Imaging device and image alignment method

Abstract

The imaging device (100) has a detection optical system (108) configured to receive detection light from the sample (102) and direct the detection light toward the main beam path (110). The imaging device (100) also has a beam splitting element (112) inserted into the main beam path (110) and configured to direct a first portion of the detection light toward the first branch beam path (114) and a second portion of the detection light toward the second branch beam path (116). At least one first filter element (122) is configured to be inserted into the first branch beam path (114). The first detection element (120) is disposed in the first branch beam path (114) and is 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), to capture a third test image when the first filter element (122) is inserted into the first branch beam path (114), and to capture a first real image. At least one second filter element (128) is configured to be inserted into the second branch beam path (116). The second detection element (126) is disposed in the second branch beam path (116) and is 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 second filter element (128) is not inserted into the second branch beam path (116), to capture a fourth test image when the second filter element (128) is inserted into the second branch beam path (116), and to capture a second real image. The imaging device (100) further has a controller configured to identify a first misalignment between the first test image and the second test image, to identify 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 to align the first real image and the second real 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. The present invention further relates to a method for aligning images and a computer program product.

Background Art

[0002] In conventional fluorescence microscopy, an apparatus with exchangeable filters and a high-sensitivity detector is used to generate an image from fluorescence. The filter is an optical element disposed in the beam path between the sample and the detector. Therefore, when the filter is changed, a deviation may occur in the beam path or distortion of the beam path may occur, and as a result, the images captured before and after the filter change no longer match with pixel accuracy. This effect is more prominent when multiple detectors are used to simultaneously capture images of different wavelengths by using different filters. In addition to the deviation caused by the filter, the detectors may also be misaligned relative to each other. Such misalignment appears as a relative deviation and / or rotation of the images generated by different detectors. In an imaging device having a plurality of detectors, any misalignment of the images generated by different detectors nullifies the advantages obtained by simultaneously capturing images. Therefore, in order to counteract misalignment, it is necessary to calibrate the imaging device so that the images captured by different detectors can be aligned.

[0003] Calibration is typically performed manually by the user. To calibrate the imaging device, the user has to find and mark the same points in different images. When different filters are used, different wavelengths are captured by different detectors, and the images generated by the detectors may vary significantly. This may consequently result in a certain degree of inaccuracy. Alternatively, appropriate calibration may be identified using a technical sample before actual image acquisition, and this calibration is then applied to the actual image data. However, such calibration has to be repeated every time a component that affects the beam path, such as a filter or a beam splitter, is changed, which consumes a great deal of time. Also, an experienced user is required to identify which hardware changes in the imaging device necessitate recalibration. Furthermore, additional bias may be caused by temperature changes, and thus the previous calibration may become invalid relatively quickly and unnoticed. Summary of the Invention Problems to be Solved by the Invention

[0004] Therefore, the problem is to provide a method for quickly and efficiently calibrating an imaging device, particularly an imaging device using more filter elements, and for aligning images. Means for Solving the Problems

[0005] The above problems are achieved by the subject matter of the independent claims. Advantageous embodiments are defined in the independent claims and the following description.

[0006] The proposed imaging device has a detection optical system configured to receive detection light from a sample and direct the detection light into a main beam path. The imaging device also has a beam splitting element inserted into the main beam path and configured to direct a first portion of the detection light into a first branched beam path and a second portion of the detection light into a second branched beam path. The imaging device also has at least one first filter element configured to be inserted into the first branched beam path. The imaging device also has a first detection element disposed in the first branched beam path. The first detection 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 branched beam path, a third test image when the first filter element is inserted into the first branched beam path, and a first real image. The imaging device also has at least one second filter element configured to be inserted into the second branched beam path. The imaging device also has a second detection element disposed in the second branched beam path. The second detection 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 branched beam path, a fourth test image when the second filter element is inserted into the second branched beam path, and a second real image. The imaging device further has a controller configured to identify a first misalignment defect between the first test image and the second test image, a second misalignment defect between the third test image and the first test image, and a third misalignment defect between the fourth test image and the second test image, and align the first real image and the second real image based on the first misalignment defect, the second misalignment defect, and the third misalignment defect.

