing prism 506 via the second surface 504b. Due to dispersion, i.e., the wavelength-dependent change in the refractive index, different wavelengths of the detected light 118 are refracted at different angles. Thus, the first surface 504a and the second surface 504b of the dispersing prism 506 form a first dispersive element 502a. The second beam path 130b includes a single reflective element 508b that directs the second portion 128b of the detected light 118 toward the first surface 504a of the dispersing prism 506. Similar to the first portion 128a, the second portion 128b of the detected light 118 passes through the dispersing prism 506 and exits the dispersing prism 506 via the second surface 504b. Thus, similarly, the first surface 504a and the second surface 504b of the dispersing prism 506 form a second dispersive element 502b. This makes the first spectrally separated direction 132a equal to the second spectrally separated direction 132b.

[0050] The number of reflective elements 508a in the first beam path 130a and the number of reflective elements 508b in the second beam path 130b are both odd numbers, i.e., three and one, respectively. This means that the mirror symmetry between the first portion 128a and the second portion 128b of the detected light 118 is maintained. Alternatively, the number of reflective elements 508a in the first beam path 130a and the number of reflective elements 508b in the second beam path 130b may both be even numbers, e.g., four and two, respectively. In this way, the parity of the images acquired by the first array detector 124a and the second array detector 124b is restored.

[0051] One of the reflecting elements 508a in the first beam path 130a is positioned so that the first portion 128a of the detection light 118 is not only mirrored but also rotated. In FIG. 2, a first arrow A1 indicates the movement of the imaged particle relative to the optical axis, and other arrows A2a and A2b indicate the direction of movement of the particle's image relative to the optical axis in the first beam path 130a and the second beam path 130b, respectively. As can be seen from FIG. 5, the images of the first array detector 124a and the second array detector 124b are not only mirrored but also rotated by an angle of 90° relative to each other. Because the first spectral separation direction 132a and the second spectral separation direction 132b are equal, the first reference direction 134a and the second reference direction 134b are therefore perpendicular to each other.

[0052] 6 is a schematic diagram of a detection apparatus 600 according to another embodiment, in which the beam splitting element 120 includes a Wollaston prism 602 that splits the detection light 118 according to polarization.

[0053] The Wollaston prism 602 splits the incident detection light 118 into two orthogonally polarized beams diverging at a specific angle, thereby generating a first portion 128a and a second portion 128b of the detection light 118. The first portion 128a of the detection light 118 is then directed to a first surface 606a of the dispersing prism 604. The second portion 128b of the detection light 118 is directed to a second surface 606b of the same dispersing prism 604. The first surface 606a and the second surface 606b illustratively form a 90° angle. Both the first portion 128a and the second portion 128b of the detection light 118 exit the dispersing prism 604 via a third surface 606c before being directed to the first array detector 124a and the second array detector 124b, respectively. 6, the first surface 606a and the third surface 606c of the dispersing prism 604 form the first dispersive element 608a, and the second surface 606b and the third surface 606c of the dispersing prism 604 form the second dispersive element 608b. Thus, in this embodiment, the first dispersive element 608a and the second dispersive element 608b can also be considered to be two dispersive prisms each having a quadrilateral base that shares a fourth side, which is shown as a dashed line in FIG. 6. Due to the arrangement of the first surface 606a and the second surface 606b, the first spectral splitting direction 132a and the second spectral splitting direction 132b are opposite to each other, with the first spectral splitting direction 132a facing downward in FIG. 6 and the second spectral splitting direction 132b facing upward in FIG. 6. In this embodiment, this also makes the first reference direction 134a and the second reference direction 134b opposite to each other.

[0054] In the embodiment shown in FIG. 6 , the dispersive prism 604 forming the dispersive element is illustratively made of two sections 610a and 610b that together form a rectangular parallelepiped. Each section 610a and 610b is made of a different material. The refractive indices of the different materials are selected so that the dispersive prism 604 effectively acts as a parallel plate for a predetermined center wavelength. This minimizes astigmatism and improves imaging quality. A similar dispersive prism combining two different materials that effectively act as parallel plates for a predetermined center wavelength can be used in any embodiment that uses a dispersive prism as the first dispersive element 122a, 202a, 502a, 608a, 710a and / or the second dispersive element 122b, 202b, 502b, 608b, 710b.

