Method for detecting nuclear translocation of receptors present in the cytoplasm, and method for detecting signal intensity in the cell nucleus and cytoplasm.
By using centroid positions and circumscribing rectangles to track cell nuclei and compare pixel values, the method quantitatively detects nuclear translocation and signal intensity changes in living cells, addressing the limitations of existing methods.
Patent Information
- Authority / Receiving Office
- JP · JP
- Patent Type
- Patents
- Current Assignee / Owner
- IWATE UNIVERSITY
- Filing Date
- 2022-09-28
- Publication Date
- 2026-07-23
AI Technical Summary
Existing methods fail to quantitatively and continuously detect the nuclear translocation of cytoplasmic receptors and the signal intensity changes in the cell nucleus and cytoplasm over time in living cells.
A method using a computer to identify and track cell nuclei through centroid positions and circumscribing rectangles, and compare average pixel values of nuclear and cytoplasmic regions in a time series to detect nuclear translocation and signal intensity changes.
Enables the acquisition of quantitative and continuous data on nuclear translocation and signal intensity changes in living cells, allowing for accurate tracking and analysis of receptor movement into the nucleus.
Smart Images
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Abstract
Description
Technical Field
[0001] The present invention relates to a method for detecting nuclear translocation of a receptor present in the cytoplasm that detects the signal intensity activated by the movement of the receptor present in the cytoplasm into the cell nucleus, and a method for detecting the signal intensities of the cell nucleus and cytoplasm that detects the signal intensities of the cell nucleus and cytoplasm over time in a time series.
Background Art
[0002] Signals such as growth factors and hormones in cells have receptors in the cells, and the signal is activated when the antigen-receptor complex that has received the antigen (ligand) moves into the nucleus. As molecules that regulate the amount of signal by the movement of receptors present in the cytoplasm into the nucleus, various pathways such as male hormone receptors, female hormone receptors, and nuclear transcription factors have been identified. This phenomenon of moving from the cytoplasm into the nucleus is called nuclear translocation. To date, nuclear translocation has been performed by the subjective judgment of the researcher's images, and there has been a need to develop a method for quantitative and continuous detection. We thought that nuclear translocation could be quantitatively determined from an image by measuring the amounts of fluorescently labeled molecules present in both the nucleus and the cytoplasm from the image. This technology enables the acquisition of continuous data on the vital phenomenon of nuclear translocation in a living cell state. Patent Document 1 proposes an apparatus that inputs a bright-field image of a tissue section in which the cell nucleus is stained and a fluorescence image of a tissue section in which a specific biological substance is stained with a fluorescent staining reagent, extracts the cell nucleus from the cell nucleus image, extracts fluorescent bright spots from the fluorescence image, and identifies the cell nucleus to which the fluorescent bright spots should belong based on the distance between the cell nucleus and the fluorescent bright spots, and assigns the fluorescent bright spots to the cell nucleus. Based on the actual distance between the cell nucleus and the fluorescent bright spots, the cell nucleus to which the fluorescent bright spots should belong can be identified, so that the fluorescent bright spots can be accurately assigned to the true cells. Patent Document 2 proposes a method for analyzing structures constituting the cell nucleus, which involves labeling one or more types of structures constituting the cell nucleus and the nucleic acids within the cell nucleus, obtaining a labeled image of the nucleic acids of the labeled cells, separately obtaining labeled images of the structures of the labeled cells for each type of labeled structure, determining the cell nucleus region based on the obtained labeled nucleic acid images, measuring the brightness value of each pixel in the determined cell nucleus region in the obtained labeled image of the structures, determining one or more regions where the brightness value per pixel is above a threshold, measuring one or more selected from a group consisting of the statistical value of the brightness value of each pixel in the region, the total area, and the total perimeter, and analyzing at least one selected from a group consisting of the amount, localization, and morphology of the structure within the cell based on the measured statistical values. Patent Document 3 proposes a cell image analysis system that can identify cells and recognize the type of cells identified while acquiring microscopic images of living cells. Patent Document 4 proposes a method for analyzing cell behavior that includes a detection step of determining whether a candidate region is a cell region for each frame of a time-lapse image using a dictionary that includes image data of cell nuclei, and a tracking step of tracking each cell using a state-space model that uses the position of the nearest neighbor cell within a certain distance from the predicted position as observation data. [Prior art documents] [Patent Documents]
[0003] [Patent Document 1] International Publication No. 2015 / 145644 [Patent Document 2] Japanese Patent Publication No. 2011-75278 [Patent Document 3] Japanese Patent Publication No. 2012-163538 [Patent Document 4] International Publication No. 2019 / 244917 [Disclosure of the Invention] [Problems that the invention aims to solve]
[0004] Patent documents 1 and 2 do not track changes in the cell nucleus under living cell conditions. Patent Document 3 identifies cells and recognizes the type of cells when acquiring microscopic images of living cells, but, like Patent Documents 1 and 2, it does not track changes in the cell nucleus under living cell conditions. Patent Document 4 detects the positions of multiple cells, but it does not detect the signal intensity activated when receptors present in the cytoplasm move into the cell nucleus, nor does it detect the signal intensity of the cell nucleus and cytoplasm in a time series.
