Imaging apparatus and method for aligning images

Abstract

An imaging device (100) includes detection optics (108) configured to receive detection light from a sample (102) and direct the detection light into a main beam path (110). The imaging device (100) further comprises a beam splitting element (112) configured to be inserted into the main beam path (110) to direct a first portion of the detection light into a first branch beam path (114) and a second portion of the detection light into a second branch beam path (116). At least one first filter element (122) is configured to be inserted into the first branched beam path (114). A first detector element (120) is arranged in the first branch beam path (114), and configured to capture at least a first test image when the beam splitting element (112) is inserted into the main beam path (110) and the first filter element (122) is not inserted into the first branch beam path (114), a third test image when the first filter element (122) is inserted into the first branch beam path (114), and a first actual image. At least one second filter element (128) is configured to be inserted into the second branched beam path (116). A second detector element (126) is arranged in the second branch beam path (116), and configured to capture at least a second test image when the beam splitting element (112) is inserted into the main beam path (110) and the second filter element (128) is not inserted into the second branch beam path (116), a fourth test image when the second filter element (128) is inserted into the second branch beam path (116), and a second actual image. The imaging device (100) also includes a controller (130) configured to determine a first misalignment between the first test image and the second test image, a second misalignment between the third test image and the first test image, and a third misalignment between the fourth test image and the second test image; and aligning the first actual image and the second actual image based on the first misalignment, the second misalignment, and the third misalignment.

Description

Technical Field

[0001] The present invention relates to an imaging device. Furthermore, the present invention relates to a method for aligning images, and a computer program product. Background Art

[0002] In fluorescence microscopy, an arrangement comprising interchangeable filters and highly sensitive detectors is used to produce images from fluorescence. The filter is an optical element arranged in the beam path between the sample and the detector. Changing the filter may therefore cause the beam path to shift or even distort, resulting in the images captured before and after changing the filter no longer matching with pixel accuracy. This effect is even more pronounced when multiple detectors are used in order to simultaneously capture images of different wavelengths by using different filters. In addition to the shift caused by the filters, the detectors may also be misaligned relative to each other. Such misalignment manifests itself as a relative shift and / or rotation of the images generated by the different detectors. In an imaging device comprising multiple detectors, any misalignment of the images generated by the different detectors will cancel out the advantage gained by capturing the images simultaneously. Therefore, in order to eliminate the misalignment, the imaging device needs to be calibrated to align the images captured by the different detectors.

[0003] Typically, this calibration is done manually by the user. To calibrate the imaging device, the user must find and mark the same points in different images. When different filters are used, different detectors capture different wavelengths, and the images produced by the detectors may differ greatly. This may lead to a certain degree of inaccuracy. Alternatively, a suitable calibration can be determined before the actual image acquisition using a technical sample, and then applied to the actual image data. However, such calibration is very time-consuming because it must be repeated every time a component that affects the beam path (such as a filter or a beam splitter) is changed. It also requires an experienced user to determine which hardware changes in the imaging device require recalibration. In addition, temperature changes may introduce additional offsets, thereby invalidating the previous calibration relatively quickly without being noticed. Summary of the invention

[0004] It is therefore an object of the present invention to provide an imaging device and a method for aligning an image, such that calibration of the imaging device can be performed quickly and efficiently, in particular when more filter elements are used.

[0005] The aforementioned objects are achieved by the subject-matter of the independent claims. Advantageous embodiments are defined in the dependent claims and in the following description.

[0006] The proposed imaging device includes a detection optical device configured to receive detection light from a sample and guide the detection light into a main beam path. The imaging device also includes a beam splitting element configured to be inserted into the main beam path to guide a first portion of the detection light into a first branch beam path and a second portion of the detection light into a second branch beam path. The imaging device also includes at least one first filter element configured to be inserted into the first branch beam path. The imaging device also includes a first detector element arranged in the first branch beam path. The first detector element is configured to capture at least a first test image when the beam splitting element is inserted into the main beam path and the first filter element is not inserted into the first branch beam path, a third test image when the first filter element is inserted into the first branch beam path, and a first actual image. The imaging device also includes at least one second filter element configured to be inserted into the second branch beam path. The imaging device also includes a second detector element arranged in the second branch beam path. The second detector element is configured to capture at least a second test image when the beam splitting element is inserted into the main beam path and the second filter element is not inserted into the second branch beam path, a fourth test image when the second filter element is inserted into the second branch beam path, and a second actual image. The imaging device also includes a controller configured to determine a first misalignment between the first test image and the second test image, a second misalignment between the third test image and the first test image, and a third misalignment between the fourth test image and the second test image; and align the first actual image with the second actual image based on the first misalignment, the second misalignment, and the third misalignment.

