Image scanning microscope and method

Abstract

To provide an image scanning microscope and a method capable of excellently determining the spatial distribution of the concentration of at least two different phosphor species in a sample.SOLUTION: The scanning unit 110 is arranged along the beam path between the excitation unit 108 and the objective 102 and directs the excitation light 118 via the objective onto different regions of the sample space 106. The image scanning microscope 100 also includes a detection device 112 that includes a spectral encoding element 128 that varies the spatial distribution of the intensity of the detected light based on the wavelength of the detected light 126, and an array detector that detects the spatial distribution. The image scanning microscope also comprises a control unit 116 for controlling the excitation unit to set an excitation mode and for determining a spatial distribution of a concentration of at least two different fluorophore species in a sample 104 arranged in the sample space on the basis of the spatial distribution and the excitation mode and / or photon arrival times detected by the time-resolved detector elements of the detection device.SELECTED DRAWING: Figure 1

Description

[Technical Field]

[0001] The present invention relates to an image scanning microscope.The present invention further relates to a method for determining the spatial distribution of the concentrations of at least two different fluorophore species in a sample. [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 traditional confocal microscopy. Traditional confocal microscopy uses a single point detector, such as a single photomultiplier tube, to detect fluorescent light emitted from a sample. In the ISM approach, this point detector is replaced with a multi-element photodetector, which includes multiple photodetector elements (pixels) arranged in a photodetector array. Each photodetector element in the array is configured to receive fluorescent light and output a detector signal. As the sample is scanned using a laser focus, each photodetector element detects a small image of the illuminated sample at each scan location. Appropriate algorithms are then used to combine the multiple scanned images to reconstruct a single high-resolution image of the sample.

[0003] Using an ISM approach, it is possible to increase spatial image resolution and signal-to-noise ratio by using information from different photodetector elements. However, existing solutions are limited in their ability to distinguish between different fluorophore species. In particular, existing solutions are limited in their ability to determine the spatial distribution of the concentrations of different fluorophore species within a sample. Summary of the Invention [Problem to be solved by the invention]

[0004] The object of the present invention is to provide an image scanning microscope and a method that allows for better determination of the spatial distribution of the concentrations of at least two different fluorophore species in a sample than known image scanning microscopes or methods. [Means for solving the problem]

[0005] The above problem is solved by the subject matter of the independent claims. Advantageous embodiments are defined in the dependent claims and the following description.

[0006] The proposed image scanning microscope includes an excitation unit configured to generate excitation light according to at least one excitation modality and an objective lens directed toward a sample space, configured to direct the excitation light toward the sample space and receive detection light from the sample space. The scanning unit of the image scanning microscope is arranged along a beam path between the excitation unit and the objective lens and configured to selectively direct the excitation light toward different regions of the sample space via the objective lens. The image scanning microscope also includes a detection device including at least one spectral encoding element configured to vary the spatial distribution of the intensity of the detection light based on the wavelength of the detection light, and at least one array detector configured to detect the spatial distribution of the intensity of the detection light. The main beam splitter of the image scanning microscope is configured to direct the excitation light toward the objective lens via the scanning unit and the detection light toward the detection device. The image scanning microscope further includes a control unit configured to control the excitation unit to set an excitation pattern and to determine the spatial distribution of concentrations of at least two different fluorophore species in the sample arranged in the sample space based on the spatial distribution of the intensity of the detected detected light and the at least one photon arrival time detected by the excitation pattern and / or at least one time-resolved detector element of the detection device.

[0007] Changes in environmental conditions can alter the emission and / or excitation characteristics of fluorophores. For example, changes in pH levels shift the emission wavelength of a fluorophore, and changes in temperature alter excitation efficiency and fluorescence lifetime. Therefore, the term fluorophore species is used herein to refer to a collection of fluorophores grouped by their emission and / or excitation properties. Two different fluorophore species may be two different fluorophores or the same fluorophore found in different regions of a sample, each of which may have different environmental conditions. These fluorophores may be exogenous fluorophores introduced into the sample and / or endogenous fluorophores naturally present in the sample.

[0008] The sample is imaged using an image scanning microscope, in which the sample is scanned using excitation light focused by an objective lens using a scanning unit. The excitation light excites different fluorophore species within the sample, causing them to emit detection light according to their inherent properties and the environmental conditions at the location of the fluorophore species within the sample. This detection light is collected by the objective lens and directed via the scanning unit to a detection device, where it is descanned. The descanned detection light is then modulated by a spectral encoding element based on the wavelength of the detected light. For example, the spectral encoding element may be a diffractive element that deflects different wavelengths by different amounts. The modulated detection light is then received by an array detector, which detects a spatial distribution of the intensity of the detected light. As the sample is scanned using the excitation light, at least one spatial distribution is detected by the array detector at each scan position. From the collection of these spatial distributions, a single high-resolution image of the sample can be reconstructed using algorithms known from image scanning microscopy (ISM).