[0005] The present invention aims to provide a method for detecting the nuclear translocation of cytoplasmic receptors, which detects the signal intensity activated when receptors present in the cytoplasm move into the cell nucleus, and a method for detecting the signal intensity of the cell nucleus and cytoplasm in a time series. [Means for solving the problem]
[0006] The nuclear translocation detection method for cytoplasmic receptors according to claim 1 of the present invention is a method for detecting nuclear translocation of cytoplasmic receptors using a plurality of frames obtained by imaging fluorescently labeled cell nuclei in a time series under living cell conditions, and detecting the signal intensity that is activated when a receptor present in the cytoplasm moves into the cell nucleus, wherein a computer performs a cell nucleus detection step S2 to identify the cell nucleus region of each cell nucleus for the imaged frames, and uses the centroid position and circumscribing rectangle of the cell nucleus region identified in the cell nucleus detection step S2 to determine the nuclear translocation of a first frame and a second frame imaged after the first frame. The method is characterized by the following steps: a cell nucleus tracking step S3, which tracks the cell nuclei by comparing the positional relationships of the cell nuclei present in the image; and a signal intensity acquisition step S4, which obtains the time change of signal intensity by comparing the average pixel values of the cell nucleus regions in the first frame and the cell nucleus regions in the second frame, which are tracked in the cell nucleus tracking step S3 and assigned the same unique number; and the detection of nuclear translocation of the receptor present in the cytoplasm from the time change of the signal intensity obtained in the signal intensity acquisition step S4. The present invention as described in claim 2 is a method for detecting the nuclear translocation of a receptor present in the cytoplasm as described in claim 1, wherein the cell nucleus tracking step S3 includes: a centroid position calculation step for each of the cell nuclei, for which the centroid position is calculated from the identified cell nucleus region; a circumscribed rectangle calculation step for each of the cell nuclei, for which the circumscribed rectangle is calculated from the identified cell nucleus region; a first frame circumscribed rectangle identification step for which the circumscribed rectangle calculated in the circumscribed rectangle calculation step is identified for the cell nuclei present in the first frame; a second frame centroid position identification step for which the centroid position calculated in the centroid position calculation step is identified for the cell nuclei present in the second frame; a first search step for which the cell nuclei whose centroid position, identified in the second frame centroid position identification step, is located inside the circumscribed rectangle identified in the first frame circumscribed rectangle identification step are designated as cell nuclei to be tracked in the first frame; and in the second frame The method is characterized by comprising: a second frame circumscribed rectangle identification step for identifying the circumscribed rectangle calculated in the circumscribed rectangle calculation step for the cell nuclei that exist; a first frame centroid position identification step for identifying the centroid position calculated in the centroid position calculation step for the cell nuclei that exist in the first frame; a second search step for identifying cell nuclei that have the centroid position identified in the first frame centroid position identification step located inside the circumscribed rectangle identified in the second frame circumscribed rectangle identification step as cell nuclei to be tracked in the second frame; and a trajectory correspondence step for assigning the unique number of the cell nuclei to be tracked in the first frame to the cell nuclei to be tracked in the second frame if the centroid position of the cell nuclei to be tracked in the second frame has the centroid position located inside the circumscribed rectangle of the cell nuclei to be tracked in the first frame, and the centroid position of the cell nuclei to be tracked in the first frame has the centroid position located inside the circumscribed rectangle of the cell nuclei to be tracked in the second frame. The present invention as described in claim 3 is a method for detecting the nuclear translocation of a receptor present in the cytoplasm as described in claim 1, wherein the cytoplasm is labeled with fluorescence different from the fluorescence for the cell nucleus, and in the signal intensity acquisition step S4, the corresponding cytoplasm is identified from the bounding rectangle, the cell nucleus region is identified as a mask image for the identified cytoplasm, the outer periphery of the mask image is identified as the cytoplasmic region, and the time change of the signal intensity is obtained by comparing the average pixel values of each cytoplasmic region by performing a grayscale conversion process on the cytoplasmic region in the first frame and the cytoplasmic region in the second frame, which are assigned the same unique number in the cell nucleus tracking step S3. The present invention as described in claim 4 is a method for detecting the nuclear translocation of a receptor present in the cytoplasm as described in claim 1, characterized in that in the cell nucleus detection step S2, a grayscale conversion process is performed on the frame, a binarization process is performed after the grayscale conversion process, and an opening process and a closing process are performed after the binarization process to identify the cell nucleus region of each cell nucleus. The present invention as described in claim 5 is a method for detecting the nuclear migration of a receptor present in the cytoplasm as described in claim 2, characterized in that, in the trajectory correspondence step, if the centroid position of the cell nucleus to be tracked in the second frame is not located inside the circumscribing rectangle of the cell nucleus to be tracked in the first frame, the unique number of the cell nucleus to be tracked in the first frame is not assigned. The present invention as described in claim 6 is a method for detecting the nuclear migration of a receptor present in the cytoplasm as described in claim 2, characterized in that, in the trajectory correspondence step, if the centroid position of the cell nucleus to be tracked in the first frame is not located inside the circumscribing rectangle of the cell nucleus to be tracked in the second frame, the unique number of the cell nucleus to be tracked in the first frame is not assigned. The present invention as described in claim 7 is a method for detecting the nuclear migration of a receptor present in the cytoplasm as described in claim 2, characterized in that, in the trajectory correspondence step, if the centroid position of the cell nucleus to be tracked in the first frame and the centroid position of a cell nucleus other than the cell nucleus to be tracked in the first frame are located inside the circumscribing rectangle of the cell nucleus to be tracked in the second frame, the unique number of the cell nucleus to be tracked in the first frame is not assigned. A method for detecting the signal intensity of a cell nucleus and cytoplasm according to claim 8 of the present invention, wherein the method for detecting the signal intensity of a cell nucleus