[0007] The imaging device is calibrated to align the first real image and the second real image. This calibration has two parts. The first part of the calibration includes capturing a first test image by a first detector and, when the beam splitter element is inserted into the main beam path and the filter element is not inserted into the first and second branch beam paths, capturing a second test image by a second detector element. Next, a first alignment defect between the first test image and the second test image is identified by a controller. The first alignment defect is an alignment defect caused by the beam splitter element. Thereby, in the first part of the calibration, the controller identifies an alignment defect between the first detector and the second detector caused by the beam splitter element.

[0008] The second part of the calibration includes capturing a third image by the first detector when a first filter element is inserted into the first branch beam path and capturing a fourth image by the second detector when a second filter element is inserted into the second branch beam path. Next, a second alignment defect between the first test image and the third test image and a third alignment defect between the second test image and the fourth test image are identified by the controller. The second alignment defect is an alignment defect caused by the first filter element, and the third alignment defect is an alignment defect caused by the second filter element. Thereby, in the second part of the calibration, the controller identifies additional alignment defects caused by 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 needs to be performed again. The third test image and the fourth test image only need to be referenced to the first test image and the second test image respectively, and do not need to be referenced to each other. Therefore, the time required for the second part of the calibration only increases linearly with the number of the first filter element and the second filter element. Further, the first part of the calibration process only needs to be performed when the system configuration of the imaging system is changed. For example, it only needs to be performed when the beam splitting element is removed from the main beam path and reinserted into the main beam path. Thereby, the calibration consisting of two parts can perform the calibration of the imaging device quickly and efficiently, especially as the number of filter elements used increases.

[0010] In one preferred embodiment, the controller is configured to detect a system configuration change of the imaging system that can cause at least one change in the first alignment defect, the second alignment defect, and the third alignment defect. The controller may be configured to control the first detection element to capture the first test image again and control the second detection element to capture the second test image again when a system configuration change is detected by the controller. The controller may further be configured to re-identify the first alignment defect, the second alignment defect, and / or the third alignment defect when a system configuration change is detected by the controller. In particular, the controller may be configured to detect a system configuration change based on a temperature change and / or based on whether the beam splitting element has been removed from and reinserted into the main beam path after the first alignment defect is identified.

[0011] In this embodiment, the controller is configured to identify whether it is necessary to re-identify a first alignment defect, a second alignment defect, and / or a third alignment defect. The controller can easily track the states 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. Therefore, the controller can detect any changes in the system configuration and determine which changes can cause at least one change in the first alignment defect, the second alignment defect, and the third alignment defect. Based on this information, the controller can identify whether it is necessary to re-identify the first alignment defect, the second alignment defect, and / or the third alignment defect. These embodiments enable even inexperienced or new users to properly use the imaging system because the users do not need to have complete knowledge about the imaging device. This makes the imaging system extremely user-friendly.

[0012] In another preferred embodiment, the imaging system has an output unit. The controller may be configured to notify the user, via the output unit, that the first alignment defect must be re-identified if a system configuration change is detected by the controller. In this embodiment, the controller notifies the user when realignment is necessary. The user may then decide to perform realignment, for example via a user input device. In this embodiment, even a less experienced or new user can make a decision after obtaining sufficient information about whether to perform realignment, thereby making the imaging device easier to use. The output unit may in particular be a monitor configured to display an image generated by the controller to the user. The output unit may also be configured to output sound and notify the user that the first alignment defect needs to be re-identified by an acoustic signal. The controller may also be configured to display the first real image and / or the second real image via the output unit.