[0009] The proposed image-scanning microscope extends the ISM approach by not only reconstructing a single high-resolution image from the spatial distribution but also determining the spatial distribution of the concentrations of at least two different fluorophore species within a sample by detecting additional information about the different fluorophore species. This determination is based on what is referred to herein as a fingerprint, which describes the system response for each fluorophore species. The fingerprint may include the temporal emission behavior and / or spectral excitation behavior of the fluorophore species reconstructed from at least one photon arrival time, i.e., the response of different fluorophore species to a particular excitation modality of the excitation light, e.g., the spectral composition and / or modulation pattern of the excitation light. Additional aspects of the fingerprint may be provided by a spectral coding element that changes the spatial distribution of the intensity of the detected light based on the wavelength of the detected light. This not only enables image-scanning microscopy with high spatial resolution and a high signal-to-noise ratio by detecting the detected light using an array detector, but also enables more robust differentiation of multiple different fluorophore species within a sample by detecting the additional fingerprints of the fluorophore species. In combination, this allows the reconstruction of the spatial distribution of the concentrations of at least two different fluorophore species within a sample with the high spatial resolution and signal-to-noise ratio offered by the ISM approach.

[0010] In another embodiment, the control unit is configured to determine at least one spectral information based on the spatial distribution of the intensity of the detected light and to determine the spatial distribution of the concentrations of at least two different fluorophore species in the sample taking the spectral information into account. In this embodiment, the fact that the spectral encoding element varies the spatial distribution of the intensity of the detected light in a predictable manner based on the wavelength of the detected light is used to extract the spectral information as an additional aspect of the fingerprint. This spectral information may then be used, for example, to distinguish between the at least two different fluorophore species. This makes the determination of the spatial distribution of the concentrations of the at least two different fluorophore species in the sample more accurate and reliable.

[0011] In another embodiment, the excitation unit is configured to generate modulated excitation light, in particular pulse-modulated excitation light. In such an embodiment, the excitation modality comprises a modulation pattern of the excitation light. By using excitation light with a known modulation pattern, additional information about different fluorophore species can be reconstructed. For example, the excitation light can be pulse-modulated. By determining the delay between the individual pulses of the excitation light and the detected photon arrival time, it is possible to determine the fluorescence decay rate and thus the fluorescence lifetime of different fluorophore species. The fluorescence lifetime can be used as an additional aspect of a fingerprint to reliably distinguish between different fluorophore species.

[0012] In another embodiment, the excitation unit includes multiple excitation light sources. At least two of these excitation light sources may be configured to generate modulated light. The excitation unit may further be configured to combine the modulated light generated by the at least two excitation light sources into the modulated excitation light. In this embodiment, two of these excitation light sources may be configured to emit modulated light at different wavelengths, for example. Each of these wavelengths may be used to excite a different fluorophore species. Furthermore, each of the two excitation light sources may be configured to generate modulated light having different temporal characteristics. This allows photons detected by the time-resolved detector elements to be associated by correlating their respective arrival times with the temporal characteristics of the excitation light. Such an arrangement allows the fluorescence lifetimes of multiple fluorophore species to be determined simultaneously.

[0013] In another embodiment, the control unit is configured to perform fluorescence lifetime measurements based on the modulation pattern of the excitation light and the photon arrival times, and to determine the spatial distribution of concentrations of at least two different fluorophore species within the sample based on the fluorescence lifetime measurements. In this embodiment, the control unit determines the fluorescence lifetimes of the different fluorophore species to assist in distinguishing between them. For example, the control unit can use an exponential fitting technique to determine a histogram of photon arrival times and determine the fluorescence lifetimes. Based on the fluorescence lifetimes determined at different scan positions, the concentrations of the different fluorophore species within the sample can be more accurately determined.

[0014] In another embodiment, the excitation unit is configured to selectively generate excitation light of at least two different spectral compositions. In such an embodiment, the excitation mode includes the spectral composition of the excitation light. The excitation unit can include, for example, multiple single-wavelength lasers. The excitation unit can also include a supercontinuum laser, also known as a white light laser, and an exchangeable filter or acousto-optical device for selecting a specific wavelength from the laser light generated by the supercontinuum laser as the excitation light. In this embodiment, it is possible to dynamically generate laser light having multiple different wavelengths as the excitation light. This allows, for example, the excitation light to be adapted to the excitation spectra of many different fluorophores, allowing different fluorophores to be excited simultaneously.