[0007] In order to align the first actual image and the second actual image, the imaging device is calibrated. The calibration includes two parts. The first part of the calibration involves capturing a first test image with a first detector and capturing a second test image with a second detector when a beam splitting element is inserted into the main beam path and no filter element is inserted into the first branch beam path and the second branch beam path. Then, the controller determines a first misalignment between the first test image and the second test image. The first misalignment is a misalignment introduced by the beam splitting element. Thus, in the first part of the calibration, the controller determines the misalignment between the first detector and the second detector caused by the beam splitting element.

[0008] The second part of the calibration involves capturing a third image with the first detector when the first filter element is inserted into the first branch beam path, and capturing a fourth image with the second detector when the second filter element is inserted into the second branch beam path. The controller then determines a second misalignment between the first test image and the third test image, and a third misalignment between the second test image and the fourth test image. The second misalignment is a misalignment introduced by the first filter element, and the third misalignment is a misalignment introduced by the second filter element. Thus, in the second part of the calibration, the controller determines an additional misalignment due to the first filter element and the second filter element.

[0009] The first and second parts of the calibration are performed independently of each other. Therefore, each time the first filter element and / or the second filter is changed, only the second part of the calibration has to be performed again. The third test image and the fourth test image only have to refer to the first test image and the second test image, respectively, and not to each other. Accordingly, the time required for the second part of the calibration increases only linearly with the number of first filter elements and second filter elements. Further, the first part of the calibration process only needs to be performed when the system configuration of the imaging system changes, for example, when the beam splitting element is removed from the main beam path and reinserted into the main beam path. Therefore, the two-part calibration enables the calibration of the imaging device to be performed quickly and efficiently, especially when more filter elements are used.

[0010] In a preferred embodiment, the controller is configured to detect a system configuration change of the imaging system, which system configuration change may cause a change in at least one of the first misalignment, the second misalignment and the third misalignment. The controller may be configured to control the first detector element to recapture the first test image and to control the second detector element to recapture the second test image when the controller has detected the system configuration change. The controller may also be configured to redetermine the first misalignment, the second misalignment and / or the third misalignment when the controller has detected the system configuration change. In particular, the controller may be configured to detect a system configuration change based on a temperature change and / or based on whether a beam splitting element has been removed from the main beam path and reinserted into the main beam path since the first misalignment was determined.

[0011] In this embodiment, the controller is configured to determine whether the first misalignment, the second misalignment, and / or the third misalignment need to be re-determined. The controller can easily track the status of all relevant components of the imaging system, for example, whether these components have been moved or whether the temperature of the imaging device has changed significantly. In other words, the controller can easily track the system configuration of the imaging system. Accordingly, the controller can detect any changes in the system configuration and determine which change may cause at least one of the first misalignment, the second misalignment, and the third misalignment to change. This information enables the controller to determine whether the first misalignment, the second misalignment, and / or the third misalignment need to be re-determined. This embodiment allows even inexperienced users or new users to use the imaging system correctly because the user does not need to have a full understanding of the imaging device. This makes the imaging system very easy to use.

[0012] In another preferred embodiment, the imaging system comprises an output unit. The controller may be configured to notify a user via the output unit that the first misalignment needs to be re-determined when the controller has detected a change in the system configuration. In this embodiment, the controller notifies the user when recalibration is required. The user may then decide to perform a recalibration, for example by making a user input via a user input device. In this embodiment, even an inexperienced user or a new user may be able to make an informed decision whether to perform a recalibration, thereby making the imaging device easier to use. In particular, the output unit may be configured as a monitor that displays an image generated by the controller to the user. The output unit may also be configured to output a sound and notify the user via a sound signal that the first misalignment needs to be re-determined. The controller may also be configured to display the first actual image and / or the second actual image via the output unit.