[0055] In general, the spectral dispersion, i.e., the size of the point spread function (PSF), achieved by the dispersive elements 122a, 122b, 202a, 202b, 502a, 502b, 608a, 608b, 710a, 710b relative to the diffraction limit can be adjusted by the choice of material and cut angle of the dispersive prism, as well as by the overall optical design of the detector 102, 200, 300, 400, 500, 600, 700, such that, for example, the focal length and pupil diameter are selected accordingly. The choice of spectral dispersion depends on the desired balance of spectral resolution and signal-to-noise ratio for the detector 102, 200, 300, 400, 500, 600, 700, and also on economic considerations regarding the number of array detectors 124a, 124b, where these array detectors 124a, 124b necessarily include readout electronics.

[0056] Figure 7 is a schematic diagram of a detection device 700 according to another embodiment. The detection device 700 according to Figure 7 differs from the detection device 600 according to Figure 6 in that the beam splitting element 702 additionally comprises an Abbe-König prism 704 with a polarizing beam splitter coating.

[0057] The Abbe-König prism 704 rotates one polarization component 118b of the detected light 118, while the other polarization component 118a passes through the Abbe-König prism 704 unrotated. The polarization that is rotated is determined by the polarizing beam splitter coating. The detected light 118 then strikes the Wollaston prism 602, which deflects the two polarization components 118a and 118b at different angles to produce first and second portions 128a and 128b of the detected light 118. The operation of the Abbe-König prism 704 is described in more detail below with reference to FIG. 8.

[0058] By way of example only, the detection device 700 according to Fig. 7 includes a dispersing prism 706 having two surfaces 708a, 708b that form a first dispersing element 710a and a second dispersing element 710b. As with the embodiment described above with reference to Fig. 5, this results in the first spectral separation direction 132a and the second spectral separation direction 132b being equal. Furthermore, the Wollaston prism 602 is positioned such that the first beam path 130a and the second beam path 130b lie in a plane parallel to the side edges of the dispersing prism 706, i.e., in a plane parallel to the third surface 708c of the triangular dispersing prism 706. This results in the first beam path 130a and the second beam path 130b intersecting the first surface 706a at the same height above the third surface 708c.

[0059] 2, a first arrow A1 indicates the movement of the imaged particle relative to the optical axis, and other arrows A2a, A2b indicate the direction of movement of the particle image relative to the optical axis for the two polarization components 118a, 118b. As can be seen in Figure 7, since the first and second spectral separation directions 132a, 132b are equal, a rotation of one of the polarization components 118a, 118b causes the first and second reference directions 134a, 134b to be opposite in direction.

[0060] 2-7 do not explicitly include a pinhole 126. However, the pinhole 126 can be located in any embodiment upstream of the beam splitting element 120, 702, i.e., in the direction from which the detection light 118 arrives.

[0061] FIG. 8 is a schematic diagram of an Abbe-König prism 704 with a polarizing beam splitter coating.

[0062] The Abbe-König prism 704 includes two sections 800a and 800b. The first section 800a has the shape of an isosceles triangle with an apex angle of 125° and is located at the bottom of FIG. 8. The second section 800b has a rectangular parallelepiped shape with a notch 802 at its base. The notch 802 is sized to receive the first section 800a without forming an air gap between the first section 800a and the second section 800b. Thus, the first section 800a and the second section 800b contact each other to form a first optical surface 804a and a second optical surface 804b that form an angle of 125°. Each of the two optical surfaces 804a and 804b is coated with a polarizing beam splitter coating.

[0063] The first polarization component 118a of the detected light 118 is shown in FIG. 8 as a dashed line, and the second polarization component 118b of the detected light 118 is shown in FIG. 8 as a solid line. The first polarization component 118a is not reflected by the polarizing beam splitter coating and therefore passes through the first optical surface 804a and the second optical surface 804b without polarization. In contrast, the second polarization component 118b is reflected by the polarizing beam splitter coating. Therefore, the second polarization component 118b is reflected by the first optical surface 804a toward 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 downward again. The second polarization component is then reflected by the second optical surface 804b, thereby becoming parallel to the first polarization component 118a again. Both the first polarization component 118a and the second polarization component 118b exit the Abbe-König prism 704 parallel, but the optical path length of the second polarization component 118b through the Abbe-König prism 704 is longer.

[0064] Figure 9 is a schematic diagram of an optical scanning microscope 900 according to another embodiment. The optical scanning microscope 900 shown in Figure 9 differs from the optical scanning microscope 100 shown in Figure 1 in that it includes an acousto-optical device 902.