and cytoplasm in a time series is performed using a plurality of frames obtained by imaging a cell nucleus and cytoplasm labeled with fluorescence of different spectra in a time series under living cell conditions, and the signal intensity of the cell nucleus and cytoplasm is detected in a time series, characterized in that a computer performs a cell nucleus detection step S2 in which it identifies the cell nucleus region of each of the imaged frames; a cell nucleus tracking step S3 in which it tracks the cell nucleus by comparing the positional relationship of the cell nucleus present in a first frame and a second frame imaged after the first frame using the centroid position and circumscribing rectangle of the cell nucleus region identified in the cell nucleus detection step S2; and a signal intensity acquisition step S4 in which it obtains the time change of the signal intensity by comparing the average pixel values of the cell nucleus region and cytoplasmic region in the first frame and the cell nucleus region and cytoplasmic region in the second frame, which are tracked in the cell nucleus tracking step S3 and assigned the same unique number. [Effects of the Invention]
[0007] According to the present invention's method for detecting the nuclear translocation of receptors present in the cytoplasm, the nuclear region of a cell is identified, the cell nucleus is tracked using the centroid position and circumscribing rectangle of the nuclear region, and the time change in signal intensity is obtained by comparing the average pixel values of each nuclear region that is tracked and assigned the same unique number, thereby detecting the nuclear translocation of receptors present in the cytoplasm. This allows for the acquisition of quantitative and continuous data on the biological phenomenon of nuclear translocation in a living cell state. Furthermore, according to the signal intensity detection method for the cell nucleus and cytoplasm of the present invention, the cell nucleus is identified, the cell nucleus is tracked using the centroid position and circumscribing rectangle of the cell nucleus, and the time change in signal intensity is obtained by comparing the average pixel values of each cell nucleus and cytoplasmic region that are tracked and assigned the same unique number. Therefore, it is possible to obtain quantitative and continuous data on the biological phenomenon of nuclear migration in a living cell state. [Brief explanation of the drawing]
[0008] [Figure 1] A flowchart illustrating a method for detecting the nuclear translocation of a cytoplasmic receptor according to one embodiment of the present invention, and a method for detecting signal intensity in the cell nucleus and cytoplasm according to this embodiment. [Figure 2] Photograph showing the cell nucleus detection step (S2) as shown in Figure 1. [Figure 3] Figure 1 is an explanatory diagram showing the cell nucleus tracking step (S3). [Figure 4] Figure 1 shows a photograph illustrating the signal intensity acquisition step (S4). [Modes for carrying out the invention]
[0009] The first embodiment of the present invention provides a method for detecting the nuclear translocation of receptors present in the cytoplasm, in which a computer performs the following steps: a nucleus detection step in which it identifies the nuclear region of each cell nucleus in an imaged frame; a nucleus tracking step in which it tracks cell nuclei by comparing the positional relationship of cell nuclei present in a first frame and a second frame imaged after the first frame using the centroid position and circumscribing rectangle of the nuclear region identified in the nucleus detection step; and a signal intensity acquisition step in which it performs grayscale conversion processing on the nuclear region in the first frame and the nuclear region in the second frame, which are tracked in the nucleus tracking step and assigned the same unique number, to compare the average pixel values of each nuclear region and acquire the time change in signal intensity. The nuclear translocation of receptors present in the cytoplasm is detected from the time change in signal intensity acquired in the signal intensity acquisition step. According to this embodiment, by identifying the cell nuclear region, tracking the cell nucleus using the centroid position and circumscribing rectangle of the cell nuclear region, and obtaining the time change in signal intensity by comparing the average pixel values of each cell nuclear region that is tracked and assigned the same unique number, it is possible to detect the nuclear translocation of receptors present in the cytoplasm. Thus, it is possible to obtain quantitative and continuous data on the biological phenomenon of nuclear translocation in a living cell state.
[0010] A second embodiment of the present invention is a method for detecting the nuclear translocation of receptors present in the cytoplasm according to the first embodiment, wherein the cell nucleus tracking step includes: a centroid position calculation step for each cell nucleus, for which the centroid position is calculated from the identified cell nucleus region; a circumscribed rectangle calculation step for each cell nucleus, for which the circumscribed rectangle is calculated from the identified cell nucleus region; a first frame circumscribed rectangle identification step for each cell nucleus present in the first frame, for which the circumscribed rectangle calculated in the circumscribed rectangle calculation step is identified; a second frame centroid position identification step for each cell nucleus present in the second frame, for which the centroid position calculated in the centroid position calculation step is identified; and a first search step for which cell nuclei whose centroid position, identified in the second frame centroid position identification step, is located inside the circumscribed rectangle identified in the first frame circumscribed rectangle identification step are designated as cell nuclei to be tracked in the first frame. The method includes: a second frame circumscribed rectangle identification step, which identifies the circumscribed rectangle calculated in the circumscribed rectangle calculation step for cell nuclei present in the second frame; a first frame centroid position identification step, which identifies the centroid position calculated in the centroid position calculation step for cell nuclei present in the first frame; a second search step, which designates cell nuclei whose centroid position, as identified in the first frame centroid position identification step, is located inside the circumscribed rectangle identified in the second frame circumscribed rectangle identification step as cell nuclei to be tracked in the second frame; and a trajectory correspondence step, which assigns the unique number of the first frame tracked cell nucleus to the second frame tracked cell nucleus if the centroid position of the second frame tracked cell nucleus is located inside the circumscribed rectangle of the first frame tracked cell nucleus, and the centroid position of the first frame tracked cell nucleus is located inside the circumscribed rectangle of the second frame tracked cell nucleus. According to this embodiment, the centroid position and circumscribing rectangle for each cell nucleus are calculated from the identified cell nucleus region, and by comparing and tracking the positional relationship of each cell nucleus between preceding and succeeding frames using these centroid positions and circumscribing rectangles, accurate tracking can be performed.