[0013] In another preferred embodiment, the controller is configured to determine the first misalignment, the second misalignment and / or the third misalignment based on the images of the edges and / or corners in the first test image and the third test image and / or in the second test image and the fourth test image. In particular, the edges and / or corners are edges or corners of a sample carrier of the imaging device or of a sample insert inserted into the sample carrier or the sample carrier, respectively. The edges or corners are visible in all four test images and can therefore be used like a dedicated reference. Further, the edges or corners are visible regardless of the filter element. Thus, in this embodiment, a simple and cost-effective means for determining the first misalignment, the second misalignment and / or the third misalignment is provided.

[0014] In another preferred embodiment, the controller is configured to determine the first misalignment, the second misalignment and / or the third misalignment based on an image of a benchmark in the first test image and the third test image and / or in the second test image and the fourth test image. The benchmark can be arranged, for example, on the sample carrier and / or on the microscope stage. Using the benchmark is a very reliable method for determining the first misalignment, the second misalignment and / or the third misalignment.

[0015] In another preferred embodiment, the controller is configured to determine a first image transformation that aligns the first test image with the second test image based on the first misalignment, determine a second image transformation that aligns the third test image with the first test image based on the second misalignment, and determine a third image transformation that aligns the fourth test image with the second test image based on the third misalignment. The controller is configured to align the first actual image with the second actual image based on the first image transformation, the second image transformation, and the third image transformation. The calibration provides information about the relative misalignment between the first detector and the second detector. In this embodiment, the information about the relative misalignment is encoded in the first image transformation, the second image transformation, and the third image transformation. Providing the information about the relative misalignment in the form of an image transformation makes the alignment of the first actual image and the second actual image more effective. For example, the first image transformation, the second image transformation, and the third image transformation can be easily stored in a storage element for later use. Further, when a system configuration change is detected and recalibration is required, the first image transformation, the second image transformation, and the third image transformation can be easily adjusted.

[0016] In another preferred embodiment, the controller is configured to determine a correlation coefficient between the aligned first actual image and the aligned second actual image, and based on the correlation coefficient, determine the quality of the alignment of the first actual image and the second actual image. When the first actual image and the second actual image are aligned, they will be highly correlated. Therefore, the correlation coefficient can be a good measure of the alignment quality of the first actual image and the second actual image. Determining the correlation coefficient is a quality control measure that improves the alignment of the first actual image and the second actual image, thereby making the imaging device more reliable. The correlation coefficient can also be used to determine when recalibration is needed.

[0017] In another preferred embodiment, the beam splitting element is configured to direct the detection light having a wavelength shorter than a predetermined center wavelength into a first branch beam path, and to direct the detection light having a wavelength longer than a predetermined center wavelength into a second branch beam path. In this embodiment, the beam splitting element acts like an edge filter, i.e., the beam splitting element directs most of the detection light having a wavelength shorter than the center wavelength into the first branch beam path. The residual light (i.e., most of the detection light having a wavelength longer than the center wavelength) is directed into the second branch beam path. Thus, at least two different fluorophores can be imaged simultaneously.

[0018] In another preferred embodiment, the imaging device comprises at least two first filter elements and / or at least two second filter elements. The first filter element and the second filter element can be arranged on a filter wheel. Thus, the filter elements can be easily replaced.

[0019] In another preferred embodiment, the first filter element and / or the second filter element is a bandpass filter. A bandpass is a filter that blocks all wavelengths of light except for a band around a central wavelength. In this embodiment, the filter element filters out most of the detection light except for the detection light emitted by the sample in a band around the central wavelength. Thus, only relevant wavelengths are selected for detection, thereby reducing background noise.

[0020] In another preferred embodiment, the imaging system comprises a housing. The beam splitting element may be arranged inside the housing. The first filter element, the second filter element, the first detector element and the second detector element may be arranged outside the housing. In this embodiment, when the first filter element or the second filter element is replaced, this change takes place outside the housing. Therefore, during the filter replacement, the optical elements arranged inside the housing remain undisturbed. This means that the first part of the calibration and the second part of the calibration are completely separated and can therefore be performed completely independently of each other. This makes the imaging device more reliable.

[0021] In another preferred embodiment, the housing comprises a first mounting piece configured to mount the first filter element and / or the first detector element outside the housing. The housing comprises a second mounting piece configured to mount the second filter element and / or the second detector element outside the housing. In particular, the first mounting piece and / or the second mounting piece may be a C-mounting piece. In this embodiment, the imaging device is designed to be modular. Both the filter element and the detector element may be easily replaceable, allowing the user to select the component that best suits their specific application.