[0013] In another preferred embodiment, the controller is configured to identify a first alignment defect, a second alignment defect and / or a third alignment defect based on the images of edges and / or corners in the first test image and the third test image, and / or the second test image and the fourth test image. In particular, each of the edge and / or corner is an edge or corner of the sample stage of the imaging device, or of a sample inserted into the sample stage or sample carrier. The edge or corner is visible in all four test images and can therefore be used like a dedicated reference. The edge or corner is further visible regardless of the filter element. Thereby, in this embodiment, a simple and cost-effective means for identifying the first alignment defect, the second alignment defect and / or the third alignment defect is provided.

[0014] In another preferred embodiment, the controller is configured to identify a first alignment defect, a second alignment defect, and / or a third alignment defect based on a reference image in the first test image and the third test image, and / or the second test image and the fourth test image. The reference may be disposed, for example, on a sample carrier and / or a microscope stage. Using the reference is a very reliable way to identify the first alignment defect, the second alignment defect, and / or the third alignment defect.

[0015] In another preferred embodiment, the controller is configured to identify a first image transformation for aligning the first test image and the second test image based on the first alignment defect, identify a second image transformation for aligning the third test image and the first test image based on the second alignment defect, and identify a third image transformation for aligning the fourth test image and the second test image based on the third alignment defect. The controller is configured to align the first real image and the second real image based on the first image transformation, the second image transformation, and the third image transformation. Calibration provides information regarding the relative alignment defect between the first detector and the second detector. In this embodiment, the information regarding the relative alignment defect is encoded in the first image transformation, the second image transformation, and the third image transformation. By providing information regarding the relative alignment defect in the form of image transformations, the alignment of the first real image and the second real image becomes more efficient. For example, the first image transformation, the second image transformation, and the third image transformation may be easily stored in a memory element for later use. Further, the first image transformation, the second image transformation, and the third image transformation can be easily adapted when a system configuration change is detected and recalibration is required.

[0016] In another preferred embodiment, the controller is configured to identify a correlation coefficient between the aligned first real image and the aligned second real image, and to identify the quality of the alignment between the first real image and the second real image based on the correlation coefficient. When the first real image and the second real image are aligned, their degree of correlation is high. Therefore, the correlation coefficient is a good measure of the quality of the alignment between the first real image and the second real image. Identifying the correlation coefficient is a quality control measure for improving the alignment between the first real image and the second real image, thereby increasing the reliability of the imaging device. Therefore, the correlation coefficient may be used to identify when recalibration is necessary.

[0017] In another preferred embodiment, the beam splitting element is configured to direct detection light having a wavelength shorter than a predetermined central wavelength to the first branched beam path, and to direct detection light having a wavelength longer than the predetermined central wavelength to the second branched beam path. In this embodiment, in the sense that the beam splitting element directs most of the detection light having a wavelength shorter than the central wavelength to the first branched beam path, the beam splitting element operates like an edge filter. The residual light, i.e., most of the detection light having a wavelength longer than the central wavelength, is directed to the second branched beam path. Thereby, at least two different fluorescent substances can be imaged simultaneously.

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

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

[0020] In another preferred embodiment, the imaging system has a housing. The beam splitting element may be disposed within the housing. The first filter element, the second filter element, the first detection element, and the second detection element may be disposed outside the housing. In this embodiment, when the first filter element or the second filter element is replaced, this change occurs outside the housing. Thereby, the optical elements disposed within the housing remain unaffected even during filter replacement. What this means is that the first part of the calibration and the second part of the calibration are completely separated and can thus be performed completely independently of each other. Thereby, the reliability of the imaging device is higher.