[0015] In another embodiment, the excitation unit is configured to generate excitation light according to at least two different excitation modalities. The control unit may be configured to cause the image scanning microscope to perform a first measurement using excitation light according to a first excitation modality and a second measurement using excitation light according to a second excitation modality. The control unit may be further configured to determine, based on the first and second excitation modalities, a spatial distribution of the concentrations of at least two different fluorophore species within the sample and a spatial distribution of the intensity of the detected light detected during the first and second measurements. In this embodiment, the different fluorophore species are distinguished by their response to the two different excitation modalities. For example, the first measurement uses excitation light having a first wavelength range, and the second measurement uses excitation light having a second wavelength range different from the first wavelength range. During the first measurement, only the first group of different fluorophore species is excited. Similarly, during the second measurement, only the second group of different fluorophore species is excited. This makes it possible to distinguish between the first and second groups of fluorophore species based on their response to different wavelengths of excitation light. The response of each fluorophore species to different wavelengths of excitation light is then part of the fingerprint of each fluorophore species. Another excitation modality that may be changed between the first and second measurements is modulation of the excitation light. The excitation light used during the first measurement may be continuous wave, whereas the excitation light used during the second measurement may be, for example, pulse-modulated light. This allows the fluorescence lifetime to be determined in the second measurement, thereby further distinguishing between the fluorophore species detected during the first measurement.

[0016] In another embodiment, the control unit is configured to determine the spatial distribution of concentrations of at least two different fluorophore species within the sample based on a database of different fluorophore species and an imaging model that parameterizes the imaging behavior of the image-scanning microscope. In this embodiment, the control unit determines which fingerprints are associated with which specific fluorophore species based on the database and the imaging model. To determine the fluorophore species present in the sample, the control unit can then, for example, minimize a cost function that characterizes the distance between the measurement data and a weighted superposition of the fingerprints determined from the database and the imaging model. The database and the imaging model may each be stored in a local or remote memory device or a cloud service.

[0017] In another embodiment, the control unit is configured to determine the spatial distribution of concentrations of at least two different fluorophore species within the sample based on previously determined calibration data. In this embodiment, the control unit determines which fingerprints are associated with which specific fluorophore species based on calibration data describing measurement data detected for the specific fluorophore species by the image scanning microscope. To determine which fluorophore species are present within the sample, the control unit can, for example, minimize a cost function characterizing the distance between the measurement data and the calibration data, similar to the above-described embodiment. The calibration data can be stored in a local or remote memory device or a cloud service.

[0018] To determine the spatial distribution of concentrations of at least two different fluorophore species within a sample, a fingerprint can be determined from both a database and an image formation model, as well as calibration data. If neither is possible, the fingerprint can be reconstructed "blindly." This can be done, for example, by extending the technique presented by Neher et al. in "Blind source separation techniques for the decomposition of multiply labeled fluorescence images" (Biophysical Journal, vol. 96, no. 9, 6 May 2009, pp. 3791-3800).

[0019] In another embodiment, the spectral encoding element includes at least one of the following: a dispersive prism, a plane grating, a volume grating, a grism, a diffractive optical element, and a wavelength-selective filter array. The wavelength-selective filter array may be, for example, a Bayer mask. The dispersive prism handles a wide wavelength range of light, including the visible spectrum and portions of the infrared and ultraviolet spectrum. Furthermore, the dispersive prism does not generate higher diffraction orders that may not be picked up by the array detector, which can occur in a diffraction grating. Therefore, using a dispersive prism as a spectral encoding element prevents light loss and improves the signal-to-noise ratio. In particular, a blazed grating provides high diffraction efficiency with a predetermined diffraction order, reducing light loss and improving the signal-to-noise ratio. A grism combines a dispersive prism and a grating and has very little chromatic aberration. Diffractive optical elements enable more complex light manipulation, allowing control of the beam shape and providing precise control over the phase and amplitude of the detected light.

[0020] In another embodiment, the at least one array detector includes at least two time-resolved detector elements configured to detect photon arrival times. In this embodiment, the at least one array detector includes an array of multiple photodetector elements. At least two of the photodetector elements are time-resolved detector elements, such as single-photon avalanche diodes or silicon photomultipliers. This allows the at least one array detector to be used to detect at least one photon arrival time. Alternatively, the detection device can include the time-resolved detector elements as separate elements from the at least one array detector.