[0011] The third embodiment of the present invention is a method for detecting nuclear translocation of a receptor present in the cytoplasm according to the first embodiment. In this method, the cytoplasm is labeled with a fluorescence different from that of the cell nucleus. In the signal intensity acquisition step, the corresponding cytoplasm is specified from the circumscribed rectangle, and for the specified cytoplasm, the cell nucleus region is used as a mask image, and the outer periphery of the mask image is specified as the cytoplasm region. For the cytoplasm region in the first frame and the cytoplasm region in the second frame, which are assigned the same unique number in the cell nucleus tracking step, grayscale conversion processing is performed to compare the average pixel values of the respective cytoplasm regions, thereby obtaining the temporal change of the signal intensity. According to this embodiment, since the temporal change of the signal intensity is obtained by comparing the average pixel values not only for the cell nucleus region but also for the cytoplasm region, more accurate data can be obtained. Further, although the cytoplasm has a large deformation amount relative to the cell nucleus and it is difficult to specify the outer contour, for the cytoplasm, by using the cell nucleus region as a mask image and specifying the outer periphery of the mask image as the cytoplasm region, the average pixel value of the accurate cytoplasm region can be obtained.
[0012] The fourth embodiment of the present invention is a method for detecting nuclear translocation of a receptor present in the cytoplasm according to the first embodiment. In the cell nucleus detection step, grayscale conversion processing is performed on the frame, binarization processing is performed after the grayscale conversion processing, and after the binarization processing, opening processing and closing processing are performed to specify the cell nucleus region of each cell nucleus. According to this embodiment, the cell nucleus region can be accurately specified.
[0013] The fifth embodiment of the present invention is a method for detecting nuclear translocation of a receptor present in the cytoplasm according to the second embodiment. In the trajectory correspondence step, when the centroid position of the cell nucleus to be tracked in the second frame does not exist inside the circumscribed rectangle of the cell nucleus to be tracked in the first frame, the unique number of the cell nucleus to be tracked in the first frame is not assigned. According to this embodiment, accurate data can be obtained by targeting only the cell nuclei that can be reliably tracked.
[0014] In the sixth embodiment of the present invention, in the method for detecting nuclear translocation of a receptor present in the cytoplasm according to the second embodiment, in the trajectory correspondence step, when the center of gravity position of the cell nucleus of a first-frame tracking target does not exist inside the circumscribed rectangle of the cell nucleus of a second-frame tracking target, the unique number of the cell nucleus of the first-frame tracking target is not assigned. According to this embodiment, accurate data can be obtained by targeting only the cell nuclei that can be reliably tracked.
[0015] In the seventh embodiment of the present invention, in the method for detecting nuclear translocation of a receptor present in the cytoplasm according to the second embodiment, in the trajectory correspondence step, when both the center of gravity position of the cell nucleus of a first-frame tracking target and the center of gravity position of cell nuclei other than the cell nucleus of the first-frame tracking target exist inside the circumscribed rectangle of the cell nucleus of a second-frame tracking target, the unique number of the cell nucleus of the first-frame tracking target is not assigned. According to this embodiment, accurate data can be obtained by excluding, from the tracking targets, phenomena in which cell nuclei are separated into multiple nuclei, such as cell division.
[0016] The eighth embodiment of the present invention provides a method for detecting the signal intensity of cell nuclei and cytoplasm, in which a computer performs the following steps: a cell nucleus detection step in which a computer identifies the cell nucleus region of each cell nucleus in an imaged frame; a cell nucleus tracking step in which a computer tracks cell nuclei by comparing the positional relationship of cell nuclei present in a first frame and a second frame imaged after the first frame using the centroid position and circumscribing rectangle of the cell nucleus region identified in the cell nucleus detection step; and a signal intensity acquisition step in which a computer acquires the time change in signal intensity by comparing the average pixel values of the cell nucleus region and cytoplasmic region in the first frame and the cell nucleus region and cytoplasmic region in the second frame, which are tracked in the cell nucleus tracking step and assigned the same unique number. According to this embodiment, since the cell nucleus region is identified, the cell nucleus is tracked using the centroid position and circumscribing rectangle of the cell nucleus region, and the time change in signal intensity is acquired by comparing the average pixel values of the cell nucleus region and cytoplasmic region, which are tracked and assigned the same unique number, it is possible to acquire quantitative and continuous data on the biological phenomenon of nuclear migration in a living cell state. [Examples]
[0017] An embodiment of the present invention will be described below with reference to the drawings. Figure 1 is a flowchart illustrating the method for detecting the nuclear translocation of receptors present in the cytoplasm according to this embodiment, and the method for detecting signal intensity in the cell nucleus and cytoplasm according to this embodiment. The method for detecting the nuclear translocation of cytoplasmic receptors according to this embodiment involves using multiple frames of fluorescently labeled cell nuclei, imaged in time series under living cell conditions, to detect the signal intensity that is activated when cytoplasmic receptors move into the cell nucleus. Furthermore, the method for detecting the signal intensity of the cell nucleus and cytoplasm according to the examples involves detecting the signal intensity of the cell nucleus and cytoplasm over time using multiple frames obtained by imaging the cell nucleus and cytoplasm, which are labeled with fluorescence of different spectra, under living cell conditions. In the method of this embodiment, the following processing is performed using multiple frames obtained by first imaging the cell nucleus and cytoplasm, which have been labeled with fluorescence of different spectra, in a time series under living cell conditions. The following describes the case where the cell nucleus is fluoresced red and the cytoplasm is fluoresced green.