[0065] The acousto-optic device 902 is illustratively located downstream of the main beam splitter 114. However, the acousto-optic device 902 may be part of the main beam splitter 114 and 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 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 in the refractive index forms a diffraction grating in the acousto-optic medium 904, the properties of which are determined by the radio frequency applied to the transducer 906. The diffraction grating here diffracts light 116, 118 arriving at the acousto-optic device 902 into multiple orders based on the frequency and amplitude of the acoustic waves, and thus the radio frequency applied to the transducer 906.

[0066] In this embodiment of the optical scanning microscope 900, the acousto-optic device 902 is used to selectively deflect portions of the detection light 118 away from the detection system 102, for example, by directing portions of the detection light 118 to a beam dump or by directing portions of the detection light 118 away from the opening of the pinhole 126. For example, wavelengths of the excitation light 116 can be deflected away from the detection system 102, thereby preventing the excitation wavelengths from leaking into the detection system 102. Similarly, the acousto-optic device 902 is operable to pass only detection light 118 having predetermined wavelengths, for example, the emission wavelengths of one or more particular fluorophores disposed within the sample 106, to the detection system 102.

[0067] Elements having the same or similar function are designated by the same reference numerals throughout the figures. As used herein, the term "and / or" includes any and all combinations of one or more of the associated listed items and may be abbreviated as " / ".

[0068] While some aspects have been described in the context of an apparatus, it will be apparent that these aspects also represent a description of a corresponding method, where a block or apparatus corresponds to a step or feature of a step, and similarly, aspects described in the context of a step also represent a description of a corresponding block or item or feature of a corresponding apparatus. [Explanation of symbols]

[0069] 100 Optical Scanning Microscope 102 Detection device 104 Objective Lens 106 samples 108 Sample Space 110 Excitation light source 112 Scanning Unit 114 Main beam splitter 116 Excitation Light 118 Detection Light 118a,118b polarization component 120 Beam splitting element 122a, 122b Dispersion elements 124a, 124b Array detector 126 Pinhole 128a, 128b Detection light portion 130a, 130b Beam path 132a, 132b Spectral separation direction 134a,134b Reference direction 200 Detection Device 202a, 202b Dispersion elements 300 Detection Device 302 Reflective element 400 Detection Device 500 Detection Device 502a, 502b Dispersion elements 504a,504b surface 506 Dispersion Prism 508a, 508b Reflective elements 600 Detection Device 602 Wollaston Prism 604 Dispersion Prism 606a,606b,606c surface 608a, 608b Dispersion elements 610a,610b part 700 Detection Device 702 Beam Splitter 704 Abbe-König Prism 706 Dispersion Prism 708a,708b,708c surface 710a, 710b Dispersion elements 800a,800b part 802 Notch 804a,804b optical surface 900 Optical Scanning Microscope 902 Acousto-optical Device 904 Acousto-optic media 906 Transducer A1, A2a, A2b arrows R rotation axis

Claims

1. A detection device (102, 200, 300, 400, 500, 600, 700) for an optical scanning microscope (100, 900), the detection device (102, 200, 300, 400, 500, 600, 700) comprising: a beam splitting element (120, 702) configured to receive the descanned detection light (118), split the detection light (118) into two portions (128a, 128b), and direct a first portion (128a) of the detection light (118) to a first beam path (130a) and a second portion (128b) of the detection light (118) to a second beam path (130b); a first beam path (130a) including a first dispersive element (122a, 202a, 502a, 608a, 710a) configured to spectrally separate the detected light (118) along a first spectral separation direction (132a), and a first array detector (124a) configured to receive the spectrally separated detected light (118); a second beam path (130b) including a second dispersive element (122b, 202b, 502b, 608b, 710b) configured to spectrally separate the detected light (118) along a second spectral separation direction (132b) and a second array detector (124b) configured to receive the spectrally separated detected light (118); Including, the beam splitting element (120, 702), the first dispersive element (122a, 202a, 502a, 608a, 710a) and the second dispersive element (122b, 202b, 502b, 608b, 710b) are configured such that a first reference direction (134a) corresponding to the first spectral separation direction (132a) that is imaged back via the first beam path (130a) onto a plane arranged in front of the beam splitting element (120, 702) and a second reference direction (134b) corresponding to the second spectral separation direction (132b) that is imaged back via the second beam path (130b) onto the plane arranged in front of the beam splitting element (120, 702) are different from each other. A detection device (102, 200, 300, 400, 500, 600, 700).

