JP7379853B2 - Microscope and how to set it up - Google Patents

Description

The present invention relates to a microscope and a method of setting the microscope .

It is known to observe clusters of cells. For example, Patent Document 1 or Patent Document 2 is known.

US201113315794A US201213364928A

According to a first aspect of the present embodiment, an illumination optical system includes an objective lens, and scans and irradiates a cell mass in a plane intersecting the optical axis direction of the objective lens via a scanning unit. a detection optical system that collects light from the cell mass onto a light receiving section via the scanning section; and a control section, the control section configured to move the light from the cell mass to a plurality of different positions along the optical axis direction. In each, when the illumination light is two-dimensionally scanned by the scanning unit to obtain a two-dimensional image, as a pre-process, without performing image acquisition to determine image acquisition conditions for each of the plurality of positions, In the process of acquiring the two-dimensional image, based on the two-dimensional image acquired at the first position of the cell mass, information on the density of the cell structure, information on the circumference of the cell structure, and the At least one piece of information is specified among the ratio information of the brightness of the cell structure and the brightness of the part other than the cell structure, and based on the specified information , the A microscope is provided that determines image acquisition conditions at a second location different from the first location.

According to the second aspect of the present embodiment, the cell cluster includes an objective lens, and a cell mass is scanned in a plane intersecting the optical axis direction of the objective lens through a scanning unit using illumination light from a light source. In a microscope comprising an illumination optical system for illuminating light, and a detection optical system for focusing light from the cell mass onto a light receiving part via the scanning part, at each of a plurality of different positions along the optical axis direction. , when the illumination light is two-dimensionally scanned by the scanning unit to obtain a two-dimensional image, as a pre-process, the above-mentioned two In the process of acquiring a dimensional image, based on the two-dimensional image acquired at the first position of the cell mass, information on the density of the cell structure, information on the perimeter of the cell structure, and information on the perimeter of the cell structure are obtained. specifying at least one piece of information among ratio information between the brightness of the structure and the brightness of a portion of the cell other than the structure; and based on the specified information , A method for setting up a microscope is provided, comprising: determining image acquisition conditions at a second position different from the first position.

FIG. 1 is a schematic diagram showing a cell mass placed in a well W of a well plate WP. FIG. 2 is a schematic diagram of the counting device 1 according to this embodiment. FIG. 3 is a flowchart showing a method for counting the number of cells contained in a cell cluster. FIG. 4 is a flowchart showing the initial value determination process. FIG. 5 is a schematic diagram showing a state when a cell mass is irradiated with a laser beam, and a diagram showing the correlation between the laser intensity I and the number of nuclei counts on a reference plane. FIG. 6 is a flowchart showing the Z stack execution process. FIG. 7 is a schematic diagram showing a cell mass containing blood vessels inside. FIG. 8 is a schematic diagram showing a neuron containing multiple dendritic spines.

In the following description, an XYZ orthogonal coordinate system is set, and the positional relationship is explained based on this XYZ orthogonal coordinate system (see FIG. 2). The Z-axis direction is set, for example, in the vertical direction, and the X-axis direction and the Y-axis direction are set, for example, in directions parallel to the horizontal direction and orthogonal to each other.

In this specification, as shown in FIG. 1, a three-dimensional aggregate of a plurality of cells is referred to as a cell cluster. Examples of cell clusters include tissue sections, cell spheroids, organoids, and the like. Note that a cell spheroid is a three-dimensional mass of three-dimensionally cultured cells, and an organoid is a collection of small and simplified cells that have some of the characteristics of an organ. Organoids can be produced three-dimensionally in vitro by, for example, using pluripotent stem cells such as iPS cells and ES cells as raw materials and differentiating the cells while controlling cell culture conditions. can. Cell clusters also include those in which two or more two-dimensionally spread cell layers are stacked (for example, cell sheets). The cell sheet may be a single layer or a plurality of laminated layers.

Compared to two-dimensional culture, three-dimensional culture allows the construction of a more complex system with a three-dimensional structure, and it is possible to construct an environment more similar to that in a living body. When applying three-dimensionally cultured cell clusters such as cell spheroids and organoids, or cell clusters such as tissue sections, in fields such as basic research, drug discovery research, and regenerative medicine, it is necessary to There is a need to accurately know the number of cells contained in a cell. For example, when injecting a drug into a cell mass and examining how much of the drug is absorbed by the cells contained within the cell mass, it is possible to accurately determine the number of cells contained within the cell mass. , the amount of drug absorbed by each cell can be accurately evaluated.