[0022] In another preferred embodiment, the imaging device is a microscope, in particular a fluorescence microscope.

[0023] The present invention also relates to a method for aligning an image using the above-mentioned imaging device. The method includes the following steps: when a beam splitting element is inserted into a main beam path and no filter element is inserted into a first branch beam path and a second branch beam path, determining a first misalignment between an image captured by a first detector element arranged in the first branch beam path and an image captured by a second detector element arranged in the second branch beam path. The beam splitting element guides a first portion of the detection light into the first branch beam path and guides a second portion of the detection light into the second branch beam path. When the first filter element is inserted into the first branch beam path, determining a second misalignment between an image captured by the first detector element and an image captured by the first detector element when no filter element is inserted into the first branch beam path. When the second filter element is inserted into the second branch beam path, determining a third misalignment between an image captured by the second detector element and an image captured by the second detector element when no filter element is inserted into the second branch beam path. Based on the first misalignment, the second misalignment and the third misalignment, aligning a first actual image captured by the first detector element and a second actual image captured by the second detector element.

[0024] The method has the same advantages as the above imaging device and can be supplemented with the features of the dependent claims directed to the imaging device.

[0025] The present invention also relates to a computer program product, comprising a program code, wherein the program code is configured to execute the above method when the computer program product runs on a processor.

[0026] The computer program product has the same advantages as the above imaging device and method and can in particular be supplemented with the features of the dependent claims directed to the positioning device. BRIEF DESCRIPTION OF THE DRAWINGS

[0027] The specific embodiments are described below with reference to the accompanying drawings, in which: Figure 1 is a schematic diagram of an imaging device according to an embodiment; Figure 2 is and according to Figure 1 A schematic diagram of a sample carrier for use with an imaging device; Figure 3 Is based on Figure 1 and Figure 2 A flowchart of a method for aligning an image with an imaging device; Figure 4 Is based on Figure 3 A flowchart of a first sub-process of a method for aligning an image with an imaging device; and Figure 5 Is based on Figure 3 A flow chart of a second sub-process of the method of aligning an image with an imaging device. DETAILED DESCRIPTION

[0028] Figure 1 is a schematic diagram of an imaging device 100 according to an embodiment.

[0029] The imaging device 100 is exemplarily formed as a microscope. More specifically, in the present embodiment, the imaging device 100 is formed as a fluorescence microscope, which is configured to image the sample 102 by fluorescence imaging. However, the imaging device 100 is not limited to a microscope, for example, the imaging device 100 may also be a slide scanner or a flow cytometer, or any other imaging device that needs to simultaneously acquire two images of the same sample.

[0030] The sample 102 to be imaged is arranged on a sample carrier 104 of the imaging device 100. The sample carrier 104 is illustratively formed as an XY stage, which is configured to move the sample 102 in two perpendicular directions. In the present embodiment, the light source unit 106 of the imaging device 100 is arranged above the sample 102 and the sample carrier 104, and is configured to emit excitation light to excite the fluorophores located in the sample 102. In other embodiments, the light source unit 106 can be arranged below the sample carrier 104. The light source unit 106 can also be configured to emit other light, such as white light, to facilitate conventional optical microscopy techniques (such as reflected light microscopy or transmitted light microscopy). In the present embodiment, the detection light is formed by the fluorescence emitted by the excited fluorophores. In other embodiments, the detection light can be formed, for example, by light passing through the sample 102 or by light reflected by the sample 102.

[0031] The imaging device 100 includes a detection optical device 108, which is exemplarily formed as a microscope objective, arranged below the sample stage 104. The detection optical device 108 captures the detection light emitted by the sample 102 and guides the detection light into a main beam path 110 of the imaging device 100. The main beam path 110 includes a beam splitting element 112, which can be inserted into and removed from the main beam path 110. This is Figure 1denoted by a double arrow P. When the beam splitting element 112 is inserted into the main beam path 110, the beam splitting element 112 divides the main beam path 110 into a first branch beam path 114 and a second branch beam path 116 by directing a first portion of the detection light into a first branch beam path 114 and directing a second portion of the detection light into a second branch beam path 116. When the beam splitting element 112 is not inserted into the main beam path 110, the main beam path 110 continues unimpeded. In the present embodiment, the beam splitting element 112 is exemplarily formed as a dichroic beam splitting cube that directs a first portion of the detection light having a first wavelength into the first branch beam path 114, and directs a second portion of the detection light having a second wavelength into the second branch beam path 116.