[0021] In another embodiment, at least one array detector includes a two-dimensional array of photodetector elements, specifically a SPAD array or a SiPM array. Each photodetector element functions as a single pixel detector, capturing a portion of the detected light at a different location within the array. Such an array allows for the detection of a two-dimensional spatial distribution of the detected light intensity. SPAD stands for single-photon avalanche diode, a type of photodetector element characterized by its high sensitivity, fast time resolution, and ability to detect single photons with high efficiency. An advantage of SPAD arrays is their ability for highly accurate time-resolved measurements, which allows them to be used as time-resolved detector elements. SiPM stands for silicon photomultiplier, another type of photodetector element based on SPAD. Advantages of SiPMs include a low signal-to-noise ratio, high gain, low operating voltage, their compact size, and their robustness. Similar to SPAD arrays, SiPM arrays may also be used as time-resolved detector elements.

[0022] The present invention further relates to a method for determining the spatial distribution of the concentrations of at least two different fluorophore species in a sample, the method comprising the following steps: a) generating excitation light according to at least one excitation modality using an excitation unit; b) selectively directing the excitation light to different regions of the sample using a scanning unit and an objective lens; c) receiving detection light from the sample using an objective lens and directing the detection light to a detector using a main beam splitter; d) varying the spatial distribution of the intensity of the detected light based on the wavelength of the detected light using a spectral encoding element of the detection device; e) detecting the spatial distribution of the intensity of the detected light using at least one array detector of the detection device; f) optionally detecting photon arrival times using at least one array detector as a time-resolved detector element or at least one separate time-resolved detector element of the detection device; g) determining the spatial distribution of the concentrations of at least two different fluorophore species in the sample based on the spatial distribution of the detected light intensity and the excitation mode and / or photon arrival time; Includes:

[0023] This method has the same advantages as the image scanning microscope described above. In particular, this method may be supplemented with the features described herein in relation to the image scanning microscope. Furthermore, the image scanning microscope described above may be supplemented with the features described herein in relation to this method.

[0024] In one embodiment, the method includes a calibration step for generating calibration data that provides a basis for determining the spatial distribution of concentrations of at least two different fluorophore species within the sample. The calibration data describes measurement data detected by the image scanning microscope for specific fluorophore species. Based on the calibration data, the spatial distribution of concentrations of the at least two different fluorophore species within the sample can be determined, for example, by minimizing a cost function that characterizes the distance between the measurement data and the calibration data, as described above.

[0025] In another embodiment, the calibration comprises performing steps a)-f) using a sample with a known spatial distribution of concentrations of at least two different fluorophore species and / or using multiple samples each containing a single fluorophore species. In this embodiment, the calibration data is generated using one or more samples with a known spatial distribution of concentrations of different fluorophore species as a reference. This results in reliable calibration data that accurately describes how different fluorophore species are imaged.

[0026] In another embodiment, the calibration data is generated from detected light received from a region of the sample containing a single fluorophore species. This step may be used when a sample with a known spatial distribution of concentrations of different fluorophore species is not available as a reference for the calibration. In this case, for example, a region of the sample prepared in this way and known to contain only a single fluorophore species is imaged.

[0027] Specific embodiments are described below with reference to the drawings. [Brief explanation of the drawings]

[0028] [Figure 1] FIG. 1 is a schematic diagram illustrating an image scanning microscope according to one embodiment. [Figure 2] 1 is a flowchart illustrating a method for determining the spatial distribution of concentrations of at least two different fluorophore species within a sample, according to one embodiment. [Figure 3] 3 is a flow chart illustrating a calibration that may be performed as part of the method according to FIG. 2; DETAILED DESCRIPTION OF THE INVENTION

[0029] 1 is a schematic diagram of an image scanning microscope 100 according to one embodiment. The image scanning microscope 100 illustratively includes a single objective lens 102 directed at a sample 104 arranged in a sample space 106. The image scanning microscope 100 further includes an excitation unit 108, a scanning unit 110, a detection device 112, a main beam splitter 114, and a control unit 116.

[0030] The pump unit 108 is configured to generate the pump light 118 according to at least one pumping style. The pumping style may be, for example, the spectral composition of the pump light 118, the intensity of the pump light 118, or modulation of the pump light 118. The pump unit 108 illustratively includes two pump light sources 120a and 120b configured to generate light that is combined into the pump light 118. In particular, each of the pump light sources 120a and 120b may be configured to generate light having a single wavelength or a narrow wavelength band. Thus, the spectral content of the pump light 118 can be varied by selecting which of the pump light sources 120a and 120b generates light. Alternatively or additionally, the pump unit 108 may include a continuum laser and an interchangeable filter array, or may include a tunable laser that selectively generates pump light 118 of different wavelengths. To vary the modulation of the excitation light 118, at least one of the excitation light sources 120a, 120b may be configured to generate modulated light, for example pulse modulated light.