With reference to FIG. 2, a counting device 1 for counting the number of cells contained inside a cell mass according to the present embodiment will be described. The counting device 1 mainly includes a microscope device 10, a control section 20, a display section 30, and an input section 40. Note that the number of cells is an example of cell information. Moreover, in this embodiment, the number of nuclei contained in a cell can be taken as the number of cells. In this case, for example, in a cell undergoing cell division, the cell may contain a plurality of nuclei. In this case, the number of cells to be counted may be changed based on the area of the nucleus of the cell and the area of the cell. Of course, the method for counting the number of cells is not limited to counting the number of cell nuclei. For example, the area of the cell may be calculated from the contour information of the cell, and the number of cells may be counted from the calculated area of the cell. Further, the nucleus of a cell is an example of the internal structure of a cell. Furthermore, the external structure of a cell based on cell outline information is an example of a cell structure.

The microscope apparatus 10 mainly includes an illumination section 100, an optical system 120, a light receiving section 140, and a movable stage 160 on which a sample S is placed. The microscope device 10 of this embodiment is a so-called confocal laser microscope. Note that the microscope device 10 is an example of an optical detection device.

As shown in FIG. 2, the illumination unit 100 includes three CW lasers 102 and an acousto-optics tunable filter 103 (hereinafter referred to as AOTF 103). The CW lasers 102 are continuous wave lasers with wavelengths of 405 nm, 488 nm, and 561 nm, respectively, and in this embodiment, a semiconductor laser is used. The AOTF 103 is an electrically controllable transmission type bandpass filter, and is capable of high-speed switching by on/off modulation and control of laser intensity by adjusting the modulation level. In the illumination unit 100 of this embodiment, the AOTF 103 can perform on/off control of the three CW lasers 102 and control the laser intensity. Note that instead of the AOTF 103, for example, an acousto-optic modulator (AOM) can be used.

The optical system 120 includes four lenses L1 to L4, a dichroic mirror DM, a galvano mirror GM, two pinholes PH1 and PH2, a pupil projection lens 122, an imaging lens 124, and an objective lens 126. . Note that the objective lens 126 is provided with a drive mechanism 128, and the position of the objective lens 126 in the Z direction can be adjusted.

The light receiving unit 140 includes a photomultiplier tube PMT, and measures the intensity of fluorescence emitted from cells included in the sample S.

The movable stage 160 includes a stage 161 having an upper surface 161U parallel to the XY plane in FIG. 2, and a moving mechanism 162 that moves the stage 161 in the X direction and the Y direction. On the upper surface 161U of the stage 161, a well plate WP containing the sample S is arranged. The well plate WP is a transparent plate-like member, and a plurality of depressions (wells W) are two-dimensionally arranged on its surface. Each well W can be used as a test tube or a petri dish. The moving mechanism 162 can adjust the position of the stage 161 in the X direction and the Y direction under the control of the control unit 20. Thereby, the position of the well plate WP placed on the upper surface 161U of the stage 161 can be adjusted, and the sample S placed in a desired well W in the well plate WP can be irradiated with laser light. .

The control unit 20 includes an adjustment unit 21 that outputs a setting signal (adjustment signal) to the microscope apparatus 10 for controlling the operation of each part of the microscope apparatus 10 (illumination unit 100, optical system 120, light receiving unit 140, movable stage 160). and an acquisition unit 22 that creates a two-dimensional image (described later) based on a signal from the light receiving unit 140 and acquires cell information such as the number of cells included in a cell cluster based on the two-dimensional image. In the following description, the two-dimensional image based on the signal from the light receiving unit 140 can be appropriately referred to as a two-dimensional mapping image. Further, the control unit 20 receives settings and instructions for each part of the microscope apparatus 10 through the input unit 40. The control unit 20 causes the display unit 30 to display a setting screen for receiving settings and instructions for each part of the microscope device 10. The input unit 40 includes a mouse, keyboard, joystick, touch panel, etc. (not shown) for inputting various instruction information. Note that the input unit 40 is not limited to input by the measurer. For example, the information to be input to the input unit 40 may be stored in a storage unit (not shown), and the information in the storage unit may be input as various instruction information as appropriate. For example, the input section 40 may have a configuration in which the measurement conditions at the previous reference position are stored in the storage section and data to be input to the adjustment section 21 is created from the storage section as appropriate. Therefore, although the input unit 40 includes a configuration for inputting instruction information such as a mouse, the input unit 40 may also include a storage unit. The display unit 30 has a setting screen, an instruction screen, a measurement screen, a display screen that displays measurement results, and the like. In this embodiment, the input section 40 and the control section 20 including the adjustment section 21 function together as a measurement condition setting device that sets measurement conditions for the microscope device 10.