[0032] The first branch beam path 114 is Figure 1 114 and includes a first filter wheel 118 and a first detector element 120. The first filter wheel 118 includes a plurality of first filter elements 122, which can be alternately introduced into the first branch beam path 114 by rotating the first filter wheel 118. The first detector element 120 is arranged after the first filter wheel 118 in the first branch beam path 114. The second branch beam path 116 is Figure 1 116 and includes a second filter wheel 124 and a second detector element 126. The second filter wheel 124 includes a plurality of second filter elements 128, which can be alternately introduced into the second branch beam path 116 by rotating the first filter wheel 118. The second detector element 126 is arranged after the second filter wheel 124 in the second branch beam path 116.

[0033] The first detector element 120 and the second detector element 126 are configured to capture detection light and generate an image from the captured detection light. In particular, since both the first branch beam path 114 and the second branch beam path 116 are branched from the main beam path 110, the first detector element 120 and the second detector element 126 can simultaneously image the sample 102. By introducing different first filter elements 122 and second filter elements 128 into the first branch beam path 114 and the second branch beam path 116, respectively, the image generated by the first detector element 120 and the second detector element 126 will include detection light of different wavelengths. This can be used to simultaneously image different structures of the sample 102 that have been stained with different fluorophores that emit fluorescence of different wavelengths.

[0034] However, in order to correlate different structures in the images generated by the first detector element 120 and the second detector element 126, the images need to be aligned. One reason for misalignment between the images is that the first detector element 120 and the second detector element 126 and the beam splitting element 112 cannot be perfectly arranged. Due to the arrangement of the first detector element 120, the second detector element 126 and the beam splitting element 112 inside the imaging device 100, the images generated by the first detector element 120 and the second detector element 126 will always be relatively offset and / or rotated. A second reason for misalignment between the images is the first filter element 122 and the second filter element 128. The first filter element 122 and the second filter element 128 are optically active elements arranged in the beam path between the sample 102 and the first detector element 120 and between the sample 102 and the second detector element 126, respectively. Therefore, the first filter element 122 and the second filter element 128 will cause the beam path to be displaced or distorted. Such displacement will result in additional misalignment between the images generated by the first detector element 120 and the second detector element 126 .

[0035] The imaging device 100 includes a controller 130 configured to execute a method of aligning an image captured by the imaging device 100. Figures 3 to 5 The method is described in detail. Controller 130 is also configured to control light source unit 106 , sample stage 104 , first filter wheel 118 and second filter wheel 124 , first detector element 120 and second detector element 126 , and beam splitting element 112 .

[0036] The imaging device 100 further comprises a housing 132, which can also be used as a microscope stand. Most of the components of the imaging device 100, in particular the light source unit 106, the sample stage 104, the detection optics and the beam splitting element 112, are surrounded by the housing 132. Thus, the housing 132 protects the enclosed components from dust and prevents stray light from entering the main beam path 110.

[0037] The housing 132 includes two mounting members 134, 136, which are exemplarily formed as C-shaped mounting members. The first mounting member 134 is Figure 1 132 and is configured to mount the first filter element 122 and the first detector element 120 outside the housing 132. The second mounting member 136 is shown as being located on the left side of the housing 132. Figure 1 132 and is shown as being located on the right side of the housing 132 and is configured to mount the second filter element 128 and the second detector element 126 outside the housing 132.

[0038] Figure 2 is and according to Figure 1Schematic diagram of a sample carrier 200 for use with the imaging device 100.

[0039] The sample carrier 200 is illustratively formed as a microscope slide on which the sample 102 is prepared. However, the sample carrier 200 can be any element suitable for mounting the sample 102, such as a culture dish, a multi-well plate, or a sample carrier 104. The sample carrier 200 includes a reference 202 disposed in the lower left corner. The reference 202 can be used to refer to the following Figures 3 to 5 The method of aligning the image captured by the imaging device 100. Alternatively, the corners 204 and / or the edges 206 of the sample carrier 200 may be used as references.

