Detection device, cascade detection device and optical scanning microscope

Abstract

To provide a detection device for an optical scanning microscope.SOLUTION: A detection device (102) for an optical scanning microscope (100) has a first beam path (130a) and a second beam path (130b), each comprising an array detector. The detection device further comprises an acousto-optic device (120) for receiving the descanned detection light (118) and for directing a first portion (128a) of the detection light into a first beam path, a second portion (128b) of the detection light into a second beam path, and a remaining portion (132) of the detection light into a third beam path (130c). The first and second portions of the detected light have at least one selected wavelength range determined by at least one frequency of the acoustic waves generated by the transducer (126) of the acousto-optic device. The detection device further comprises a control unit (134) for controlling the transducer to determine the at least one selected wavelength range.SELECTED DRAWING: Figure 1

Description

[Technical Field]

[0001] The present invention relates to a detection device for an optical scanning microscope and to a cascade detection device for an optical scanning microscope.The present 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 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 of the present invention is to provide a detection device for a light scanning microscope, a cascade detection device for a light scanning microscope, and a light scanning microscope that enable spectral imaging with high spatial resolution and a high signal-to-noise ratio. In particular, the object of the present invention is to ameliorate the limitations of the current availability of photodetectors for quantifying emitted fluorescence in terms of the spectral content of current image scanning microscopes. [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 apparatus for an optical scanning microscope comprises a first beam path including a first array detector. The detection apparatus further comprises an acousto-optical device configured to receive descanned detection light and direct a first portion of the detection light into the first beam path. The first portion of the detection light has at least one selected wavelength or at least one selected wavelength range determined by at least one frequency of an acoustic wave generated by a transducer of the acousto-optical device. A control unit is configured to control the transducer of the acousto-optical device to determine the at least one selected wavelength or at least one selected wavelength range.

[0007] The acousto-optic device can be configured to generate only one diffraction order, for example, as an acousto-optic device based on isotropic diffraction. In such a device, two polarizations of a given wavelength range or wavelength are diffracted into a single diffraction order. Thus, there will be one angularly dispersed diffracted output and a zeroth order transmitted output, e.g., the remaining portion of the detected light. Such an acousto-optic device can be an acousto-optic deflector (AOD).

[0008] Preferably, the detection apparatus further comprises a second beam path including a second array detector. The acousto-optic device is configurable to direct a second portion of the detection light into the second beam path. The second portion of the detection light can have at least one selected wavelength range or at least one selected wavelength determined by at least one frequency of the acoustic waves generated by the transducer of the acousto-optic device.

[0009] When an unpolarized light beam strikes an acousto-optical device, the light, whose wavelength range is determined by one or more radio frequencies applied to the transducer, is deflected into two beams, each with one of two orthogonal polarizations. These two light beams are also referred to as diffracted beams, or the +1st and -1st diffraction orders. The remaining light passes through the acousto-optical device substantially undiffracted. This remaining light is also referred to as the 0th order beam. The descanned detected light typically corresponds to an image of a small particle that is emitting detected light, e.g., fluorescent light, as a result of being scanned by a focused excitation light beam, e.g., a laser focus, used to excite the fluorophore. Such particles can be considered point sources of detected light. The detected light is typically unpolarized; for example, fluorescent light is typically unpolarized.

[0010] In the proposed detection system, an acousto-optical device is used to generate two diffracted beams, each containing a first and second portion of the detection light. Each of the two portions has a wavelength range determined by at least one radio frequency applied to the transducer and is one of two orthogonal polarizations. The remaining portion of the detection light passes through the acousto-optical device as a zeroth-order beam and is directed to a third beam path. The relative angle of the diffracted beam with respect to the zeroth-order beam increases as the wavelength of the diffracted light decreases. On the other hand, a small shift in the incident angle of the detection light causes an opposite change in the angle of the diffracted beam with respect to the zeroth-order beam. This means that as the angle between the zeroth-order beam and the +1st-order beam increases, the angle between the -1st-order beam and the zeroth-order beam decreases. The increase or decrease in the relative angle is detected as a change in the position where the diffracted light strikes an array detector, which can be a two-dimensional array of photodetector elements. The asymmetry between the relative angle changes can be used to infer whether the incident angle or wavelength of the detection light has changed. For example, the three-dimensional position and wavelength of the detection light source in the sample space can be determined using numerical inverse calculations of the optical scanning microscope's optics. This not only enables image scanning microscopy with high spatial resolution and a high signal-to-noise ratio by detecting the detection light using two array detectors, but also allows for the reconstruction of spectral information about the detection light without significant loss of the detection light, which would impair the signal-to-noise ratio. Furthermore, an acousto-optical device can be used to limit the range of wavelengths reaching the first and second array detectors, thereby excluding the wavelength range of the excitation light. In this case, the array detectors can prevent detection of the excitation light, which could create a strong background and lead to erroneous or incomplete extraction of the actual fluorescence signal. The ability to dynamically limit the wavelength range based on the currently used excitation light increases the selection of excitation wavelengths and enhances the flexibility of the detection system.

[0011] The detector is configured to receive detection light from the descanned device and can therefore be used as a detector on an existing confocal laser scanning microscope or image scanning microscope, for example.