[0018] In this embodiment, the method for detecting the nuclear translocation of receptors present in the cytoplasm involves the computer extracting a target frame from a red fluorescence video (S1), detecting cell nuclei in the extracted target frame (S2), tracking the detected cell nuclei (S3), obtaining the signal intensity of the tracked cell nuclei (S4), and detecting the nuclear translocation of receptors present in the cytoplasm from the time change in the signal intensity of the cell nuclei. The red fluorescence video may also consist of multiple still images of cell nuclei captured in a time series. In the cell nucleus detection step (S2), the nuclear region of each cell nucleus is identified for the extracted target frame. In the cell nucleus tracking step (S3), the cell nuclei are tracked by comparing the positional relationship of cell nuclei present in the first frame and the second frame, which was captured after the first frame, using the centroid position and circumscribing rectangle of the cell nucleus region identified in the cell nucleus detection step (S2).
[0019] Furthermore, if the first frame in chronological order is designated as the reference frame, a unique number will be assigned to each cell nuclear region identified within that reference frame. If the reference frame is designated as the first frame, the cell nuclei are tracked by comparing the positional relationship between the first frame and the second frame, which was captured after the first frame. The cell nuclei in the second frame that were tracked are assigned a unique number that was assigned in the first frame (reference frame). Between the second frame and the third frame, which was captured after the second frame, the cell nuclei are tracked by comparing the positional relationship of the cell nuclei present in the second and third frames. The cell nuclei present in the third frame that are tracked are assigned a unique number that was assigned in the second frame.
[0020] In the signal intensity acquisition step (S4), the average pixel values of the cell nucleus regions in the first frame and the cell nucleus regions in the second frame are compared by performing a grayscale conversion process to obtain the time change in signal intensity. Thus, according to this embodiment, the method for detecting the nuclear translocation of receptors present in the cytoplasm involves identifying the cell nuclear region, tracking the cell nucleus using the centroid position and circumscribing rectangle of the cell nuclear region, and obtaining the time change in signal intensity by comparing the average pixel values of each cell nuclear region that is tracked and assigned the same unique number. This allows for the detection of the nuclear translocation of receptors present in the cytoplasm, thereby enabling the acquisition of quantitative and continuous data on the biological phenomenon of nuclear translocation in living cells.
[0021] In the cell nucleus detection step (S2), the frame is subjected to a grayscale conversion process, followed by a binarization process, and then opening and closing processes are performed to identify the nuclear region of each cell nucleus. Therefore, the nuclear region can be accurately identified.
[0022] The cell nucleus tracking step (S3) further comprises a centroid position calculation step, a circumscribed rectangle calculation step, a first frame circumscribed rectangle identification step, a second frame centroid position identification step, a first search step, a second frame circumscribed rectangle identification step, a first frame centroid position identification step, a second search step, and a trajectory correspondence step. In the centroid position calculation step, the centroid position is calculated for each cell nucleus from the identified nuclear region. In the circumscribed rectangle calculation step, a circumscribed rectangle is calculated for each cell nucleus from the identified nuclear region. In the first frame circumscribed rectangle identification step, the circumscribed rectangle calculated in the circumscribed rectangle calculation step is identified for the cell nuclei present in the first frame. In the second frame centroid position determination step, the centroid position calculated in the centroid position calculation step is determined for the cell nuclei present in the second frame. In the first search step, cell nuclei whose centroid position, as determined in the second frame centroid position determination step, is located inside the circumscribing rectangle determined in the first frame determination step are designated as cell nuclei to be tracked in the first frame. In the second frame circumscribed rectangle identification step, the circumscribed rectangle calculated in the circumscribed rectangle calculation step is identified for the cell nuclei present in the second frame. In the first frame centroid position determination step, the centroid position calculated in the centroid position calculation step is determined for the cell nuclei present in the first frame. In the second search step, cell nuclei whose centroid position, as identified in the first frame centroid position identification step, is located inside the circumscribed rectangle identified in the second frame circumscribed rectangle identification step are designated as cell nuclei to be tracked in the second frame. In the trajectory matching step, if the centroid of the cell nucleus being tracked in the second frame lies inside the circumscribing rectangle of the cell nucleus being tracked in the first frame, and the centroid of the cell nucleus being tracked in the first frame lies inside the circumscribing rectangle of the cell nucleus being tracked in the second frame, then the unique number of the cell nucleus being tracked in the first frame is assigned to the cell nucleus being tracked in the second frame.
[0023] In this way, by calculating the centroid position and circumscribing rectangle for each cell nucleus from the identified cell nucleus region, and then using these centroid positions and circumscribing rectangles to compare and track the positional relationship of each cell nucleus between preceding and succeeding frames, accurate tracking can be performed. In the trajectory tracking step, if the centroid of the cell nucleus to be tracked in the second frame does not lie within the circumscribing rectangle of the cell nucleus tracked in the first frame, a unique number is not assigned to the cell nucleus tracked in the first frame. By targeting only cell nuclei that can be reliably tracked in this way, accurate data can be obtained. Furthermore, in the trajectory matching step, if the centroid position of the cell nucleus tracked in the first frame does not lie inside the circumscribing rectangle of the cell nucleus tracked in the second frame, a unique number is not assigned to the cell nucleus tracked in the first frame. By targeting only cell nuclei that can be reliably tracked in this way, accurate data can be obtained. Furthermore, in the trajectory matching step, if the centroid position of the cell nucleus tracked in the first frame and the centroid position of a cell nucleus other than the cell nucleus tracked in the first frame are located inside the circumscribing rectangle of the cell nucleus tracked in the second frame, a unique number is not assigned to the cell nucleus tracked in the first frame. Therefore, by excluding phenomena in which a cell nucleus separates into multiple parts, such as cell division, from the tracking target, accurate data can be obtained.