[0021] In another preferred embodiment, the housing has a first mount configured to mount the first filter element and / or the first detection element outside the housing. The housing may have a second mount configured to mount the second filter element and / or the second detection element outside the housing. The first mount and / or the second mount may in particular be a C-mount. In this embodiment, the imaging device is designed modularly. By enabling easy replacement of both the filter element and the detection element, the user can select the components most suitable for 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 images by the above-described imaging device. The method includes the following steps: that is, when the beam splitting element is inserted into the main beam path and the filter element is not inserted into the first branch beam path and the second branch beam path, the image captured by the first detection element disposed in the first branch beam path and the image captured by the second detection element disposed in the second branch beam path, a step of identifying a first misalignment therebetween, the beam splitting element directing a first portion of the detection light to the first branch beam path and a second portion of the detection light to the second branch beam path. The method further includes, when a first filter element is inserted into the first branch beam path, the image captured by the first detection element and the image captured by the first detection element when the filter element is not inserted into the first branch beam path, a step of identifying a second misalignment therebetween, and when a second filter element is inserted into the second branch beam path, the image captured by the second detection element and the image captured by the second detection element when the filter element is not inserted into the second branch beam path, a step of identifying a third misalignment therebetween, and based on the first misalignment, the second misalignment and the third misalignment, aligning a first actual image captured by the first detection element and a second actual image captured by the second detection element.

[0024] The method has the same advantages as the above-described imaging device and may be supplemented using the features of the dependent claims related to the imaging device.

[0025] The present invention further relates to a computer program product having program code configured to implement the above-described method when the computer program product operates on a processor.

[0026] The computer program product has the same advantages as the above-described imaging device and method, and in particular, may be supplemented using the features of the dependent claims related to the imaging device.

[0027] Hereinafter, specific embodiments will be described with reference to the drawings.

Brief Description of the Drawings

[0028]

Figure 1

Figure 2

Figure 3

Figure 4

Figure 5

Modes for Carrying Out the Invention

[0029] FIG. 1 is a schematic diagram of an imaging device 100 according to one embodiment.

[0030] The imaging device 100 is exemplarily formed as a microscope. More specifically, in this embodiment, the imaging device 100 is formed as a fluorescence microscope configured to image a sample 102 by fluorescence imaging. However, the imaging device 100 is not limited to a microscope. For example, the imaging device 100 may be a slide scanner or a flow cytometer, or any other imaging device where two simultaneous images of the same sample are desired.

[0031] The sample 102 to be imaged is placed on the sample stage 104 of the imaging device 100. The sample stage 104 is exemplarily formed as an X-Y table configured to move the sample 102 along two perpendicular directions. In this embodiment, the light source unit 106 of the imaging device 100 is disposed above the sample 102 and the sample stage 104, and is configured to emit excitation light for exciting the fluorescent substance located within the sample 102. In another embodiment, the light source unit 106 may be disposed below the sample stage 104. The light source unit 106 may be configured to emit another light, such as white light, in order to facilitate conventional optical microscopy techniques, such as reflection optical microscopy or transmission optical microscopy, etc. In this embodiment, the detection light is formed by the fluorescence emitted by the excited fluorescent substance. In another embodiment, the detection light may be formed by, for example, the light passing through the sample 102 or the light reflected by the sample 102.

[0032] The imaging device 100 has a detection optical system 108 disposed below a sample stage 104, which is exemplarily formed as a microscope objective lens. The detection optical system 108 captures the detection light emitted by the sample 102, and directs the detection light into the main beam path 110 of the imaging device 100. The main beam path 110 has a beam splitting element 112, and the beam splitting element 112 can be inserted into and removed from the main beam path 110. This is indicated by the double-headed arrow P in FIG. 1. When the beam splitting element 112 is inserted into the main beam path 110, the beam splitting element 112 directs the first portion of the detection light to the first branched beam path 114 and the second portion of the detection light to the second branched beam path 116, thereby splitting the main beam path 110 into the first branched beam path 114 and the second branched beam path 116. When the beam splitting element 112 is not inserted into the main beam path 110, the main beam path 110 remains unobstructed. In this embodiment, the beam splitting element 112 is exemplarily formed as a dichroic beam splitting cube that directs the first portion of the detection light having a first wavelength to the first branched beam path 114 and the second portion of the detection light having a second wavelength to the second branched beam path 116.