[0012] In one embodiment, the remaining portion of the detected light is directed to a third beam path. The third beam path can include a detector element, particularly a third array detector or a non-array detector. The third beam path can also include a focusing element configured to focus the remaining portion of the detected light onto the detector element. The non-array detector is preferably a non-imaging detector, also referred to as a single pixel detector, such as a photomultiplier tube (PMT), photodiode, avalanche photodiode (APD), or a hybrid detector that combines the high sensitivity of a photomultiplier tube with the low noise and high quantum efficiency of an avalanche photodiode. The third array detector can be used to perform image scanning microscopy without reconstructing spectral information from the remaining detected light. This spectral information can be used to supplement the information provided by the first and second array detectors. For example, because the remaining detected light does not diffract, changes in the position at which the remaining detected light, i.e., the zero-order beam, strikes the third array detector are due solely to changes in the angle of incidence of the detected light. Therefore, the shift in the image of the remaining detected light in the third array detector can be used to separate the shift caused by the color change of the first detected light and the second detected light in the first array detector and the second array detector, respectively.

[0013] In another embodiment, the third beam path includes a beam dump. In such an embodiment, the remaining detected light is discarded. The beam dump safely absorbs and dissipates all of the remaining detected light and leaked excitation light, preventing damage to the detection device. Preferably, the beam dump is configured to be removable. In this way, the beam dump can be replaced with a detector element.

[0014] In another embodiment, the third beam path includes a pinhole disposed in front of the detector element. According to this embodiment, the detector element disposed in the third beam path is preferably a non-array detector. Such a configuration with a pinhole and a non-array detector enables the detection device to be used in confocal microscopy. This makes it possible to extract information from the remaining detected light that is complementary in wavelength to the information extracted from the first and second portions of the detected light. Alternatively, the acousto-optical device can be operated so that the detected light passes through the acousto-optical device without being affected, and the detection device can be used to perform confocal microscopy using the detected light.

[0015] In another embodiment, the detector element is a spectrally unmixed detector element that can extract spectral information from the remaining detected light that can complement the spectral information obtained by the first and second array detectors. An exemplary spectrally unmixed detector element is disclosed in U.S. Patent No. 5,886,784.

[0016] In another embodiment, the detection arrangement comprises a pinhole arranged in front of the acousto-optical device, in which the pinhole is arranged in front of the acousto-optical device in the propagation direction of the detection light, i.e., the detection light passes through the pinhole before reaching the acousto-optical device, and the pinhole blocks out-of-focus light and prevents scattered light from reaching the acousto-optical device.

[0017] In another embodiment, the first beam path includes at least one first beam deflecting element, particularly a first dispersive element configured to spectrally separate the first portion of the detection light. Alternatively or additionally, the second beam path includes at least one second beam deflecting element, particularly a second dispersive element configured to spectrally separate the second portion of the detection light. The angle between the two diffracted beams containing the first and second portions of the detection light may be too small to accommodate the array detector, but this maximizes the efficient use of the array detector's surface. The beam deflecting element can be used to deflect the first and second portions of the detection light to the first and second array detectors, thereby optimizing or utilizing the spatial arrangement of the detection device. Additionally, the dispersive element can be used to increase the difference in deflection angle between different wavelengths, thereby making the angle change more pronounced. This not only allows for more efficient use of the array detector's surface, but also makes color changes easier to detect. For example, the dispersion of the dispersive element can be selected so that the entire surface of the array detector can be covered by the net dispersion provided by the acousto-optic device and wavelengths in the range of 400 nm to 850 nm.

[0018] 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.

[0019] In another embodiment, the detection apparatus includes a detector element having a first region forming at least a portion of a first array detector and a second region forming at least a portion of a second array detector. The first beam path can include at least one first reflective element configured to direct a first portion of the detection light toward the first region of the detector element. The second beam path can include at least one second reflective element configured to direct a second portion of the detection light toward the second region of the detector element. In this embodiment, the first array detector and the second array detector are implemented by a single detector element. The first array detector is implemented by the first region of the detector element, and the second array detector is implemented by the second region of the detector element. Such an optical arrangement can be particularly compact, minimizing the overall footprint of the detection apparatus. Furthermore, using a single detector element can be more cost-effective than using two dedicated array detectors.

[0020] In another embodiment, the first beam path includes at least one first focusing element configured to focus a first portion of the detection light onto the first array detector. Alternatively or additionally, the second beam path includes at least one second focusing element configured to focus a second portion of the detection light onto the second array detector. The first focusing element can be used to form an image at the first array detector from the first portion of the detection light. For example, the image can be an image of a point source of the detection light. From a collection of these images, each corresponding to a different scan position, a single high-resolution image of the sample can be reconstructed using an appropriate algorithm. Similarly, the second focusing element can be used to form an image at the second array detector from the second portion of the detection light. Alternatively, at least one focusing element can be disposed upstream of the acousto-optical device to focus the first portion of the detection light onto the first array detector and the second portion of the detection light onto the second array detector.

[0021] The invention also relates to a cascade detection system, which comprises at least two of the above-described detection systems, each of which is arranged one after the other, and each detection system, except for the first detection system, is arranged in the third beam path of the preceding detection system.

[0022] In a cascaded detection system, the remaining portion of the detected light from a preceding detection system serves as the detected light for a subsequent detection system. Prior art acousto-optical devices can separate up to eight different wavelength ranges. Therefore, a single detection system with up to eight different wavelength ranges can be used for simultaneous inspections. Cascaded detection systems allow for this increased number. In addition to this, cascaded detection systems have the same advantages as the detection systems described above. In particular, cascaded detection systems can be complemented by the features described herein in relation to the detection systems. Furthermore, the detection systems and cascaded detection systems described above can also be complemented by the features described herein in relation to cascaded detection systems.

[0023] The present invention also relates to an optical scanning microscope having an excitation light source configured to generate excitation light and 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. The optical scanning microscope also includes a scanning unit disposed 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 different regions of the sample space, and a detection device or cascade detection device as described above. The scanning optical microscope further includes 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 or cascade detection device.