The control unit 20 may be formed by dedicated hardware, or may be formed by installing a predetermined program in a personal computer. The control unit 20 and the microscope device 10 do not necessarily have to be formed as an integrated device. For example, the control unit 20 may be a computer communicatively connected to the microscope apparatus 10 by wire or wirelessly.

Next, the optical path of the laser beam of the microscope device 10 will be explained. The laser beams emitted from the three CW lasers 102 all enter the AOTF 103. AOTF 103 selectively transmits laser light of a desired wavelength. Further, the AOTF 103 adjusts the intensity of laser light (also simply referred to as laser intensity). The laser light emitted from the AOTF 103 travels straight toward the lens L1. A pinhole PH1 is placed at the focal point of the lens L1. The laser beam that has passed through the lens L1 and pinhole PH1 enters the dichroic mirror DM after passing through the lens L2. The dichroic mirror DM has a characteristic of reflecting light in the wavelength band of the laser light from the CW laser 102 and transmitting light other than that. Therefore, the laser beam is reflected by the dichroic mirror DM and enters the galvanometer mirror GM. The galvanometer mirror GM is a scanning mirror that can swing back and forth, and is controlled by the control unit 20. The galvanometer mirror GM can scan the laser beam in one predetermined direction (for example, the X direction). Although only one galvano mirror GM is illustrated in FIG. 2, two galvano mirrors can be used to scan the laser beam in two directions (for example, the XY direction). Moreover, a resonant scanner can also be used in place of the galvanometer mirror.

The laser beam exiting the galvanometer mirror GM passes through the pupil projection lens 122 and the imaging lens 124, and then enters the objective lens 126. Note that the galvanometer mirror GM is arranged to be conjugate with the pupil position (exit pupil position) of the objective lens 126. The laser beam exiting the objective lens 126 is irradiated onto the sample S placed on the movable stage 160. As mentioned above, the sample S contains a mass of cells. The nucleus of each cell included in the sample S is stained with a dye in advance, and emits fluorescence when irradiated with laser light. In this embodiment, it is stained with 4',6-diamidino-2-phenylindole (DAPI). Note that the staining may be performed by staining only the nucleus of the cell, or by staining the nucleus of the cell and other structures. Moreover, the cells may be in an unstained state. When cells are not stained, for example, autofluorescence from internal structures of cells may be used.

The fluorescence that has returned from the sample S follows the objective lens 126, the imaging lens 124, the pupil projection lens 122, and the galvano mirror GM in the reverse direction and enters the dichroic mirror DM. The fluorescent light passes through the dichroic mirror DM, travels straight, and enters the lens L3. A pinhole PH2 is arranged at the focal point of the lens L3. After passing through the pinhole PH2, the fluorescence passes through the lens L4, enters the light receiving section 140, and is detected. Here, the pinholes PH1 and PH2 are arranged at positions conjugate with the focal position of the objective lens 126. Thereby, the pinhole PH2 can selectively transmit only the light (fluorescence, scattered light) from the focal position of the objective lens 126. Thereby, it is possible to block the light that causes the blurred image, and it is possible to detect only the light from the focal position.

The control unit 20 can scan the laser beam irradiation position (focal position) in the XY direction by controlling the drive of the galvano mirror GM. Thereby, when the control unit 20 irradiates each position on the XY plane with the laser beam, the light receiving unit 140 receives the intensity of fluorescence at each position. Therefore, the intensity distribution on the XY plane can be obtained, and the intensity distribution on the XY plane can be obtained as a two-dimensional image. Furthermore, the control unit 20 can adjust the position of the objective lens 126 in the Z direction by controlling a drive mechanism 128 provided in the objective lens 126. In other words, the control unit 20 can adjust the position of the focal plane of the laser beam in the Z direction (hereinafter simply referred to as the Z position). Thereby, the control unit 20 can obtain a two-dimensional mapping of the fluorescence intensity distribution on the XY plane at each Z position while adjusting the Z position of the focal plane of the laser beam. In the following description, setting the Z position of the focal plane of the laser beam and acquiring a two-dimensional mapping image of the fluorescence intensity distribution on the XY plane at the Z position is referred to as executing a Z slice. In the following description, a two-dimensional image on the XY plane at each Z position will be referred to as a Z slice image. Further, executing Z slices at a plurality of Z positions while moving the Z position of the focal plane of the laser beam over a predetermined range is called executing Z stack. Therefore, by performing Z stacking, it is possible to obtain a plurality of Z slice images at different Z positions.