[0033] The first branched beam path 114 is shown as branching to the left in FIG. 1 and has a first filter wheel 118 and a first detection element 120. The first filter wheel 118 has a plurality of first filter elements 122 that can be alternately introduced into the first branched beam path 114 by rotating the first filter wheel 118. The first detection element 120 is disposed in the first branched beam path 114 following the first filter wheel 118. The second branched beam path 116 is shown as branching to the right in FIG. 1 and has a second filter wheel 124 and a second detection element 126. The second filter wheel 124 has a plurality of second filter elements 128 that can be alternately introduced into the second branched beam path 116 by rotating the second filter wheel 124. The second detection element 126 is disposed in the second branched beam path 116 following the second filter wheel 124.

[0034] The first detection element 120 and the second detection element 126 are configured to capture detection light and generate an image from the captured detection light. In particular, since both the first branching beam path 114 and the second branching beam path 116 are split from the main beam path 110, the first detection element 120 and the second detection element 126 can image the sample 102 simultaneously. By introducing different first filter elements 122 and second filter elements 128 into the first branching beam path 114 and the second branching beam path 116 respectively, the images generated by the first detection element 120 and the second detection element 126 will have different wavelengths of the detection light. This can be used to simultaneously image different structures of the sample 102 stained with different fluorescent substances that emit fluorescence of different wavelengths.

[0035] However, it is necessary to align the images in order to associate different structures in the images generated by the first detection element 120 and the second detection element 126. One cause of misalignment between the images is that it is impossible to place the first detection element 120, the second detection element 126, and the beam splitting element 112 perfectly. Due to the arrangement of the first detection element 120, the second detection element 126, and the beam splitting element 112 within the imaging device 100, there will always be a relative bias and / or rotation of the images generated by the first detection element 120 and the second detection element 126. The second cause of 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 optical active elements arranged in the beam path between the sample 102 and the first detection element 120 and the second detection element 126 respectively. Therefore, the beam path is shifted or deformed by the first filter element 122 and the second filter element 128. As a result of this shift, additional misalignment between the images generated by the first detection element 120 and the second detection element 126 will occur.

[0036] Imaging device 100 has a controller 130 configured to implement a method for aligning an image captured by the imaging device 100. Hereinafter, this method will be described in detail with reference to FIGS. 3 to 5. The controller 130 is further configured to control a light source unit 106, a sample stage 104, a first filter wheel 118, a second filter wheel 124, a first detection element 120, a second detection element 126, and a beam splitting element 112.

[0037] The imaging device 100 further has a housing 132 that also functions as a microscope stage. Most of the components of the imaging device 100, particularly the light source unit 106, the sample stage 104, the detection optical system, and the beam splitting element 112, are sealed by the housing 132. Thereby, the housing 132 protects the sealed components from dust and prevents stray light from entering the main beam path 110.

[0038] The housing 132 has two mounts 134, 136, exemplarily formed as a c-mount. The first mount 134 is shown on the left side of the housing 132 in FIG. 1 and is configured to mount a first filter element 122 and a first detection element 120 outside the housing 132. The second mount 136 is shown on the right side of the housing 132 in FIG. 1 and is configured to mount a second filter element 128 and a second detection element 126 outside the housing 132.

[0039] FIG. 2 is a schematic view of a sample carrier 200 used in the imaging device 100 according to FIG. 1.

[0040] The sample carrier 200 is exemplarily formed as a microscope slide on which the sample 102 is prepared. However, the sample carrier 200 may be any element suitable for mounting the sample 102, such as a Petri dish, a multi-well plate, or the sample stage 104. The sample carrier 200 has a reference 202 disposed at the lower left corner. The reference 202 can be used to align the alignment image captured by the imaging device 100 by the method described below with reference to FIGS. 3 to 5. Alternatively, the corners 204 and / or edges 206 of the sample carrier 200 can also be used as references.

[0041] FIG. 3 is a flowchart of a method for aligning images by the imaging device 100 according to FIGS. 1 and 2.