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

[0025] In one embodiment, the main beam splitter includes at least one of an acousto-optic beam splitter and a dichroic beam splitter. The acousto-optic beam splitter can be configured to receive the descanned detection light and selectively direct a portion of the detection light to the detection device (or cascaded detection device) and not to 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. Similar to the acousto-optic device in the detection device, the acousto-optic beam splitter can be controlled to selectively deflect specific wavelengths or wavelength bands. In this embodiment, the above properties can be used to deflect the leaked excitation light away from the detection device. Alternatively, a dichroic beam splitter can be used.

[0026] In another embodiment, the excitation light source includes a supercontinuum laser. The excitation light source may further include an exchangeable filter or acousto-optical device to select a specific wavelength from the laser light generated by the supercontinuum laser as the excitation light. In this embodiment, laser light having multiple different wavelengths can be dynamically generated as the excitation light. This allows the excitation light to be adapted to the excitation spectra of many different fluorophores, making the optical scanning microscope even more versatile. In addition to or instead of a supercontinuum laser, the excitation light source may include multiple single-wavelength lasers.

[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] FIG. 10 is a schematic diagram illustrating a detection apparatus including additional detector elements, according to one embodiment. [Figure 3] FIG. 10 is a schematic diagram illustrating a detection apparatus according to another embodiment, in which the additional detector elements are array detectors. [Figure 4] FIG. 10 is a schematic diagram illustrating a detection device with a beam dump according to another embodiment. [Figure 5] 10 is a schematic diagram illustrating a detection device with a beam deflection element according to another embodiment. [Figure 6] 6 is a schematic diagram showing the positions of various focal spots on the array detector of the detection arrangement according to FIG. 5; [Figure 7] 6 is a graph showing the relative position of the focal spot for different wavelengths in a detection arrangement according to FIG. 5; [Figure 8] 6 is a graph showing the relative position of the focal spot for different radio frequencies applied to the transducer of the acousto-optic device of the detection arrangement according to FIG. 5; [Figure 9] 6 is a graph showing the wavelength deflected by the acousto-optical device for various radio frequencies applied to the transducer of the acousto-optical device of the detection arrangement according to FIG. 5; [Figure 10] 6 is a graph showing the relative position of the focal spot corresponding to a wavelength of 540 nm for various tilt angles of the incident detection light of the detection device according to FIG. 5; [Figure 11] 6 is a graph showing the relative position of the focal spot corresponding to a wavelength of 440 nm for various tilt angles of the incident detection light of the detection device according to FIG. 5; [Figure 12] FIG. 10 is a schematic top view illustrating a detection apparatus including detector elements forming two array detectors according to another embodiment. [Figure 13] FIG. 13 is a schematic perspective view of the detection device according to FIG. [Figure 14]FIG. 1 is a schematic diagram illustrating a cascade detection device according to one embodiment. [Figure 15] 10 is a schematic diagram showing a detection device with only one beam path according to another embodiment; 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 one or more single wavelengths or narrow wavelength bands. The excitation light source 110 may include one or more lasers that generate laser light as the excitation light 116. In particular, the excitation light source 110 may 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 may also include additional optical elements, such as lenses and apertures (not shown in FIG. 1 ) that shape 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. The scanning unit 112 may include, for example, one or more galvanometric mirrors or acousto-optic deflectors to deflect the excitation light 116. Arrow A1 in Figure 1 indicates the beam path and light propagation direction of the excitation light 116.

[0031] The excitation light 116 is used to illuminate the sample 106, generating the detected light 118. In FIG. 1 , it is assumed, purely for illustrative purposes, that the detected light 118 is unpolarized fluorescent light from two different types of fluorophores. Thus, the detected light contains two different components, each with a different wavelength range. The detected 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 relative to the detected light 118. Now, regardless of the deflection angle of the scanning unit 112, the detected light 118 is directed toward a single point. The detected light 118 is, in effect, descanned. The descanned detected light 118 is then directed toward the detector 102 by the main beam splitter 114. Arrow A2 in FIG. 1 indicates the beam paths and light propagation directions of two different components of detection light 118 (and, for example, excitation light reflected by the sample) leaving sample 106.

[0032] The detection apparatus 102 includes an acousto-optic device 120, a first array detector 122a, and a second array detector 122b. Detection light 118 is received by the detection apparatus 102 through the acousto-optic device 120, which includes an acousto-optic medium 124 and a transducer 126. The acousto-optic device 120 splits the incident detection light 118 into one or more beam pairs, also referred to as diffraction orders. Each beam pair contains light in a wavelength range determined by one of the radio frequencies applied to the transducer 126, where each beam in each pair is of one of two orthogonal polarizations. When a radio frequency is applied to the transducer 126, the transducer 126 generates acoustic waves in the acousto-optic medium 124. These acoustic waves locally modulate the refractive index within the acousto-optic medium 124. This refractive index modulation effectively forms a diffraction grating within the acousto-optic medium 124, the properties of which are determined by the radio frequency applied to the transducer 126. The diffraction grating diffracts the incident detection light 118 into diffraction orders that are based on the frequency and amplitude of the acoustic wave and, therefore, the radio frequency applied to the transducer 126. The radio frequency applied to the transducer 126 can be controlled using a control unit 134, which can be used to control which wavelength range or ranges are deflected by the acousto-optic device 120.