Z stacking is performed to cover the entire cell cluster in the Z direction, and from the obtained multiple Z slice images, the number of cells included in the cell cluster can be counted as described below. . In order to accurately count the number of cells from a Z slice image at each Z position, the obtained Z slice image must be clear enough to distinguish the nucleus of each cell. Inside a cell mass, the scattering of incident light differs depending on the Z position of the cell mass, so the setting parameters were adjusted for each Z position. For example, in order to make the Z-slice image clearer, the user adjusts the setting parameters of the microscope apparatus 10, such as the laser intensity and the gain of the photomultiplier tube PMT, each time the user executes each Z-slice. Details of the setting parameters will be described later. However, it is time consuming to adjust the configuration parameters each time each Z slice is executed. In addition, when adjusting the Z-slice image, since there is no index for adjustment, the sharpness of the Z-slice image obtained may vary greatly depending on the difference in the skill level of the user, and the quantitative nature of the analysis result may be affected. The problem was that it was not guaranteed.

On the other hand, since the counting device 1 according to the present embodiment includes a control section 20 having an adjustment section 21 and an acquisition section 22, the setting parameters of the microscope device 10 are automatically set as shown below. can do.

Next, a method for counting the number of cells included in a cell mass using the counting device 1 will be described with reference to FIG. 3. The well plate WP containing the cell mass in the well W is placed on the stage 161 (S101). A variable i is set to 1 (S102), and the X and Y positions of the stage 161 are adjusted so that the mass of cells placed in the i-th well W can be measured (S103). When the variable i is 1 (S104: Yes), the Z position of the objective lens 126 is adjusted so that the focal plane of the laser beam is the same as the reference plane BP described below (S105).

Note that, as shown in FIG. 1, the portion of the cell mass that contacts the well W spreads outward along the well W. Therefore, the cell mass has a constricted shape slightly above the portion that contacts the well W. In this embodiment, as will be described later, the initial value of the variable parameter Pv of the microscope device 10 is determined at the reference plane BP. From the viewpoint of ensuring the intensity of the laser beam, it is conceivable to set the reference plane BP at a position closest to the objective lens 126 and in contact with the well W of the cell mass. However, the state of the cells contained in the portion of the cell mass that contacts the well W may be changed compared to the cells contained in other portions. Therefore, in this embodiment, the reference plane BP is set to pass through a constricted part of the cell mass slightly above the part that contacts the well W. The reference plane BP is set at a Z position below, for example, 10 μm from the portion of the cell mass that contacts the well W. The Z position of the reference plane is not limited to below 10 μm, and other Z positions may be used as long as the intensity of the laser beam can be ensured. Further, the reference plane BP is set at an upper Z position that is, for example, more than 5 μm away from the part of the cell mass that contacts the well W. In addition, if the details of the part of the cell mass that contacts the well W are not different from the state of cells contained in other parts, the part of the cell mass that contacts the well W may be set as the reference plane BP. I do not care. Alternatively, a position where more cell nuclei can be imaged may be set as the reference plane BP.

Note that when setting the reference plane BP, the outline of the cell mass at each Z position may be obtained by executing Z stacking with appropriate setting parameters to determine the outer shape of the cell mass. In this case, since it is sufficient to obtain the outline of a cluster of cells at each Z position, there is no problem even if it is not possible to obtain a Z slice image clear enough to distinguish the nuclei contained in each cell. Alternatively, the cell mass may be estimated from the measured contour, and the cell reference plane BP may be estimated from the estimated cell mass. It does not matter if part of the outline of a cell cluster cannot be measured.

After adjusting the Z position of the objective lens 126 so that the focal plane of the laser beam is the same as the reference plane BP, the initial value of the variable parameter Pv of the microscope device 10 is determined by the procedure described below (S106). Next, the Z position of the focal plane of the laser beam is set to the start position of the Z stack (S107), and the Z stack is performed on the cell mass placed in the i-th well W by the procedure described below ( S108). When the Z-stack is completed, it is determined whether the previous Z-stack was performed on the last well W (S109). If the Z stack for the last well W has not been completed (S109: No), the variable i is incremented and the process returns to step S103. Note that if the variable i is not 1 in step S104, steps S105 and S106 are omitted and the process proceeds to step S107. In step S109, if it is determined that the Z stack for the last well W has been completed (S109: Yes), the measurement is ended.