[0042] The method described with reference to FIG. 3 includes calibration of the imaging device 100. By this calibration, the images captured by the first detection element 120 and the second detection element 126 can be aligned.

[0043] The process is started in step S300. In step S302, a first sub-process is performed. In the first sub-process, the controller 130 identifies a first misalignment between the first detector and the second detector due to the beam splitting element 112. The first sub-process will be described in more detail below with reference to FIG. 4. In step 304, a second sub-process is performed. In the second sub-process, the controller 130 identifies additional misalignments due to the first filter element 122 and the second filter element 128. In particular, the controller 130 identifies a second misalignment caused by introducing one of the first filter elements 122 into the first branched beam path 114 and a third misalignment caused by introducing one of the second filter elements 128 into the second branched beam path 116. In step S306, based on the first misalignment, the second misalignment, and the third misalignment, the first real image captured by the first detection element 120 and the second real image captured by the second detection element 126 are aligned. The first real image and the second real image are images of the sample 102 captured in the context of an actual experiment, as opposed to images captured for the purpose of calibrating the imaging device 100. The alignment of the first real image and the second real image is performed in particular by combining a plurality of image transformations identified during the first sub-process and the second sub-process. This will be described in more detail below with reference to FIGS. 4 and 5. This process is terminated in step S216.

[0044] The first sub-process and the second sub-process, namely steps S302 and S304, are calibrations of the imaging device 100. The complete calibration process only needs to be performed once. After that initial calibration, the first sub-process and the second sub-process can be performed separately from each other. For example, when the first filter element 122 and / or the second filter is changed, it is sufficient to repeat the second sub-process in order to re-identify the second alignment defect and / or the third alignment defect respectively. The first sub-process only needs to be repeated when the system configuration of the imaging system changes. For example, it can be repeated after the beam splitting element 112 is removed from the main beam path 110 and re-inserted into the main beam path 110.

[0045] FIG. 4 is a flowchart of the first sub-process of a method for aligning an image by the imaging device 100 according to FIG. 3.

[0046] In step S400, the first sub-process is started. In step S402, when the beam splitting element 112 is inserted into the main beam path 110 and the filter element is not inserted into the first branch beam path 114 and the second branch beam path 116, the first detection element 120 captures a first test image and the second detection element 126 captures a second test image. In step S404, the controller 130 identifies a first misalignment between the first test image and the second test image. When capturing the first test image and the second test image 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 between the first test image and the second test image, that is, the first misalignment, is mainly caused by the misalignment caused by the arrangement of the first detection element 120 and the second detection element 126 and the beam splitting element 112. In step S404 in particular, the controller 130 identifies a first image transformation for aligning the first test image with the second test image. This first image transformation is saved and used later to align the first real image and the second real image. In step S406, the first sub-process is terminated.

[0047] FIG. 5 is a flowchart of a second sub-process of the method according to FIG. 3 for aligning an image using the imaging device 100.

[0048] In step S500, a first sub-process is started. In step S502, when one of the first filter elements 122 is inserted into the first branch beam path 114, a third test image is captured by the first detection element 120. Step S502 can be repeated to capture additional third test images for each of the remaining first filter elements 122. In step S404, the controller 130 identifies a second misalignment defect between the first test image and the third test image. When the first test image is captured, no filter element is arranged in the first branch beam path 114. By comparing the first test image and the third test image, the effect of the first filter element 122 in the first branch beam path 114 can be observed. Therefore, the second misalignment defect is a misalignment defect caused by the introduction of the first filter element 122 into the first branch beam path 114. If additional third test images are captured, step S504 may be repeated for the additional third test images. In step S504, the controller 130 can also identify a second image transformation for aligning the third test image with the first test image, and the second image transformation can be saved and used later to align the first real image and the second real image.