[0040] Figure 3 Is based on Figure 1 and Figure 2 Flow chart of a method of aligning an image by the imaging device 100.

[0041] refer to Figure 3 The method comprises calibration of the imaging device 100. The calibration enables the images captured by the first detector element 120 and the second detector element 126 to be aligned.

[0042] The process starts at step S300. In step S302, a first sub-process is performed. In the first sub-process, the controller 130 determines a first misalignment between the first detector and the second detector caused by the beam splitting element 112. Figure 4 The first sub-process is described in more detail. In step 304, a second sub-process is performed. In the second sub-process, the controller 130 determines additional misalignments caused by the first filter element 122 and the second filter element 128. In particular, the controller 130 determines a second misalignment caused by introducing one of the first filter elements 122 into the first branch beam path 114, and a third misalignment caused by introducing one of the second filter elements 128 into the second branch beam path 116. In step S306, based on the first misalignment, the second misalignment, and the third misalignment, a first actual image captured by the first detector element 120 and a second actual image captured by the second detector element 126 are aligned. The first actual image and the second actual image are images of the sample 102 captured in the context of an actual experiment, rather than images captured for the purpose of calibrating the imaging device 100. In particular, the alignment of the first actual image and the second actual image is performed by combining multiple image transformations determined during the first sub-process and the second sub-process. Referring to FIG. 1 , FIG. 1 , FIG. 1 , and FIG. 1 , Figure 4 and Figure 5 This is described in more detail. The process ends at step S308.

[0043] The first sub-process and the second sub-process (i.e., steps S302 and S304) are calibrations of the imaging device 100. The complete calibration process only needs to be performed once. After the initial calibration, the first sub-process and the second sub-process can be performed independently of each other. For example, when the first filter element 122 and / or the second filter are changed, only the second sub-process needs to be repeated to redetermine the second misalignment and / or the third misalignment, respectively. The first sub-process only needs to be repeated when the system configuration of the imaging system changes, for example, after the beam splitting element 112 has been removed from the main beam path 110 and reinserted into the main beam path.

[0044] Figure 4 Is based on Figure 3 Flow chart of a first sub-process of the method of aligning an image by the imaging device 100.

[0045] The first sub-process starts from step S400. In step S402, when the beam splitting element 112 is inserted into the main beam path 110 and no filter element is inserted into the first branch beam path and the second branch beam path 116, a first test image is captured by the first detector element 120, and a second test image is captured by the second detector element 126. In step S404, the controller 130 determines a first misalignment between the first test image and the second test image. When the first test image and the second test image are captured in step S402, no filter element is arranged in the first branch beam path 114 and the second branch beam path 116. Therefore, the misalignment of the first test image with the second test (i.e., the first misalignment) is mainly caused by the arrangement of the first detector element 120 and the second detector element 126 and the displacement introduced by the beam splitting element 112. In particular, in step S404, the controller 130 determines a first image transformation that aligns the first test image with the second test image. The first image transformation is saved and used later to align the first actual image and the second actual image. The first sub-process ends at step S406.

[0046] Figure 5 Is based on Figure 3 Flow chart of a second sub-process of the method of aligning an image by the imaging device 100.

[0047] The first subprocess begins at step S500. In step S502, a third test image is captured by the first detector element 120 when one of the first filter elements 122 is inserted into the first branch beam path 114. Step S502 may be repeated to capture additional third test images for each of the remaining first filter elements 122. In step S504, the controller 130 determines a second misalignment between the first test image and the third test image. When the first test image was captured, no filter element was arranged in the first branch beam path 114. By comparing the first test image with the third test image, the effect of the first filter element 122 on the first branch beam path 114 can be seen. Accordingly, the second misalignment is a misalignment caused by the introduction of the first filter element 122 into the first branch beam path 114. When additional third test images have been captured, step S504 may be repeated for these additional third test images. In step S504 , the controller 130 may also determine a second image transformation that aligns the third test image with the first test image, which second image transformation may be saved and later used to align the first actual image with the second actual image.