Next, the process of step S106 described above, that is, the process for determining the initial value of the variable parameter Pv of the microscope apparatus 10 (hereinafter referred to as initial value determination process) will be described with reference to FIGS. 4 and 5.

Setting parameters of the microscope device 10 include, for example, laser intensity, imaging speed, number of averaging, and gain. The laser intensity is the intensity of the laser light emitted from the illumination unit 100, and can be adjusted using the AOTF 103 as described above. The higher the laser intensity, the brighter the fluorescence, and the clearer the resulting Z-slice image. However, if the laser intensity is set too high, there is a risk of damaging the cell mass irradiated with the laser light. The photographing speed corresponds to the scanning speed of the galvanometer mirror GM. When the scanning speed of the galvanometer mirror GM is low, the number of photons (photon number) that enters the photomultiplier tube PMT during a predetermined measurement time increases, so that the obtained Z slice image becomes clearer. However, if the imaging speed is lowered, the time required to execute Z slices (and Z stacks) becomes longer. The number of averages is the number of Z slice images that are averaged in one Z slice. By averaging the Z slice images for the number of times of averaging, the resulting Z slice image can be made clearer. If the number of times of averaging is too large, the time required to execute Z slice (and Z stack) will also become longer. The gain is the sensitivity of the photomultiplier tube PMT, and can be adjusted by the applied voltage applied to the photomultiplier tube PMT. By increasing the gain, it becomes possible to detect even when the fluorescence intensity is weak (when the number of photons is small), and by decreasing the gain, it is possible to prevent brightness saturation even when the fluorescence intensity is strong. These setting parameters are set by the adjustment section 21. Note that all of these setting parameters may be set by the adjustment section 21, or at least one of these setting parameters may be set by the adjustment section 21.

As shown in FIG. 4, in the initial value determination process, the adjustment unit 21 first selects the variable parameter Pv from the above-mentioned setting parameters (S201). Setting parameters other than the selected variable parameter Pv are fixed parameters and set to appropriate values. In the following description, it is assumed that the laser intensity I is selected as the variable parameter Pv.

Next, the adjustment unit 21 determines the upper limit value, lower limit value, and change width Δ for the selected variable parameter Pv (S202), and sets the value of the variable parameter Pv to the lower limit value (S203). Here, the adjustment unit 21 may determine the upper limit value, lower limit value, and variation width Δ of the selected variable parameter Pv based on a table stored in a memory (not shown) of the control unit 20. Alternatively, the adjustment unit 21 may determine the upper limit value, lower limit value, and variation range Δ of the selected variable parameter Pv based on values input by the user through the input unit 40. Next, the adjustment unit 21 causes the microscope device 10 to perform a Z slice, and the acquisition unit 22 acquires a Z slice image on the reference plane BP (S204). The acquisition unit 22 then counts the number of nuclei based on the acquired Z slice image (S205). For example, the acquisition unit 22 can create contour lines of brightness in the Z slice image and determine that a portion with a certain brightness or higher corresponds to a nucleus, and the portion corresponding to the nucleus (hereinafter simply referred to as a nuclear portion) The number of nuclei can be counted by counting the number of .

Next, the adjustment unit 21 changes the value of the variable parameter Pv by the change width Δ (S206), and determines whether the value of the variable parameter Pv is less than or equal to the upper limit value (S207). If the value of the variable parameter is less than or equal to the upper limit value (S207: YES), the adjustment unit 21 causes the microscope device 10 to perform a Z slice using the value of the variable parameter Pv changed by the change width Δ in step S206. The acquisition unit 22 acquires the Z slice image on the reference plane BP again (S208). The acquisition unit 22 then counts the number of nuclei based on the acquired Z slice image (S209). Next, the acquisition unit 22 compares the number of nuclei calculated based on the previous Z slice and the number of nuclei calculated based on the current Z slice, and determines whether these changes are zero. It is determined whether or not (S210). Note that if the user has previously counted the number of nuclei using another method, in the process of step S210, the acquisition unit 22 calculates the number of nuclei based on the number of nuclei counted by the user and the current Z stack. It may also be determined whether the number of nuclei obtained is the same.