[0031] The light generated by the two excitation light sources 120a, 120b is combined into the excitation light 118 using, for example, a mirror 122 and a dichroic beam splitter 124. The light generated by the first excitation light source 120a is directed by the dichroic beam splitter 124 to the main beam splitter 114. The light generated by the second excitation light source 120b is directed by the mirror 122 through the dichroic beam splitter 124 to the main beam splitter 114. Thus, the dichroic beam splitter 124 combines the light generated by the two excitation light sources 120a, 120b into the excitation light 118. Although not shown in FIG. 1 , the excitation unit 108 may include additional optical elements, such as lenses and diaphragms, for forming a beam from the excitation light 118.

[0032] The excitation light 118 generated by the excitation unit 108 is directed to the scanning unit 110 by the main beam splitter 114. The scanning unit 110 is configured to deflect the excitation light 118 and selectively direct the excitation light 118, for example, in a serpentine pattern, to different regions of the sample space 106 via the objective lens 102. This allows scanning of the sample 104 using the excitation light 118 focused by the objective lens 102. To deflect the excitation light 118, the scanning unit 110 may include, for example, one or more galvanometric mirrors or acousto-optical deflectors. The beam path of the excitation light 118 is shown in FIG. 1 using dashed lines originating from the excitation light sources 120a and 120b to the sample 104.

[0033] The detection light 126 is generated by illuminating the sample 104 with the excitation light 118. In particular, the excitation light 118 excites fluorophores arranged within the sample 104, which then emit fluorescent light as the detection light 126. Depending on the excitation mode of the excitation light 118, different fluorophore species may be excited. For example, the excitation light 118 may have a narrow wavelength band, so that only a portion of the fluorophore species arranged within the sample 104 are excited. The detection light 126 is collected by the objective lens 102 and directed back toward the main beam splitter 114 via the scanning unit 110. Because the scanning unit 110 is arranged between the main beam splitter 114 and the objective lens 102, the excitation light 118 is deflected in the opposite direction relative to the detection light 126. This directs the detection light 126 to a single point regardless of the deflection angle of the scanning unit 110. In other words, the detection light 126 is descanned, so to speak. The descanned detection light 126 is then directed by the main beam splitter 114 to the detector 112. The beam path of the detection light 126 is shown in Figure 1 using a dotted line originating from the sample 104.

[0034] The detection device 112 illustratively includes a spectral encoding element 128 and two array detectors 130a and 130b. The spectral encoding element 128 varies the spatial distribution of the intensity of the detected light 126 based on the wavelength of the detected light 126. For example, the spectral encoding element 128 may include at least one dispersive prism that deflects shorter wavelengths more than longer wavelengths. The spectral encoding element 128 may also include a grating, such as a planar grating or a volume grating, that disperses the detected light 126 into its constituent wavelengths by diffraction, generating multiple diffraction orders. The spatial distribution of the intensity of the detected light 126 may be varied using an array of wavelength-selective filters, such as a Bayer mask, positioned upstream of the array detectors 130a and 130b. The array of wavelength-selective filters allows only certain wavelengths of the detected light 126 to pass through specific regions of the array detectors 130a and 130b, thereby varying the intensity pattern across the surface of the array detectors 130a and 130b.

[0035] The detected light 126 that passes through the spectral encoding element 128 is then received by array detectors 130a, 130b. Each of the array detectors 130a, 130b 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 photodetector element functions as a single pixel detector that captures a portion of the detected light 126 at a different position within the array. Thus, the array detectors 130a, 130b enable the detection of a spatial distribution of the intensity of the detected light 126. As the sample 104 is scanned with the excitation light 118, at least one spatial distribution is detected by each of the array detectors 130a, 130b at each scanning position. From the collection of these spatial distributions, a single high-resolution image of the sample 104 can be reconstructed. This imaging technique for a single high-resolution image of a sample is known as image scanning microscopy (ISM), which provides improved spatial resolution and signal-to-noise ratio compared to conventional confocal laser scanning microscopy (CLSM). Because the spatial distribution of the intensity of the detected light 126 varies based on the wavelength of the detected light 126, it is also possible to determine spectral information about the detected light 126, such as the spectral composition of the detected light 126. Furthermore, two or more photodetector elements of at least one of the array detectors 130a, 130b may be configured to detect photon arrival times. Alternatively, one of the two array detectors 130a, 130b may be a non-imaging or single-pixel detector element capable of specifically detecting photon arrival times, instead of a time-resolved detector element.