[0048] In step S506, a fourth test image is captured by the second detector element 126 when one of the second filter elements 128 is inserted into the second branch beam path 116. Step S506 may be repeated to capture additional fourth test images for each of the remaining second filter elements 128. In step S508, the controller 130 determines a third misalignment between the second test image and the fourth test image. When additional third test images have been captured, step S508 may be repeated for these additional fourth test images. When the second test image is captured, no filter element is arranged in the second branch beam path 116. By comparing the second test image with the fourth test image, the effect of the second filter element 128 on the second branch beam path 116 can be seen. Accordingly, the third misalignment is a misalignment caused by the introduction of the second filter element 128 into the second branch beam path 116. In step S508, the controller 130 may also determine a third image transformation that aligns the fourth test image with the second test image, which may be saved and later used to align the first actual image with the second actual image. The second sub-process ends in step S510.

[0049] Steps S502 and S506 may be performed simultaneously or in any order. Similarly, steps S504 and S508 may be performed simultaneously or in any order.

[0050] Figure 3 , Figure 4 and Figure 5 A method for aligning a first actual image and a second actual image captured by an imaging device 100 based on calibration of the imaging device 100 is described. The calibration provides information about the misalignment of the first actual image and the second actual image, which is quantified in a first image transformation, a second image transformation, and a third image transformation. In order to align the first actual image and the second actual image, the second image transformation is applied to the first actual image, and the third image transformation is applied to the second actual image. This eliminates the misalignment caused by the first filter element 122 and the second filter element 128 being arranged in the first branch beam path 114 and the second branch beam path 116, respectively. Thereafter, the first image transformation is applied to the transformed first actual image. This eliminates the misalignment caused by inserting the beam splitting element 112 into the main beam path 110. After applying the image transformation, the first actual image and the second actual image are aligned.

[0051] In all figures, the same or similarly functioning elements are indicated by the same reference numerals. As used herein, the term "and / or" includes any and all combinations of one or more associated listed items and may be abbreviated as " / ". The individual features of the embodiments and all combinations of the individual features of the embodiments with each other and with the individual features or feature groups of the previous description and / or claims are considered to be disclosed.

[0052] Although some aspects have been described in the context of an apparatus, it is apparent that these aspects also represent a description of a corresponding method, wherein a block or device corresponds to a method step or a feature of a method step. Similarly, aspects described in the context of a method step also represent a description of a corresponding block or item or feature of a corresponding apparatus.

[0053] Reference numerals list 100 Imaging Device 102 samples 104 Sample stage 106 Light source unit 108 Detection Optics 110 Main beam path 112 beam splitting element 114, 116 Branched beam paths 118 Filter Wheel 120 Detector element 122 filter element 124 Filter Wheel 126 Detector element 128 filter elements 130 Controller 132 Housing 134, 136 Mounting parts 200 Sample carriers 202 Benchmark 204 Corner 206 Edge P Arrow

Claims

1. An imaging device (100), comprising: detection optics (108) configured to receive detection light from the sample (102) and direct the detection light into a main beam path (110); a beam splitting element (112) configured to be inserted into the main beam path (110) to direct a first portion of the detection light into a first branch beam path (114) and to direct a second portion of the detection light into a second branch beam path (116); at least one first filter element (122) configured to be inserted into the first branch beam path (114); a first detector element (120) disposed in the first branch beam path (114) and configured to capture at least a first test image when the beam splitting element (112) is inserted into the main beam path (110) and the first filter element (122) is not inserted into the first branch beam path (114), a third test image when the first filter element (122) is inserted into the first branch beam path (114), and a first actual image; at least one second filter element (128) configured to be inserted into the second branch beam path (116); a second detector element (126) disposed in the second branch beam path (116) and configured to capture at least a second test image when the beam splitting element (112) is inserted into the main beam path (110) and the second filter element (128) is not inserted into the second branch beam path (116), a fourth test image when the second filter element (128) is inserted into the second branch beam path (116), and a second actual image; and a controller (130) configured to determine a first misalignment between the first test image and the second test image, a second misalignment between the third test image and the first test image, and a third misalignment between the fourth test image and the second test image; And aligning the first actual image with the second actual image according to the first misalignment, the second misalignment and the third misalignment.

2. The imaging system according to claim 1, wherein: The controller (130) is configured to detect a system configuration change of the imaging system, the system configuration change causing a change in at least one of the first misalignment, the second misalignment, and the third misalignment; control the first detector element (120) to recapture the first test image and control the second detector element (126) to recapture the second test image when the controller (130) has detected the system configuration change; and redetermine the first misalignment, the second misalignment, and / or the third misalignment when the controller (130) has detected the system configuration change.