[0024] In the method for detecting the nuclear translocation of receptors present in the cytoplasm according to this embodiment, it is preferable to obtain the signal intensity in the cytoplasmic region along with the signal intensity in the cell nucleus. To obtain the signal intensity of the cytoplasmic region, in S1, the computer extracts a green fluorescence animation corresponding to the red fluorescence animation as the target frame to be extracted. In the signal intensity acquisition step (S4), the computer identifies the corresponding cytoplasm from the bounding rectangle of the cell nucleus. For the identified cytoplasm, the computer uses the cell nucleus region as a mask image and identifies the cytoplasmic region as the outer periphery of the mask image. In the cell nucleus tracking step (S3), the computer performs a grayscale conversion on the cytoplasmic region in the first frame and the cytoplasmic region in the second frame, which are assigned the same unique number, and compares the average pixel values of each cytoplasmic region to obtain the time change in signal intensity. In this way, by comparing the average pixel values not only for the nuclear region but also for the cytoplasmic region, we can obtain the time-dependent changes in signal intensity, thus acquiring more accurate data. Furthermore, although the cytoplasm is more deformed than the nucleus and difficult to define its outline, by using the nuclear region as a mask image and identifying the outer periphery of the mask image as the cytoplasmic region, we can obtain accurate average pixel values for the cytoplasmic region.
[0025] In the method for detecting the signal intensity of the cell nucleus and cytoplasm according to this embodiment, a target frame is extracted from the red fluorescence video and the green fluorescence video (S1), the cell nucleus is detected in the extracted target frame (S2), the detected cell nucleus is tracked (S3), and the signal intensity of the tracked cell nucleus is obtained (S4). In the signal intensity acquisition step (S4), the time change in signal intensity is acquired by comparing the average pixel values of the cell nuclear region and cytoplasmic region in the first frame and the cell nuclear region and cytoplasmic region in the second frame, for the cell nuclear region and cytoplasmic region in the second frame that were tracked in the cell nuclear tracking step (S3) and assigned the same unique number. In this way, by identifying the cell nuclear region, tracking the cell nucleus using the centroid position and circumscribing rectangle of the cell nuclear region, and comparing the average pixel values of each cell nuclear region and cytoplasmic region that are tracked and assigned the same unique number, it is possible to obtain temporal changes in signal intensity. Thus, it is possible to obtain quantitative and continuous data on the biological phenomenon of nuclear migration in living cells. After the signal intensity acquisition step (S4), a new frame is selected as the target frame (S1), cell nuclei are detected in the selected target frame (S2), the detected cell nuclei are tracked (S3), and the signal intensity of the tracked cell nuclei is acquired (S4). For example, after a cell nucleus present in the second frame is assigned a unique number that was assigned in the first frame, a third frame, which was captured after the second frame, is extracted as a new frame. After the cell nuclei present in the third frame are assigned unique numbers, a fourth frame, which was captured after the third frame, is extracted as a new frame.
[0026] Figure 2 is a photograph showing the cell nucleus detection step (S2) shown in Figure 1. Figure 2(a) shows the target frame (original frame) from the red fluorescence video extracted in S1, Figure 2(b) shows the original frame after grayscale conversion, Figure 2(c) shows the grayscale conversion followed by binarization and noise reduction (opening and closing), and Figure 2(d) shows the state in which unique numbers have been assigned to the identified cell nucleus regions. In the cell nucleus detection step, as shown in Figure 2(a), processing is applied to one frame of a previously acquired video image of a cell nucleus that has been fluorescently colored red. First, a grayscale conversion process is applied to the acquired frames (Figure 2(b)). Then, a binarization process is performed, and noise is removed by applying opening and closing processes to the binarized frames (Figure 2(c)). Finally, a unique number is assigned to each cell nucleus region through a labeling process (Figure 2(d)). In this way, cell nuclei are detected by obtaining information such as the centroid position and the bounding rectangle. The threshold used for binarization is dynamically determined to be the optimal value for that frame. Binarization is applied to each value while varying the threshold from the largest possible pixel value to the smallest possible pixel value in the grayscale image. For the binarized result, the average of the threshold at which noise first increased sharply and the threshold at which the most cell nuclei were detected is calculated and used as the optimal threshold. The areas counted as noise are removed by the opening and closing processes. The areas counted as cell nuclei are those remaining after the opening and closing processes.
[0027] Figure 3 is an explanatory diagram illustrating the cell nucleus tracking step (S3) shown in Figure 1. Figure 3(a) shows the circumscribed rectangle of the cell nucleus in the previous frame (first frame), Figure 3(b) shows the centroid position of the cell nucleus in the current frame (second frame), and Figure 3(c) shows a comparison between the circumscribed rectangle of Figure 3(a) and the centroid position of Figure 3(b). Furthermore, Figure 3(d) shows the centroid position of the cell nucleus in the previous frame (first frame), Figure 3(e) shows the circumscribed rectangle of the cell nucleus in the current frame (second frame), and Figure 3(f) shows a comparison between the centroid position of Figure 3(d) and the circumscribed rectangle of Figure 3(e). In the cell nucleus tracking step, the movement of the cell nucleus is tracked frame by frame based on the information of each cell nucleus region obtained during cell nucleus detection. For a given cell nucleus region in the current frame, we search for the cell nucleus region being tracked in the previous frame that has a bounding rectangle containing the centroid position inside, as shown in Figures 3(a) to 3(c). Next, for the same cell nucleus region in the current frame, we search for the cell nucleus region being tracked in the previous frame whose centroid lies inside the circumscribed rectangle, as shown in Figures 3(d) to 3(f). If the search yields a single cell nuclear region from the previous frame, that cell nuclear region is mapped to the target cell nuclear region in the current frame as a trajectory. If multiple results are found, or if no results are found, no mapping is performed. This process is performed for all cells in the current frame. The accuracy of such cell nucleus detection and tracking processes can be improved using genetic algorithms.