[0036] The control unit 116 is configured to control the excitation unit 108, the scanning unit 110, and the array detectors 130a, 130b, receive image data from the array detectors 130a, 130b, and process the image data. The control unit 116 is further configured to cause the image scanning microscope 100 to perform a method for determining the spatial distribution of concentrations of at least two different fluorophore species in the sample 104. Specifically, the control unit 116 controls the excitation unit 108 to set an excitation mode. The control unit 116 then determines the spatial distribution of concentrations of the different fluorophore species in the sample 104 based on the spatial distribution of the intensity of the detected detection light 126 and the excitation mode and / or photon arrival time. This method is described in more detail below with reference to FIG. 2.

[0037] 2 is a flowchart of a method for determining the spatial distribution of concentrations of at least two different fluorophore species in a sample 104. The method is described, by way of example only, as being performed using the image scanning microscope 100 according to FIG. 1. Prior to starting the method, the sample 104 may be prepared by introducing fluorophores, such as fluorescent dyes, proteins, or quantum dots, into the sample 104. Alternatively, the method may be performed using only endogenous fluorophores.

[0038] The method starts in step S200. In optional step S202, calibration is performed to generate calibration data. This calibration is described in more detail below with reference to FIG. 3. In step S204, excitation light 118 is generated according to an excitation format. The excitation format may be the spectral content of the excitation light 118, for example, the wavelength range of the excitation light 118. The excitation format may also be the intensity of the excitation light 118 and / or modulation of the excitation light 118. The excitation light 118 may be, for example, pulse-modulated. In one example, the control unit 116 controls the excitation unit 108 to generate the excitation light 118 according to the excitation format. Then, in step 206, the excitation light 118 is directed toward the sample 104. For example, the excitation light 118 is focused at a scan position within the sample 104 using the scanning unit 110 and the objective lens 102.

[0039] The excitation light 118 then excites fluorophores present in the sample 104, which also produce fluorescent light as detected light 126. Both the emission and excitation characteristics of fluorophores can vary based on the fluorophores' intrinsic properties, such as the molecule used and its composition, as well as environmental conditions such as pH level and temperature. For example, the same fluorophore may have different emission spectra based on the pH level of its immediate surroundings. Thus, the term fluorophore species is used to distinguish between groups of fluorophores with different emission and / or excitation characteristics. Then, in step S208, the detected light 126 emitted by the different fluorophore species is received, for example, by the objective lens 102. In step S210, the spatial distribution of the intensity of the detected light 126 is varied based on the wavelength of the detected light 126, for example, using a spectral encoding element 128. This can involve diffracting the detected light 126, i.e., polarizing different wavelengths by different amounts. It can also involve filtering specific wavelengths or wavelength ranges. In step S212, the spatial distribution of the intensity of the altered detected light 126 is detected using at least one array detector 130a, 130b. This spatial distribution is a convolution of a point spread function (PSF) depending on the optical configuration of an imaging system, e.g., image scanning microscope 100, and the source of the detected light 126, which may be assumed to be point-like. Additionally, in optional step S214, the arrival time of at least one photon of the detected light 126 is detected, e.g., using array detector 130a, 130b or a dedicated time-resolved detector element.

[0040] Steps S206-S214 are repeated until the region of interest of the sample 104 is scanned. Each time, the excitation light 118 is directed to a different region of the sample 104, thereby illuminating a different scan position with the excitation light 118. In one example, the sample 104 is scanned in a serpentine pattern with the excitation light 118 to illuminate the region of interest. For example, the excitation light 118 is selectively deflected to different positions of the entrance pupil of the objective lens 102, which then focuses the excitation light 118 onto different regions of the sample 104. This results in a collection of spatial distributions of the intensity of the detection light 126, each associated with a specific scan position. From this collection of spatial distributions, a single high-resolution image of the sample 104 can be reconstructed using algorithms known from ISM. When step S214 is performed, a collection of photon arrival times is also obtained, each associated with a specific scan position.

[0041] Furthermore, steps S204-S214 may be repeated, each time using a different excitation modality, to obtain multiple light collections with spatial distributions, each associated with a different excitation modality. For example, in a first measurement, the region of interest is scanned using excitation light 118 according to a first excitation modality, such as a first wavelength range. In a second measurement, the region of interest is again scanned using excitation light 118 according to a second excitation modality, such as a second wavelength range different from the first wavelength range.