2. The first reference direction (134a) and the second reference direction (134b) are opposite to each other or perpendicular to each other.

2. The detection device (102, 200, 300, 400, 500, 600, 700) of claim 1.

3. the beam splitting element (120) is configured to mirror a second portion (128b) of the detection light (118) directed onto the second beam path (130b); Detecting device (102, 200, 300, 400, 500) according to claim 1 or 2.

4. the beam splitting element (702) is configured to rotate a second portion (128b) of the detection light (118) that is directed onto the second beam path (130b); Detecting device (700) according to any one of claims 1 to 3.

5. the beam splitting element (120, 702) is configured to direct a first polarization component of the detection light (118) as the first portion (128a) into the first beam path (130a) and direct a second polarization component of the detection light (118) as the second portion (128b) into the second beam path (130b); Detecting device (600, 700) according to any one of claims 1 to 4.

6. the beam splitting element (702) includes an Abbe-König prism (704) having a polarizing beam splitter coating configured to transmit a first polarization component (118a) of the detection light (118) and reflect a second polarization component (118b) of the detection light (118); The detection device (700) of claim 5.

7. The beam splitting element (702) includes a Wollaston prism (602).

7. The detection device (700) of claim 5 or 6.

8. 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); 8. A detection device (102, 200, 300, 400, 500, 600, 700) according to any one of claims 1 to 7.

9. the first dispersive element (122a, 202a) includes a first dispersive prism, and the second dispersive element (122b, 202b) includes a second dispersive prism; the orientation of the first dispersion prism and the orientation of the second dispersion prism are related by a single rotation about a rotation axis (R); The detection device (102, 200, 400) of claim 8.

10. the first dispersive element (202a) includes a first dispersive prism, and the second dispersive element (202b) includes a second dispersive prism; the second dispersing prism is rotated about its optical axis relative to the first dispersing prism; 10. The detection device (400) of claim 8 or 9.

11. the first dispersive element (608a) and the second dispersive element (608b) are realized by different surfaces (606a, 606b, 606c) of a single dispersive prism (604); Detecting device (600) according to any one of claims 8 to 10.

12. the first dispersive element (502a) and the second dispersive element (502b) are realized by the same surfaces (504a, 504b) of a single dispersive prism (506); Detecting device (500) according to any one of claims 5 to 10.

13. at least one of the first dispersive element (122a) and the second dispersive element (122b) comprises at least one of a planar grating, preferably a blazed grating, a grism, and a diffractive optical element; 9. The detection device (102) according to any one of claims 1 to 8.

14. the first beam path (130a) does not include a first reflective element (508a) or includes at least one first reflective element (508a), and the second beam path (130b) does not include a second reflective element (508b) or includes at least one second reflective element (508b); The number of the first reflective elements (508a) and the number of the second reflective elements (508b) are both even or both odd. Detecting device (102, 200, 400, 500, 600, 700) according to any one of claims 1 to 13.

15. The detection device (102) has a pinhole (126). Detecting device (102) according to any one of claims 1 to 14.

16. An optical scanning microscope (100, 900), comprising: an excitation light source (110) configured to generate excitation light (116); an objective lens (104) directed toward a sample space (108) and configured to direct excitation light (116) into the sample space (108) and receive detection light (118) from the sample space (108); a scanning unit (112) disposed along a beam path between the excitation light source (110) and the objective lens (104) and configured to selectively direct excitation light (116) through the objective lens (104) to various regions of the sample space (108); A detection device (102, 200, 300, 400, 500, 600, 700) according to any one of claims 1 to 15, a main beam splitter (114) configured to direct excitation light (116) through the scanning unit (112) to the objective lens (104) and direct detection light (118) to the detector (102, 200, 300, 400, 500, 600, 700); Optical scanning microscope (100,900) including.

17. The optical scanning microscope (900) further includes an acousto-optical device (902) located downstream of or part of the main beam splitter (114); the acousto-optic device (902) is configured to receive the descanned detection light (118) and selectively direct a portion of the detection light (118) to the detection arrangement (102, 200, 300, 400, 500, 600) and not direct another portion of the detection light (118) to the detection arrangement (102, 200, 300, 400, 500, 600, 700).

17. The optical scanning microscope (900) of claim 16.

Images