[0033] In FIG. 1 , two beam pairs are generated, one for each component of the detection light 118. The first beam of each beam pair is referred to as the +1st-order diffracted beam and has a first polarization. The +1st-order diffracted beam forms a first portion 128a of the detection light 118, which is deflected to a first beam path 130a including the first array detector 122a in FIG. 1 . The second beam of each beam pair is referred to as the −1st-order diffracted beam and has a second polarization orthogonal to the first polarization. The −1st-order diffracted beam forms a second portion 128b of the detection light 118, which is deflected to a second beam path 130b including the second array detector 122b in the lower part of FIG. 1 . The remaining portion 132 of the detection light 118 is referred to as the zeroth-order beam. The zeroth-order beam passes through the acousto-optic device 120 and is directed to a third beam path 130c.

[0034] The first and second portions 128a and 128b of the detected light 118 are received by the array detectors 122a and 122b. Because both diffraction orders and thus both polarization directions of the detected light 118 are detected, the detection device 102 can operate on at least the specific selected wavelength range of the detected light 118 without significant loss of the detected light 118. Each of the array detectors 122a and 122b includes an array of photodetector elements, preferably a two-dimensional array of photodetector elements, such as photodiodes, such as single-photon avalanche diodes (SPADs), or photomultiplier tubes, such as gallium arsenide phosphide (GaAsP) photomultiplier tubes (PMTs). Each photodetector element functions as a single pixel detector that captures a portion of the detected light 118 at a different location within the array. Thus, the array detectors 122a and 122b can detect the spatial distribution of the intensity of the detected light 118. As the sample 106 is scanned by the excitation light 116, at least one spatial distribution is detected by each array detector 122a, 122b at each scanning position. From the collection of spatial distributions, a single high-resolution image of the sample 106 can be reconstructed, for example, using pixel reassignment. Although not explicitly shown in FIG. 1 , a focusing element, such as a lens, can be disposed in the first beam path 130a to focus the first portion 128a of the detection light 118 onto the first array detector 122a. Correspondingly, another focusing element can be disposed in the second beam path 130b to focus the second portion 128b of the detection light 118 onto the second array detector 122b. This imaging technique, known as image scanning microscopy (ISM), can be applied in combination with conventional confocal laser scanning microscopy (CLSM) to increase spatial resolution and signal-to-noise ratio compared to conventional confocal laser scanning microscopy.

[0035] During scanning of the sample 106, a single particle, e.g., a single fluorophore, can be imaged multiple times in successive steps. The angle at which the descanned detection light 118, e.g., emission from an infinitesimal fluorescent-emitting particle, enters the acousto-optic device 120 depends on the particle's position relative to the center of the excitation spot. When the particle is at the center of the excitation spot (excitation Airy disk), the descanned detection light 118 propagates parallel to the descanned reflected excitation light 116. On the other hand, when the particle is displaced from the center of the excitation spot, the descanned emission light (detection light 118) propagates at a non-zero angle α relative to the descanned reflected excitation light 116. In a chromatically corrected optical system, this angle between the descanned detection light 118 and the descanned reflected excitation light 116 is independent of the color of the detection light 118 or emission light 118.

[0036] 1 can be used to decode not only the relative position of the emitting particle with respect to the center of the excitation spot, but also the color of the particle's emission radiation. For a given wavelength propagating along the direction of the reflected excitation light, i.e., with α=0, the geometry of the acousto-optic arrangement results in specific angles at which the two diffraction orders appear at the output of the acousto-optic device 120. In other words, for a given emission wavelength of the detection light 118, there is a nominal angle between the 0th order beam and the +1st diffracted beam, and a nominal angle between the 0th order beam and the −1st diffracted beam.

[0037] When α is not 0, the angle between the 0th order beam and the +1st order diffracted beam increases (or decreases), and the angle between the −1st order diffracted beam and the 0th order beam decreases (or increases).

[0038] It should be pointed out that in scanning microscopy, infinitesimal fluorescence-emitting particles are typically excited by a scanned excitation light beam, and the infinitesimal fluorescence-emitting particles typically have a fixed localization state. However, after the detection light has been descanned, the same considerations as above still apply to the detection light, since they depend on the relative position between the localization state of the excitation spot and the localization state of the tiny fluorescence-emitting particles as a function of time during scanning.

[0039] On the other hand, when the emission wavelength of the microparticles changes, the angle between the 0th order beam and the +1st order diffracted beam increases (or decreases), and the angle between the -1st order diffracted beam and the 0th order beam increases (or decreases).

[0040] When focusing lenses are used to focus the diffracted beams emerging from the acousto-optic device onto the respective array detectors, the change in the closing angle in the two scenarios described above is recorded as a shift in the image of a single particle on the array detectors 122a and 122b. By distinguishing between these two types of shifts, spectral information can be extracted, extending the capabilities of ISM technology.

[0041] The detected light 118 may include fluorescence light 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. To address this issue, the main beam splitter 114 may include an acousto-optic beam splitter.

[0042] The acousto-optic beam splitter may be used to selectively deflect portions of the detection light 118 away from the detector 102, for example, by directing the portion to a beam dump or away from a pinhole aperture. For example, the wavelength of the excitation light 116 may be deflected away from the detector 102, thereby preventing the excitation wavelength from leaking into the detector 102. Similarly, the acousto-optic device 120 may be operated to deflect only the detection light 118 in one or more predetermined wavelength ranges to the array detectors 122a, 122b, for example, to deflect a selected range of emission wavelengths of one or more particular fluorophores disposed within the sample 106.