[0042] In optional step S216, at least one spectral information is determined based on the spatial distribution of the intensity of the detected light 126 determined in the iterative step S212. In the iterative step S210, the detected light 126 is modulated based on the wavelength of the detected light 126, so that spectral information, e.g., the spectrum of the detected light 126, can be reconstructed from the spatial distribution. For this purpose, known algorithms from spectral ISM may be used. The spectral information may be determined, for example, by the control unit 116. In step S218, which is optionally performed during the iterative step S214, a fluorescence lifetime measurement is performed. This fluorescence lifetime measurement is based on the photon arrival times detected in the iterative step S214 and the modulation of the excitation light 118. For example, the excitation light 118 is pulse-modulated. The delay between individual pulses of the excitation light 118 and the detected photon arrival times are determined. From the determined delay, a fluorescence lifetime may be determined using exponential fitting. The fluorescence lifetime measurement may be performed, for example, by the control unit 116.

[0043] In step S220, the spatial distribution of concentrations of at least two different fluorophore species within the sample 104 is determined, which can be calculated by:

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[0044] As an example, an algorithm to reduce the dimensionality of a measured spatial distribution to the dimension of interest:

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[0045] Measured spatial distribution

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[0046] This model

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[0047] Different phosphor species in sample 104 f If the fingerprints of different fluorophores j in the sample 104 are known, fThe spatial distribution of the concentration of can be achieved using maximum likelihood estimation (MLE) or the equivalent negative log-likelihood (NLL) minimization method. In either case, this determination is based on the measured spatial distribution

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[0048] Assuming there is Gaussian noise in the measurement data, the cost function:

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[0049] Assuming Gaussian noise, the fingerprint

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[0050] Fingerprint

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[0051] Phosphor type f Fingerprint for

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[0052] Step S220 may be performed, for example, by the control unit 116. The method then ends in step S222.

[0053] Figure 3 is a flow chart of a calibration that may be performed as part of the method according to Figure 2. Before the calibration and the method of Figure 2 are performed, one or more reference samples may be prepared, whereby the different fluorophores j in the reference samples may be measured. f Spatial distribution of

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[0054] Calibration begins in step S300. In step S302, calibration data is acquired from a reference sample and / or one of the fluorophores j using the imaging system used for the actual measurement (i.e., steps S204-S220) or an equivalent imaging system for the measurement (e.g., the same model imaging system). f The light is collected by imaging areas of the sample 104 that each contain only the .lambda.

[0055] Then, in step S304, the phosphor species j present in the reference sample, or specially prepared area of ​​the sample 104, is detected. f Fingerprint

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[0056] spatial distribution

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[0057] The calibration then ends in step S306.

[0058] Fingerprint

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[0059] All spatial distributions

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[0060] In all figures, elements performing the same or similar operations are designated by the same reference numerals. 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 " / ".

[0061] 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]

[0062] 100-image scanning microscope 102 Objective Lens 104 samples 106 Sample Space 108 Excitation Unit 110 Scanning Unit 112 Detection Device 114 Main beam splitter 116 Control Unit 118 Excitation Light 120a, 120b Excitation light source 122 Mirror 124 Dichroic Beam Splitter 126 Detected Light 128 Spectral Encoding Elements 130a, 130b Array detector

Claims

1. An imaging scanning microscope (100), comprising: an excitation unit (108) configured to generate excitation light (118) according to at least one excitation modality; an objective lens (102) directed toward a sample space (106) and configured to direct the excitation light (118) toward the sample space (106) and receive detection light (126) from the sample space (106); a scanning unit (110) arranged along a beam path between the excitation unit (108) and the objective lens (102), the scanning unit (110) configured to selectively direct the excitation light (118) through the objective lens (102) to different regions of the sample space (106); a detection device (112) including at least one spectral coding element (128) configured to vary a spatial distribution of the intensity of the detected light (126) based on the wavelength of the detected light (126), and at least one array detector (130a, 130b) configured to detect the spatial distribution of the intensity of the detected light (126); a main beam splitter (114) configured to direct the excitation light (118) to the objective lens (102) via the scanning unit (110) and direct the detection light (126) to the detector (112); a control unit (116) configured to control the excitation unit (108) to set the excitation pattern and to determine a spatial distribution of concentrations of at least two different fluorophore species in a sample (104) arranged in the sample space (106) based on a spatial distribution of the intensity of the detected detection light (126) and on at least one photon arrival time detected by the excitation pattern and / or at least one time-resolved detector element of the detection device (112); An imaging scanning microscope (100) comprising:

2. the control unit (116) is configured to determine at least one spectral information based on the spatial distribution of the intensity of the detected light (126) and to determine the spatial distribution of concentrations of at least two different fluorophore species in the sample (104) taking into account the spectral information. The imaging microscope (100) of claim 1.