3. The imaging system according to claim 2, wherein: The controller (130) is configured to detect the system configuration change based on a temperature change and / or based on whether the beam splitting element (112) has been removed from the main beam path (110) and reinserted into the main beam path (110) since the first misalignment was determined.

4. The imaging system according to claim 2 or 3, comprising an output unit; wherein, The controller (130) is configured to notify a user via the output unit that the first misalignment needs to be re-determined when the controller (130) has detected the system configuration change.

5. The imaging device (100) according to any one of the preceding claims, wherein: The controller (130) is configured to determine the first misalignment, the second misalignment and / or the third misalignment based on images of edges (206) and / or corners (204) in the first test image and the third test image and / or in the second test image and the fourth test image.

6. The imaging device (100) according to claim 5, wherein: The edge (206) and / or the corner (204) are respectively the edge (206) and / or the corner (204) of the sample carrier (104) of the imaging device (100), or the edge (206) or the corner (204) of the sample (102) insert inserted into the sample carrier (104) or the sample carrier (200).

7. The imaging device (100) according to any one of the preceding claims, wherein: The controller (130) is configured to determine the first misalignment, the second misalignment, and / or the third misalignment based on images of a fiducial (202) in the first and third test images and / or in the second and fourth test images.

8. The imaging device (100) according to any one of the preceding claims, wherein: The controller (130) is configured to determine a first image transformation for aligning the first test image and the second test image based on the first misalignment, determine a second image transformation for aligning the third test image and the first test image based on the second misalignment, and determine a third image transformation for aligning the fourth test image and the second test image based on the third misalignment; and wherein the controller (130) is configured to align the first actual image and the second actual image based on the first image transformation, the second image transformation and the third image transformation.

9. The imaging device (100) according to any one of the preceding claims, wherein: The controller (130) is configured to determine a correlation coefficient between the aligned first actual image and the aligned second actual image, and to determine a quality of the alignment of the first actual image and the second actual image based on the correlation coefficient.

10. The imaging device (100) according to any one of the preceding claims, wherein: The beam splitting element (112) is configured to guide detection light having a wavelength shorter than a predetermined central wavelength into the first branch beam path (114), and to guide detection light having a wavelength longer than the predetermined central wavelength into the second branch beam path (116).

11. The imaging device (100) according to any one of the preceding claims, comprising a housing (132); wherein The beam splitting element (112) is arranged inside the housing (132); and wherein the first filter element (122), the second filter element (128), the first detector element (120) and the second detector element (126) are arranged outside the housing (132).

12. The imaging device (100) according to claim 11, wherein: The housing (132) includes a first mounting member (134) configured to mount the first filter element (122) and / or the first detector element (120) outside the housing (132); and wherein the housing (132) includes a second mounting member (136) configured to mount the second filter element (128) and / or the second detector element (126) outside the housing (132).

13. The imaging device (100) according to any one of the preceding claims, wherein: The imaging device (100) is a microscope, in particular a fluorescence microscope.

14. A method for aligning an image captured by an imaging device (100), the method comprising the following steps: a) determining a first misalignment between an image captured by a first detector element (120) arranged in the first branch beam path (114) and an image captured by a second detector element (126) arranged in the second branch beam path (116) when a beam splitting element (112) is inserted into a main beam path (110) and no filter element is inserted into a first branch beam path and a second branch beam path (116), wherein the beam splitting element (112) directs a first portion of the detection light into the first branch beam path (114) and directs a second portion of the detection light into the second branch beam path (116); b) determining a second misalignment between an image captured by the first detector element (120) when a first filter element (122) is inserted into the first branch beam path (114) and an image captured by the first detector element (120) when no filter element is inserted into the first branch beam path (114); c) determining a third misalignment between an image captured by the second detector element (126) when a second filter element (128) is inserted into the second branch beam path (116) and an image captured by the second detector element (126) when no filter element is inserted into the second branch beam path (116); and d) aligning a first actual image captured by the first detector element (120) and a second actual image captured by the second detector element (126) based on the first misalignment, the second misalignment and the third misalignment.

15. A computer program product comprising a program code, the program code being configured to perform the method according to claim 14 when the computer program product is run on a processor.

Images