[0043] FIG. 2 is a schematic diagram of a detection apparatus 200 according to one embodiment. The detection apparatus 200 according to FIG. 2 differs from the detection apparatus according to FIG. 1 in that it includes additional focusing elements 202a and 202b arranged in the first and second beam paths 130a and 130b. The first focusing element 202a is arranged in the first beam path 130a and focuses the first portion 128a of the detection light 118 onto the first array detector 122a, thereby generating, for example, an image of the light source of the detection light 118. Similarly, the second focusing element 202b is arranged in the second beam path 130b and focuses the second portion 128b of the detection light 118 onto the second array detector 122b. As the sample 106 is scanned by the excitation light 116, at least one image is acquired at each scanning position by each of the array detectors 122a and 122b. From these images, a single high-resolution image of the sample 106 can be reconstructed using suitable algorithms known from image scanning microscopy, for example pixel reallocation.

[0044] 2 further includes a third focusing element 202c and a detector element 204 disposed in a third beam path 130c. The third focusing element 202c is configured to focus the remaining portion 132 of the detected light 118 onto the detector element 204. The detector element 204 may be a third array detector, which allows the detection apparatus 200 to perform image scanning microscopy using the remaining detected light 132. Furthermore, the acousto-optical device 120 is operable to direct all of the detected light 118 onto the third beam path 130c. This allows the detection apparatus 200 to be used for conventional image scanning microscopy without extracting any spectral information.

[0045] Figure 3 is a schematic diagram of another embodiment of a detection apparatus 300. The detection apparatus 300 shown in Figure 3 differs from the detection apparatus 200 shown in Figure 2 in that the detector elements 302 are non-array detectors and pinholes 304 are positioned in front of the detector elements 302.

[0046] In this embodiment, the detection apparatus 300 is configured to perform confocal imaging using the remaining detected light 132. This allows the detection apparatus 300 to perform image scanning microscopy and confocal imaging simultaneously. Similar to the embodiment described above with reference to FIG. 2, the acousto-optical device 120 can also be operated so that all of the detected light 118 is directed to the third beam path 130c. This allows a user to selectively use the detection apparatus 300 for either image scanning microscopy or confocal imaging. The detector elements 302 can be detector elements 302 that perform spectral resolution. This allows the detection apparatus 300 to also be used for spectrally resolved confocal imaging.

[0047] Figure 4 is a schematic diagram of a detection apparatus 400 according to another embodiment. The detection apparatus 400 according to Figure 4 differs from the detection apparatus 200 according to Figure 2 in that the third beam path 130c has a beam dump 402. According to this embodiment, the remaining detected light 132, i.e., the detected light 118 that is not acquired by the first array detector 122a and the second array detector 122b, is discarded by being directed to the beam dump 402, where it is absorbed and dissipated.

[0048] The detector elements 204, 302 (shown in Figures 2 and 3, respectively) and the beam dump 402 located in the third beam path 130c may be removable, allowing them to be replaced by one of the beam dumps 402 or detector elements 204, 302. The detector elements 204, 302 and the beam dump 402 may be interchangeable with another detection arrangement 102, 200, 300, thereby forming a cascaded detection arrangement 1400, which is described below with reference to Figure 14.

[0049] Figure 5 is a schematic diagram of a detection device 500 according to another embodiment. The detection device 500 according to Figure 5 differs from the detection device 200 according to Figure 2 in that the first beam path 130a and the second beam path 130b include beam deflection elements 502a, 502b.

[0050] The first beam deflecting element 502a is disposed in the first beam path 130a and deflects the first portion 128a of the detection light 118 away from the third beam path 130c. The first beam deflecting element 502a is formed, for example, as a dispersive element, more specifically, as a first dispersive prism. The second beam deflecting element 502b is disposed in the second beam path 130b and deflects the second portion 128b of the detection light 118 away from the third beam path 130c. Like the first beam deflecting element 502a, the second beam deflecting element 502b is also formed, for example, as a dispersive element, more specifically, as a second dispersive prism. Because the deflecting elements 502a and 502b are formed as dispersive prisms, they spectrally separate the first portion 128a and the second portion 128b of the detection light 118, further enhancing the spectral separation generated by the acousto-optic device 120. The dispersion produced by the dispersing prisms is selected such that in the first beam path 130a, the net dispersion provided by the acousto-optic device 120 and the dispersing prism 502a allows wavelengths in the range of 400 nm to 850 nm to cover the entire surface of the first array detector 122a, and in the second beam path 130b, the net dispersion provided by the acousto-optic device 120 and the dispersing prism 502b allows wavelengths in the range of 400 nm to 850 nm to cover the entire surface of the second array detector 122b.

[0051] 5, the first beam path 130a, the second beam path 130b, and the third beam path 130c illustratively each include one focusing element 504a, 504b, and 504c, each illustratively formed as a lens. The first focusing element 504a is disposed in the first beam path 130a between the first beam deflecting element 502a and the first array detector 122a. Similarly, the second focusing element 504b is disposed in the second beam path 130b between the second beam deflecting element 502b and the second array detector 122b. The third focusing element 504c is disposed in the third beam path 130c between the acousto-optical device 120 and the detector element 302.

[0052] FIG. 6 is a schematic diagram of the positions of various focal spots 600 on the first array detector 122a and the second array detector 122b of the detection apparatus 500 according to FIG. 5. Each focal spot 600 corresponds to a different wavelength and is labeled accordingly. The first and second portions 128a and 128b of the detected light 118 are spectrally separated by the acousto-optic device 120 and the beam deflection elements 502a and 502b, resulting in spatially separated focal spots 600 on the surfaces of the array detectors 122a and 122b. The left side of FIG. 6 shows the surface of the first array detector 122a. The right side of FIG. 6 shows the surface of the second array detector 122b. Each surface is shown in FIG. 6 with a coordinate system indicating the x and y positions of the focal spots. Five focal spots 600 are shown on each surface, corresponding to wavelengths of 440 nm, 540 nm, 640 nm, 740 nm, and 840 nm, respectively. Each focal spot 600 is located on a diagonal 602a, 602b of the respective surface. As can be seen from Figure 6, the focal spots 600 are well separated from each other, which means that different wavelengths can be clearly distinguished.