[0049] In step S506, when one of the second filter elements 128 is inserted into the second branch beam path 116, the second detection element 126 captures a fourth test image. Step S506 can be repeated to capture additional fourth test images for each of the remaining second filter elements 128. In step S508, the controller 130 identifies a third misalignment defect between the second test image and the fourth test image. If additional third test images have been captured, step S508 may be repeated for the additional fourth test images. When capturing the second test image, no filter element is disposed 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 in the second branch beam path 116 can be observed. Thus, the third misalignment defect is a misalignment defect caused by introducing the second filter element 128 into the second branch beam path 116. In step S508, the controller 130 can also identify a third image transformation for aligning the fourth test image with the second test image, and the third image transformation can be saved and later used to align the first real image and the second real image. The second sub-process ends in step S510.

[0050] Steps S502 and S506 can be performed simultaneously or sequentially in any order. Similarly, steps S504 and S508 can be performed simultaneously or sequentially in any order.

[0051] Figures 3, 4, and 5 illustrate a method of aligning a first actual image and a second actual image captured by the imaging device 100 based on calibration of the imaging device 100. This calibration supplies information regarding misalignment between the first actual image and the second actual image, which is quantified in the first image conversion, the second image conversion, and the third image conversion. To align the first actual image and the second actual image, the second image conversion is applied to the first actual image and the third image conversion is applied to the second actual image. This cancels misalignment caused by the first filter element 122 disposed in the first branch beam path 114 and the second filter element 128 disposed in the second branch beam path 116, respectively. Thereafter, the first image conversion is applied to the already-converted first actual image. This cancels misalignment caused by the beam splitting element 112 inserted into the main beam path 110. After applying the image conversions, the first actual image and the second actual image are aligned.

[0052] In all the figures, the same reference numerals are assigned to the same elements or elements of similar operations. 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 " / ". All combinations of the individual features of the embodiments, as well as combinations of the individual features of the embodiments in combination with each other, or with the individual features or groups of features of the preceding description and / or the claims, are considered to be disclosed.

[0053] Although some aspects have been described in the context of an apparatus, it is clear that these aspects also represent a description of corresponding methods, where a block or apparatus corresponds to a step or a feature of a step. Similarly, aspects described in the context of a step also represent a description of corresponding blocks or items or features of a corresponding apparatus.

Description of Reference Numerals

[0054] 100 Imaging device 102 Sample 104 Sample stage 106 Light source unit 108 Detection optical system 110 Main beam path 112 Beam splitting element 114, 116 Branch beam paths 118 Filter wheel 124 Filter wheel 126 Detection element 128 Filter element 130 Controller 132 Housing 134, 136 Mounts 200 Sample carrier 202 Reference 204 Corner 206 Edge P arrow

Claims

1. An imaging device (100), wherein the imaging device (100) comprises: A detection optical system (108) configured to receive detection light from a sample (102) and direct the detection light toward a main beam path (110); A beam splitting element (112) inserted into the main beam path (110) and configured to direct a first portion of the detection light toward a first branch beam path (114) and a second portion of the detection light toward 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 detection element (120) disposed in the first branch beam path (114), the first detection element (120) being 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), to capture a third test image when the first filter element (122) is inserted into the first branch beam path (114), and to capture a first real image; At least one second filter element (128) configured to be inserted into the second branch beam path (116); A second detection element (126) disposed in the second branch beam path (116), the second detection element (126) being 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), to capture a fourth test image when the second filter element (128) is inserted into the second branch beam path (116), and to capture a second real image; A controller (130); And The controller (130) is configured to: Identify 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; Align the first real image and the second real image based on the first misalignment, the second misalignment, and the third misalignment. Imaging device (100).

2. The controller (130) detects a system configuration change of the imaging device (100) that can cause at least one change among the first alignment defect, the second alignment defect, and the third alignment defect, controls the first detection element (120) to re-capture the first test image, controls the second detection element (126) to re-capture the second test image when the system configuration change is detected by the controller (130), and is configured to re-identify the first alignment defect, the second alignment defect, and / or the third alignment defect when the system configuration change is detected by the controller (130). The imaging device (100) according to claim 1.