[0028] Figure 4 is a photograph showing the signal intensity acquisition step (S4) shown in Figure 1. Figure 4(a) shows one frame of a moving image of a cell that has been fluorescently colored green, Figure 4(b) shows the nuclear region of the target cell nucleus, and Figure 4(c) shows the cytoplasm of the cell nucleus in Figure 4(b). Figure 4(d) shows a mask image, with the left image being the cytoplasmic region and the right image being the nuclear region. Figure 4(e) shows the state where the outer edge of the mask image has been identified as the cytoplasmic region, with the left image being the cytoplasmic region and the right image being the nuclear region. In the signal intensity acquisition step (S4), data is acquired using the information obtained from cell nucleus detection for one frame of a previously acquired video image of a green-fluorescent cell. First, for a given cell, the corresponding cell region is obtained from the green fluorescence frame based on the bounding rectangle. Next, a mask image is created from the information of the cell nuclear region obtained during detection. Furthermore, this mask image is expanded using techniques such as dilation and region expansion, and the region of the original mask image is removed to prepare a mask image of the cytoplasmic region. Using these mask images, the cell nucleus and cytoplasmic regions are extracted from the acquired cell regions, and the signal intensity of each is determined. The signal intensity is the average of the pixel values of each region after grayscale conversion.
[0029] As described above, the present invention allows for the tracing of each cell, live imaging, and real-time quantification, enabling the visual visualization of the reaction between intracellular receptors and their antibodies, and quantitative detection of intracellular nuclear translocation. Cellular nuclear receptors play a crucial role in ligand-dependent transcriptional regulation, and androgen receptors (ARs) are known to be nuclear receptors associated with various diseases, including prostate cancer and androgenetic alopecia (AGA). According to the present invention, the nuclear translocation dynamics of ARs after ligand stimulation can be quantitatively detected using raw imaging and unstable immortalized dermal papilla cells (DPCs). By measuring the amount of fluorescently labeled molecules present in both the nucleus and cytoplasm of a cell from images, the state of nuclear migration can be quantitatively determined from images. This enables the acquisition of continuous and quantitative data on the biological phenomenon of nuclear migration in living cells. This invention allows for the automatic output of quantitative observation results using a computer, by preparing two types of moving images of cells fluorescently stained with fluorescent proteins (a video of the fluorescently stained cells and a video of the same cells after binarization).
[0030] In the detection of cell nuclei in this invention, a binarization method is used to extract the outline of the cell nucleus, and the detection accuracy is improved by searching for an appropriate binarization threshold in each frame. Furthermore, in the cell nucleus tracking method of this invention, the positional relationship of each cell nucleus is compared and tracked between preceding and succeeding frames, based on information such as the centroid and circumscribing rectangle of each cell nucleus region obtained from the detection of cell nuclei in time-lapse images. Furthermore, in obtaining signal intensity in this invention, color information is acquired by live imaging inside and outside the cell nucleus based on information about the cell nucleus region obtained by detecting the cell nucleus, and signal intensity is obtained. By combining the obtained signal intensity data with the cell nucleus tracking results, the temporal change in signal intensity can be obtained. Furthermore, in the cell nucleus detection method of this invention, the average of the threshold at which noise first increases sharply compared with the previous threshold result for the binarized result, and the threshold at which the cell nucleus region is detected most frequently, is calculated and used as the optimal threshold. In this process, the region to be counted as noise is removed by opening and closing processes. Furthermore, in the cell nucleus tracking method of this invention, the movement of the cell nucleus is tracked frame by frame based on the information of each cell nucleus region obtained during cell nucleus detection. The accuracy of this cell nucleus detection and tracking process can be improved by a genetic algorithm. Furthermore, in obtaining signal intensity in this invention, data is acquired using information obtained from cell nucleus detection for one frame of a previously acquired video image of a green-fluorescent cell. First, the region of the cell corresponding to the cell is obtained from the green-fluorescent frame based on the bounding rectangle of the arbitrary cell. Next, a mask image is created from the cell nucleus region obtained during detection. This mask image is expanded using methods such as expansion processing or region expansion processing, and the region of the original mask image is removed to prepare a mask image of the cytoplasmic region. Then, using these mask images, the cell nucleus region and cytoplasmic region are extracted from the acquired cell region, and the signal intensity of each is determined. These signal intensities are the average of the pixel values of each region converted to grayscale. By determining the signal intensity for each cell nucleus through the above operations, the amount of fluorescently labeled expression molecules can be quantitatively determined from the image processing results. This invention allows for the processing of two types of moving images of cells fluorescently illuminated with fluorescent proteins using a computer, thus reducing costs compared to other cell observation techniques. Furthermore, this invention automatically outputs quantitative observation results by inputting two types of moving images, eliminating the need for user operation and reducing the burden on the user. [Industrial applicability]
[0031] This invention makes it possible to develop compounds that inhibit the nuclear translocation of specific molecules using a large library of small molecules. Furthermore, it makes it possible to calculate the speed of nuclear translocation and quantitatively detect whether nuclear translocation is delayed or inhibited. [Explanation of symbols]
[0032] S1 Target frame extraction step S2 Cell nucleus detection step S3 Cell nuclear tracking step S4 Signal Intensity Acquisition Step
Claims
1. A method for detecting the nuclear translocation of cytoplasmic receptors, which involves using multiple frames of fluorescently labeled cell nuclei imaged in time series under living cell conditions to detect the signal intensity that is activated when a receptor present in the cytoplasm moves into the cell nucleus, Computers A cell nucleus detection step that identifies the cell nucleus region of each cell nucleus in the imaged frame, A cell nucleus tracking step involves tracking the cell nucleus by comparing the positional relationship of the cell nucleus present in the first frame and the second frame, which is imaged after the first frame, using the centroid position and the circumscribed rectangle of the cell nucleus region identified in the cell nucleus detection step, A signal intensity acquisition step is performed to obtain the time change in signal intensity by comparing the average pixel values of each cell nuclear region by performing a grayscale conversion process on the cell nuclear region in the first frame and the cell nuclear region in the second frame, which are tracked in the cell nuclear tracking step and assigned the same unique number; Perform Nuclear translocation of the receptor present in the cytoplasm is detected from the time change of the signal intensity obtained in the signal intensity acquisition step. A method for detecting the nuclear translocation of receptors present in the cytoplasm, characterized by the following features.