[0053] The well-separated focal spots 600 can also be seen from Figure 7, which is a graph 700 having the relative positions of the focal spots 600 on the vertical axis 702 and the wavelength on the horizontal axis 704 for the detection device 500 according to Figure 5. The relative positions are shown in meters, and the wavelengths in nanometers. The positions of the focal spots 600 corresponding to a wavelength of 540 nm were selected as a reference for the relative positions. A first curve 706a interpolates the relative positions of the focal spots 600 on the first array detector 122a, and a second curve 706b interpolates the relative positions of the focal spots 600 on the second array detector 122b. Both curves 706a and 706b are flat for longer wavelengths, which means that the distance between the focal spots 600 in that wavelength region is shorter than for shorter wavelengths.

[0054] FIG. 8 is a graph 800 having the relative position of the focal spot 600 on the vertical axis 802 and the radio frequency applied to the transducer 126 of the acousto-optic device 120 on the horizontal axis 804 for the detection apparatus 500 according to FIG. 5. The relative position is shown in meters and the radio frequency is shown in megahertz. As in FIG. 7, a first curve 806a interpolates the relative position of the focal spot 600 on the first array detector 122a, and a second curve 806b interpolates the relative position of the focal spot 600 on the second array detector 122b. As can be seen from FIG. 8, the relationship between the applied radio frequency and the relative position of the focal spot 600 is approximately linear over the range of radio frequencies shown in FIG. 8.

[0055] Graph 800 shown in FIG. 8 is best understood when read in conjunction with FIG. 9, which is a graph 900 having the radio frequency applied to the transducer 126 of the acousto-optic device 120 on the vertical axis 902 and the wavelength deflected by the acousto-optic device 120 on the horizontal axis 904 of the detection apparatus 500 according to FIG. 5. Curve 906 interpolates the radio frequency. The radio frequency is given in megahertz, and the wavelength deflected by the acousto-optic device 120 is given in nanometers. As can be seen from FIG. 9, to deflect shorter wavelengths, a higher radio frequency must be applied to the transducer 126. Similarly, to deflect longer wavelengths, a lower radio frequency must be applied to the transducer 126.

[0056] 5-9, the spacing between focal spots 600 corresponding to wavelengths in the range of 640 nm to 840 nm is smaller than the spacing between focal spots 600 corresponding to wavelengths in the range of 440 nm to 640 nm. If the spacing between focal spots 600 corresponding to wavelengths in the range of 640 nm to 840 nm is not large enough to perform robust pixel reallocation, a cascaded detection arrangement 1400 including multiple detection arrangements 500 in series can be used. The cascaded detection arrangement 1400 is described in more detail below with reference to FIG. 14.

[0057] 10 is a graph 1000 having the relative position of the focal spot 600 on the vertical axis 1002 and the tilt angle of the incident detection light 118 with respect to the optical axis 1004 for the detection device 500 according to FIG. 5. The relative position is shown in meters and the tilt angle in radians. The position of the focal spot 600 corresponding to an incident angle of 0 rad is chosen as a reference for the relative position. The focal spot 600 corresponds to a wavelength of 540 nm.

[0058] The amount of tilt depends on the amount of deviation of the point source of the detection light 118 from the optical axis of the objective lens 104. The amount of tilt also depends on the optical properties of the optical scanning microscope 100 between the sample 106 and the acousto-optical device 120, such as the magnification. In FIG. 10, it is assumed that the tilt angle of the incident detection light 118 incident on the acousto-optical device 120 varies from −4.5 mrad to +4.5 mrad. A first curve 1006a interpolates the relative position of the focal spot 600 on the first array detector 122a, and a second curve 1006b interpolates the relative position of the focal spot 600 on the second array detector 122b. As can be seen from FIG. 10, the two curves are nearly identical (the second curve 1006b is slightly steeper). This means that as the angle of incidence changes, the focal spot 600 moves in the same direction on the surfaces of the first array detector 122a and the second array detector 122b.

[0059] Figure 11 is another graph 1100 with the relative position of the focal spot 600 on the vertical axis 1102 and the tilt angle of the incident detection light 118 with respect to the optical axis 1104 for the detection device 500 according to Figure 5. As in Figure 10, the relative position is shown in meters and the tilt angle in radians. The position of the focal spot 600 corresponding to a wavelength of 540 nm and an angle of incidence of 0 rad is chosen as the reference for the relative position. In contrast to Figure 10, in Figure 11 the focal spot 600 corresponds to a wavelength of 440 nm.

[0060] The first curve 1106a interpolates the relative position of the focal spot 600 on the first array detector 122a, and the second curve 1106b interpolates the relative position of the focal spot 600 on the second array detector 122b. As can be seen from FIG. 11 , the two curves are spaced approximately 0.3 mm apart in the y-direction. This spacing is due to the fact that as the wavelength of the detected light 118 decreases, the angle between the zeroth order beam and both the +1st and −1st order diffracted beams increases. However, the slopes of the two curves 1106a and 1106b are nearly identical, indicating that the focal spot 600 moves in the same direction on the surfaces of the first and second array detectors 122a and 122b as the angle of incidence changes.

[0061] Fig. 12 is a schematic plan view of a detection device 1200 according to another embodiment. The detection device 1200 shown in Fig. 12 differs from the detection device 500 shown in Fig. 5 in that two array detectors 122a and 122b are formed by the surface of a single detector element 1202.