3. the excitation unit (108) is configured to generate modulated excitation light (118), in particular pulse-modulated excitation light (118), and the excitation format comprises a modulation pattern of the excitation light (118); 3. The imaging scanning microscope (100) according to claim 1 or 2.

4. the pumping unit (108) includes a plurality of pumping light sources (120a, 120b), at least two of which are configured to generate modulated light, and the pumping unit (108) is further configured to combine the modulated light generated by the at least two pumping light sources (120a, 120b) into the modulated pumping light (118). The imaging microscope (100) of claim 3.

5. the control unit (116) is configured to perform fluorescence lifetime measurements based on the modulation pattern of the excitation light (118) and the photon arrival times, and to determine a spatial distribution of concentrations of at least two different fluorophore species in the sample (104) based on the fluorescence lifetime measurements.

5. The imaging microscope (100) according to claim 3 or 4.

6. the excitation unit (108) is configured to selectively generate excitation light (118) of at least two different spectral compositions, and the excitation format comprises a spectral composition of the excitation light (118); The imaging scanning microscope (100) according to any one of claims 1 to 5.

7. the excitation unit (108) is configured to generate excitation light (118) according to at least two different excitation modalities; the control unit (116) is configured to cause the image scanning microscope (100) to perform a first measurement using excitation light (118) according to a first excitation modality and a second measurement using excitation light (118) according to a second excitation modality; the control unit (116) is further configured to determine, based on the first and second excitation modalities, a spatial distribution of concentrations of at least two different fluorophore species within the sample (104) and a spatial distribution of intensities of detected light (126) detected during the first and second measurements. The imaging microscope (100) according to any one of claims 1 to 6.

8. the control unit (116) is configured to determine the spatial distribution of concentrations of at least two different fluorophore species in the sample (104) based on a database of different fluorophore species and an image formation model that parameterizes the imaging behavior of the image scanning microscope (100). The imaging scanning microscope (100) according to any one of claims 1 to 7.

9. the control unit (116) is configured to determine a spatial distribution of concentrations of at least two different fluorophore species within the sample (104) based on previously determined calibration data; The imaging scanning microscope (100) according to any one of claims 1 to 8.

10. the spectral encoding element (128) comprises at least one of the following: a dispersive prism, a plane grating, a volume grating, a grism, a diffractive optical element, and a wavelength-selective filter array; The imaging microscope (100) according to any one of claims 1 to 9.

11. the at least one array detector (130a, 130b) includes at least two time-resolved detector elements configured to detect photon arrival times; The imaging scanning microscope (100) according to any one of claims 1 to 10.

12. the at least one array detector (130a, 130b) comprises a two-dimensional array of photodetector elements, in particular a SPAD array or a SiPM array; 12. The imaging scanning microscope (100) according to any one of claims 1 to 11.

13. 1. A method for determining the spatial distribution of concentrations of at least two different fluorophore species in a sample (104), the method comprising: a) generating excitation light (118) according to at least one excitation modality using an excitation unit (108); b) selectively directing the excitation light (118) to different regions of the sample (104) using a scanning unit (110) and an objective lens (102); c) receiving detection light (126) from the sample (104) using the objective lens (102) and directing the detection light (126) to a detector (112) using a main beam splitter (114); d) varying the spatial distribution of the intensity of the detected light (126) based on the wavelength of the detected light (126) using a spectral encoding element (128) of the detection device (112); e) detecting a spatial distribution of the intensity of the detected light (126) using at least one array detector (130a, 130b) of the detection device (112); f) optionally detecting photon arrival times using the at least one array detector (130a, 130b) as a time-resolved detector element or using at least one separate time-resolved detector element of the detection arrangement (112); g) determining the spatial distribution of the concentrations of at least two different fluorophore species in the sample (104) based on the spatial distribution of the intensity of the detected light (126) and on the excitation mode and / or photon arrival time; A method comprising:

14. The method includes calibrating to generate calibration data that is a basis for determining the spatial distribution of concentrations of at least two different fluorophore species within the sample (104).

14. The method of claim 13.

15. the calibration includes performing steps a) to f) using a sample with a known spatial distribution of concentrations of at least two different fluorophore species and / or using multiple samples each containing a single fluorophore species; 15. The method according to claim 13 or 14.

16. the calibration data is generated from detected light (126) received from an area of ​​the sample (104) containing a single fluorophore species; 16. The method according to any one of claims 13 to 15.

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