2. In the aforementioned cell nucleus tracking step, For each of the aforementioned cell nuclei, a centroid position calculation step is performed to calculate the centroid position from the identified cell nuclear region, For each of the aforementioned cell nuclei, a circumscribed rectangle calculation step is performed to calculate the circumscribed rectangle from the identified cell nuclear region, A first frame bounding rectangle identification step for identifying the bounding rectangle calculated in the bounding rectangle calculation step for the cell nuclei present in the first frame, A second frame centroid position identification step, which identifies the centroid position calculated in the centroid position calculation step with respect to the cell nuclei present in the second frame, A first search step in which the cell nuclei whose centroid position, as identified in the second frame centroid position identification step, is located inside the circumscribed rectangle identified in the first frame circumscribed rectangle identification step are designated as the cell nuclei to be tracked in the first frame, A second frame circumscribed rectangle identification step, which identifies the circumscribed rectangle calculated in the circumscribed rectangle calculation step for the cell nuclei present in the second frame, A first frame centroid position identification step, which identifies the centroid position calculated in the centroid position calculation step with respect to the cell nuclei present in the first frame, A second search step in which the cell nucleus whose centroid position, as identified in the first frame centroid position identification step, is located inside the circumscribed rectangle identified in the second frame circumscribed rectangle identification step is designated as the cell nucleus to be tracked in the second frame, If the centroid position of the second frame-tracking target cell nucleus lies inside the circumscribing rectangle of the first frame-tracking target cell nucleus, and the centroid position of the first frame-tracking target cell nucleus lies inside the circumscribing rectangle of the second frame-tracking target cell nucleus, then a trajectory correspondence step is performed in which the unique number of the first frame-tracking target cell nucleus is assigned to the second frame-tracking target cell nucleus. has The method for detecting the nuclear translocation of a receptor present in the cytoplasm, as described in feature 1.
3. The cytoplasm is labeled with fluorescence different from the fluorescence to the cell nucleus, In the signal intensity acquisition step, The corresponding cytoplasm is identified from the aforementioned circumscribing rectangle, For the identified cytoplasm, the cell nuclear region is used as a mask image, and the outer periphery of the mask image is identified as the cytoplasmic region. The time-dependent change in signal intensity is obtained by comparing the average pixel values of the cytoplasmic regions in the first frame and the second frame, which are assigned the same unique number in the cell nucleus tracking step, through a grayscale conversion process. The method for detecting the nuclear translocation of a receptor present in the cytoplasm, as described in feature 1.
4. In the cell nucleus detection step, A grayscale conversion process is performed on the aforementioned frame. After the grayscale conversion process, a binarization process is performed. After the binarization process, an opening process and a closing process are performed. Identify the nuclear region of each of the aforementioned cell nuclei. The method for detecting the nuclear translocation of a receptor present in the cytoplasm, as described in feature 1.
5. In the aforementioned trajectory correspondence step, If the centroid position of the cell nucleus to be tracked in the second frame is not located inside the circumscribing rectangle of the cell nucleus to be tracked in the first frame, the unique number of the cell nucleus to be tracked in the first frame will not be assigned. The method for detecting the nuclear translocation of a receptor present in the cytoplasm, as described in feature 2.
6. In the aforementioned trajectory correspondence step, If the centroid position of the cell nucleus being tracked in the first frame is not located inside the circumscribing rectangle of the cell nucleus being tracked in the second frame, the unique number of the cell nucleus being tracked in the first frame will not be assigned. The method for detecting the nuclear translocation of a receptor present in the cytoplasm, as described in feature 2.
7. In the aforementioned trajectory correspondence step, If the centroid position of the first frame-tracked cell nucleus and the centroid positions of cell nuclei other than the first frame-tracked cell nucleus are located inside the circumscribing rectangle of the second frame-tracked cell nucleus, then the unique number of the first frame-tracked cell nucleus will not be assigned. The method for detecting the nuclear translocation of a receptor present in the cytoplasm, as described in feature 2.
8. A method for detecting the signal intensity of a cell nucleus and cytoplasm, comprising detecting the signal intensity of the cell nucleus and cytoplasm over time using multiple frames obtained by imaging the cell nucleus and cytoplasm in a time series under living cell conditions, wherein the signal intensity of the cell nucleus and cytoplasm is detected over time. Computers A cell nucleus detection step that identifies the cell nucleus region of each cell nucleus in the imaged frame, A cell nucleus tracking step involves tracking the cell nucleus by comparing the positional relationship of the cell nucleus present in the first frame and the second frame, which is imaged after the first frame, using the centroid position and the circumscribed rectangle of the cell nucleus region identified in the cell nucleus detection step, A signal intensity acquisition step is performed to obtain the time change of the signal intensity by comparing the average pixel values of the cell nuclear region and cytoplasmic region in the first frame and the cell nuclear region and cytoplasmic region in the second frame, which are tracked in the cell nuclear tracking step and assigned the same unique number. A method for detecting signal intensity in the cell nucleus and cytoplasm, characterized by the above.