3. 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 and re-inserted into the main beam path (110) after the first alignment defect is identified. The imaging device (100) according to claim 2.

4. The imaging device (100) has an output unit, and the controller (130) is configured to notify the user, via the output unit, that the first alignment defect must be re-identified when the system configuration change is detected by the controller (130). The imaging device (100) according to claim 2 or 3.

5. The controller (130) is configured to identify the first alignment defect, the second alignment defect, and / or the third alignment defect based on the first test image and the third test image, and / or based on the images of edges (206) and / or corners (204) in the second test image and the fourth test image. The imaging device (100) according to any one of claims 1 to 4.

6. The edge (206) and / or the corner (204) is respectively an edge (206) or a corner (204) of the sample stage (104) of the imaging device (100), or of a sample (102) inserted into the sample stage (104) or the sample carrier (200). The imaging device (100) according to claim 5.

7. The controller (130) is configured to identify the first misalignment, the second misalignment, and / or the third misalignment based on the reference (202) images in the first test image and the third test image, and / or the second test image and the fourth test image. The imaging device (100) according to any one of claims 1 to 6.

8. The controller (130) is configured to identify a first image transformation for aligning the first test image and the second test image based on the first misalignment, identify a second image transformation for aligning the third test image and the first test image based on the second misalignment, and identify a third image transformation for aligning the fourth test image and the second test image based on the third misalignment. The controller (130) is configured to align the first real image and the second real image based on the first image transformation, the second image transformation, and the third image transformation. The imaging device (100) according to any one of claims 1 to 7.

9. The controller (130) is configured to identify a correlation coefficient between the aligned first real image and the aligned second real image, and identify the quality of the alignment between the first real image and the second real image based on the correlation coefficient. The imaging device (100) according to any one of claims 1 to 8.

10. The beam splitting element (112) is configured to direct detection light having a wavelength shorter than a predetermined center wavelength toward the first branched beam path (114), and direct detection light having a wavelength longer than the predetermined center wavelength toward the second branched beam path (116). The imaging device (100) according to any one of claims 1 to 9.

11. The imaging device (100) has a housing (132), and the beam splitting element (112) is disposed within the housing (132), and the first filter element (122), the second filter element (128), the first detection element (120), and the second detection element (126) are disposed outside the housing (132). The imaging device (100) according to any one of claims 1 to 10.

12. The housing (132) has a first mount (134) configured to mount the first filter element (122) and / or the first detection element (120) outside the housing (132), and the housing (132) has a second mount (136) configured to mount the second filter element (128) and / or the second detection element (126) outside the housing (132). The imaging device (100) according to claim 11.

13. The imaging device (100) is a microscope, particularly a fluorescence microscope. The imaging device (100) according to any one of claims 1 to 12.

14. A method for aligning an image captured by an imaging device (100), the method comprising: a) When the beam splitting element (112) is inserted into the main beam path (110) and the filter element is not inserted into the first branch beam path (114) and the second branch beam path (116), identifying a first misalignment between an image captured by the first detection element (120) disposed in the first branch beam path (114) and an image captured by the second detection element (126) disposed in the second branch beam path (116), the step of directing a first portion of the detection light to the first branch beam path (114) and a second portion of the detection light to the second branch beam path (116) by the beam splitting element (112); b) Identifying a second misalignment between an image captured by the first detection element (120) when the first filter element (122) is inserted into the first branch beam path (114) and an image captured by the first detection element (120) when the filter element is not inserted into the first branch beam path (114). c) identifying a third misalignment between an image captured by the second detection element (126) when the second filter element (128) is inserted into the second branched beam path (116) and an image captured by the second detection element (126) when the filter element is not inserted into the second branched beam path (116); d) aligning a first actual image captured by the first detection element (120) and a second actual image captured by the second detection element (126) based on the first misalignment, the second misalignment, and the third misalignment; A method comprising the steps of: **Claim 15** A computer program product having program code configured to implement the method according to claim 14 when the computer program product is run on a processor.

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