[0062] 5, the two array detectors 122a, 122b are separate elements of the detection apparatus 500, whereas in this embodiment, the two array detectors 122a, 122b are formed by a single detector element 1202. More specifically, the first array detector 122a is formed by a first region of the detector elements 1202, and the second array detector 122b is formed by a second region of the detector elements 1202. The first beam path 130a includes a first reflective element 1204a to direct the first portion 128a of the detected light 118 to the first region of the detector elements 1202. Similarly, the second beam path 130b includes a second reflective element 1204b to direct the second portion 128b of the detected light 118 to the second region of the detector elements 1202.

[0063] The detector elements 1202 are arranged, for example, on top of two beam-deflecting elements, as can be seen in Fig. 13, which is a schematic perspective view of the detector arrangement 1200 according to Fig. 12. Such an arrangement of the elements is particularly compact.

[0064] 14 is a schematic diagram of a cascaded detection system 1400, according to one embodiment. The cascaded detection system 1400 includes a first detection system 1402 and a second detection system 1404 arranged in series.

[0065] The first detector 1402 includes a first acousto-optical device 1406, a first array detector 122a, and a second array detector 122b. The detection light 118 is received by the first detector 1402 via the first acousto-optical device 1406, and the first detector 1402 directs 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. Similar to the detector 200 described above with reference to FIG. 2, the first beam path 130a and the second beam path 130b of the first detector 1402 include focusing elements 202a and 202b and array detectors 122a and 122b. However, the first detector 1402 is shown similarly to the detector 200 according to FIG. 2 merely by way of example. The first detector 1402 may be any of the detectors 102, 200, 300, 500, 1200 described above with reference to Figures 1 to 3, 5, 12 and 13, i.e., any embodiment that does not include a beam dump 402 located in the third beam path 130c.

[0066] The second detector 1404 is disposed in the third beam path 130c of the first detector 1402 and includes a second acousto-optical device 1408, a third array detector 1410c, a fourth array detector 1410a, and a fifth array detector 1410b. The second detector 1404 receives the remaining portion 132 of the detected light 118 from the first detector 1402. In other words, the remaining portion 132 of the detected light 118 plays the role of the detected light 118 in the first detector 1402 in the second detector 1404.

[0067] The remaining portion 132 of the detection light 118 is received by the second detector 1404 via the second acousto-optic device 1408, which directs the fourth portion 1412a of the detection light 118 to a fourth beam path 1414a and the fifth portion 1412b of the detection light 118 to a fifth beam path 1414b. What remains of the detection light 118, i.e., the detection light 118 minus the first portion 128a, the second portion 128b, the third portion 1412a, and the fourth portion 1412b, is referred to as a second remaining portion 1416 of the detection light 118, and is directed by the second acousto-optic device 1408 to a sixth beam path 1414c.

[0068] Similar to the first detector 1402, the fourth beam path 1414a of the second detector 1404 includes a focusing element 1418a and an array detector 1410a, the fifth beam path 1414b includes a focusing element 1418b and an array detector 1410b, and the sixth beam path includes a focusing element 1418c and an array detector 1410c, respectively. Thus, the second detector 1404 is similar to the detector 1200 of FIG. 2. However, the second detector 1404 may also be any of the detectors 102, 200, 300, 400, 500, and 1200 described above with reference to FIGS. 1-13.

[0069] Prior art acousto-optic devices operating at near-ultraviolet, visible, and near-infrared wavelengths can operate at up to eight different wavelength ranges. That is, by applying up to eight different radio frequencies to the transducer 126, up to eight different wavelength ranges can be diffracted from the array of one of the two array detectors 122 a, 122 b. The cascaded detection system 1400 extends this capability by directing the remaining detected light 132 to a second detector 1404, thereby extracting up to eight additional wavelength ranges from the detected light 118 that would otherwise be lost.

[0070] The cascaded detector 1400 can also be used to increase the separation of the focal spots 600 for longer wavelengths (see FIGS. 5-9). Specifically, the first detector 1402 of the cascaded detector 1400 can include a beam-deflecting element including a dispersive prism and can be used to detect wavelengths in the range of 440 nm to 640 nm. The second detector 1404 of the cascaded detector 1400 can be used to detect wavelengths in the range of 640 nm to 840 nm. The second detector 1404 can include a beam-deflecting element including a dispersive prism that differs in its apex angle and / or glass from the dispersive prism used in the first detector 1402 to ensure greater dispersion in the second detector 1404. This ensures that the wavelength separation of the second detector 1404 to the array detectors 122a, 122b enables better performance of the pixel reassignment procedure.

[0071] FIG. 15 shows a schematic diagram of a detection apparatus 1500 according to another embodiment. The detection apparatus 1500 differs from the detection apparatus 102 shown in FIG. 1 in that the acousto-optic device 1502 is configured to generate only the first portion 128a of the detection light 118, which is received by the array detector 122a. The second portion 128b of the detection light 118 and the second array detector 122b are not present. The remaining portion of the detection light not diffracted by the acousto-optic device 120 is directed into the third beam path 130c. Apart from these differences, the functionality of the embodiment according to FIG. 15 corresponds to the embodiment shown in FIG. 1.

[0072] 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 " / ".

[0073] 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]

[0074] 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 120 Acousto-optical Devices 122a, 122b Array detector 124 Acousto-optic media 126 Converter 128a, 128b Detection light portion 130a, 130b, 130c beam path 132 Detection light part 134 Control Unit 200 Detection Device 202a, 202b, 202c focusing elements 204 detector elements 300 Detection Device 302 Detector element 304 Pinhole 400 Detection Device 402 Beam Dump 500 Detection Device 502a, 502b Beam deflection elements 504a, 504b, 504c focusing elements 600 focal spots 602a,602b diagonal 700 graphs 702 vertical axis 704 Horizontal axis 706a,706b curve 800 graphs 802 vertical axis 804 horizontal axis 806a,806b curve 900 graphs 902 vertical axis 904 Horizontal axis 906 curve 1000 graphs 1002 vertical axis 1004 horizontal axis 1006a,1006b curve 1100 graphs 1102 Vertical axis 1104 Horizontal axis 1106a,1106b curve 1200 Detector 1202 detector element 1204a, 1204b Reflective elements 1400 Cascade Detector 1402 Detection device 1404 Detector 1406,1408 Acousto-optical devices 1410a, 1410b, 1410c Array detector 1412a, 1412b Detected light portion 1414a, 1414b, 1414c Beam path 1416 Detection light part 1418a, 1418b, 1418c focusing elements 1500 Detector 1502 Acousto-optical devices

Claims

1. A detection device (102, 200, 300, 400, 500, 1200, 1402, 1404) for an optical scanning microscope (100), the detection device (102, 200, 300, 400, 500, 1200, 1402, 1404) comprising: a first beam path (130a) including a first array detector (122a); an acousto-optic device (120, 1406, 1408) configured to receive the descanned detection light (118) and direct a first portion (128a) of the detection light (118) into the first beam path (130a); a control unit (134); Equipped with the first portion of the detection light (118) has at least one selected wavelength range determined by at least one frequency of an acoustic wave generated by a transducer (126) of the acousto-optic device (120, 1406, 1408); the control unit (134) is configured to control a transducer (126) of the acousto-optic device (120, 1406, 1408) to determine the at least one selected wavelength range. A detection device (102, 200, 300, 400, 500, 1200, 1402, 1404).

2. the detection system (200, 300, 500, 1200, 1404) further comprises a second beam path (130b) including a second array detector (122b); the acousto-optical device (120, 1406, 1408) is configured to direct a second portion (128b) of the detection light (118) into the second beam path (130b), the second portion of the detection light (118) having the at least one selected wavelength range; 2. The detection device (200, 300, 500, 1200, 1404) of claim 1.

3. The remaining portion (132) of the detected light (118) is directed into a third beam path (130c). Detector device (200, 300, 500, 1200, 1404) according to claim 1 or 2.

4. the third beam path (130c) includes a detector element (204, 302), in particular a third array detector (204) or a non-array detector (302), or the third beam path (130c) includes a beam dump (402); The detection device (300) of claim 3.

5. The third beam path (130c) has a pinhole (304) disposed in front of the detector element (302). The detection device (300) of claim 4.

6. the detector elements (204, 302) are detector elements that perform spectral decomposition; 6. A detection device (200, 300, 500, 1200, 1404) according to claim 4 or 5.

7. A pinhole (304) is disposed in front of the acousto-optical device (120, 1406, 1408). The detection device (300) according to any one of claims 1 to 6.

8. the first beam path (130a) includes at least one first beam deflecting element (502a), in particular a first dispersive element configured to spectrally separate the first portion (128a) of the detection light (118); and / or the second beam path (130b) includes at least one second beam deflecting element (502b), in particular a second dispersive element configured to spectrally separate the second portion (128b) of the detection light (118); 8. The detection device (500) according to any one of claims 1 to 7.

9. at least one of the first dispersive element and the second dispersive element includes at least one dispersive prism; The detection device (500) of claim 8.

10. the detection system (1200) comprises a detector element (1202) having a first region forming at least a portion of the first array detector (122a) and a second region forming at least a portion of the second array detector (122b); the first beam path (130a) includes at least one first reflecting element configured to direct a first portion (128a) of the detection light (118) to a first region of the detector element (1202), and at least one second reflecting element configured to direct a second portion (128b) of the detection light (118) to a second region of the detector element (1202); The detection device (1200) according to any one of claims 1 to 9.

11. the first beam path (130a) includes at least one first focusing element (202a, 504a) configured to focus a first portion (128a) of the detection light (118) onto the first array detector (122a); and / or the second beam path (130b) includes at least one second focusing element (202b, 504b) configured to focus a second portion (128b) of the detection light (118) onto the second array detector (122b); Detecting device (200, 300, 400, 500, 1200, 1402, 1404) according to any one of claims 1 to 10.

12. A cascade detection device (1400) comprising at least two detection devices (102, 200, 300, 400, 500, 1200, 1402, 1404) according to any one of claims 1 to 11, The detection devices (102, 200, 300, 400, 500, 1200, 1402, 1404) are arranged in tandem, each detector (102, 200, 300, 400, 500, 1200, 1402, 1404), except for the first detector (1402), is disposed in the third beam path (130c) of the preceding detector (102, 200, 300, 400, 500, 1200, 1404); Cascade detection device (1400).

13. An optical scanning microscope (100), 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) arranged 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 different regions of the sample space (108); A detection device (102, 200, 300, 400, 500, 1200, 1402, 1404) according to any one of claims 1 to 11 or a cascade detection device (1400) according to claim 12, a main beam splitter (114) configured to direct excitation light (116) to the objective lens (104) through the scanning unit (112) and direct detection light (118) to the detector (102, 200, 300, 400, 500, 1200, 1402, 1404) or the cascade detector (1400); An optical scanning microscope (100) comprising:

14. the main beam splitter (114) comprises at least one of an acousto-optic beam splitter and a dichroic beam splitter; The optical scanning microscope (100) of claim 13.

15. the excitation light source (110) comprises a supercontinuum laser and / or multiple single wavelength lasers; 15. An optical scanning microscope (100) according to claim 13 or 14.

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