As described above, in step S201, the adjustment unit 21 selects the laser intensity I as the variable parameter Pv. In this case, as shown in FIG. 5, as the laser intensity I is increased, the number of nuclei calculated based on the Z slice converges to a constant value. This is because by increasing the laser intensity I, the Z slice image acquired by the Z slice becomes clearer, and the accuracy of nuclear detection increases. However, once the laser intensity I becomes strong enough to detect the nuclei of all cells included in the Z slice image, the number of nuclei detected in the Z slice image remains constant even if the laser intensity I is increased further. . Note that if a cell mass is irradiated with a laser beam with an unnecessarily strong laser intensity, the cells contained in the cell mass may be damaged. Therefore, the number of nuclei calculated based on the previous Z slice and the number of nuclei calculated based on the current Z slice are compared, and if these changes are zero (S210: YES), Then, the adjustment unit 21 determines the value of the variable parameter Pv used when executing the previous Z slice as the initial value of the variable parameter Pv (S211), and ends the initial value determination process.

Note that the number of nuclei calculated based on the previous Z slice and the number of nuclei calculated based on the current Z slice are compared, and if these changes are not zero (S210: NO), , returning to the process of step S206, the adjustment unit 21 changes the value of the variable parameter Pv by the change width Δ (S206). Then, the adjustment unit 21 determines again whether the value of the variable parameter Pv is less than or equal to the upper limit value (S207). If the value of the variable parameter exceeds the upper limit (S207: NO), the process proceeds to step S211, and the adjustment unit 21 adjusts the value of the variable parameter Pv used when executing the previous (last) Z slice. , the initial value of the variable parameter Pv is determined (S211), and the initial value determination process ends. Alternatively, to further improve the counting accuracy, change the parameters by the upper limit, lower limit, and change width Δ determined in step S202, take multiple images, and set the laser intensity and nucleus count as shown in the lower diagram of FIG. The initial value determination process may be completed by actually creating a relationship diagram, determining the minimum laser intensity I at which the number of nuclear counts becomes constant from the relationship diagram as the initial value. The user may specify the laser intensity by displaying a diagram of the relationship between the laser intensity and the nuclear count.

By executing such an initial value determination process, the initial value of the variable parameter Pv is automatically determined regardless of the difference in skill level of the users. Finally, the standard nuclear density is calculated from the Z slice image of the BP plane taken with the initial value of the Pv parameter. Nuclear density is a value obtained by dividing the number of nuclei calculated from a Z slice image by the cross-sectional area of a cell mass calculated from the same Z slice image. Since the nuclear density of general spheroids and organoids is constant regardless of location, if the nuclear density deviates significantly from the standard nuclear density, it can be determined that an appropriate image has not been captured. Before proceeding to S108, an appropriate range of nuclear density is determined.

Next, the process of step S108 described above (hereinafter referred to as Z stack execution process) will be explained with reference to FIG. First, the adjustment unit 21 adjusts the Z position of the objective lens 126 so that the focal plane position becomes the Z stack start position (S301). Note that when the Z stack is executed, the adjustment unit 21 shifts the Z position of the focal plane in a direction away from the interface between the cell mass and the well W (the upper surface of the well W). 21 sets the starting position of the Z stack near the interface between the cell mass and the well W.

Next, the adjustment unit 21 causes the microscope apparatus 10 to perform a Z slice, and the acquisition unit 22 acquires a Z slice image at the set Z position (S302). The acquisition unit 22 creates brightness contour lines in the acquired Z slice image, discriminates the core portion, and calculates the average value of the brightness of the core portion (S303). The adjustment unit 21 determines the laser intensity I in the next Z slice based on the calculated average value of the luminance of the core portion (S304). As described above, the initial value of the variable parameter Pv is determined at the reference plane BP that is not far away from the interface between the cell mass and the well W. Since the first Z slice is executed using the initial value of the variable parameter Pv determined on the reference plane BP, the average value of the brightness of the core portion in the obtained Z slice image is sufficiently large. Therefore, the adjustment unit 21 sets the laser intensity I in the second Z slice to the same value as the laser intensity I in the first Z slice.

Next, the acquisition unit 22 calculates the nuclear density based on the acquired Z slice image, and determines whether the calculated nuclear density is within an appropriate range (S305). Here, too, the first Z slice is executed using the initial value of the variable parameter Pv determined on the reference plane BP, so the calculated nuclear density is within the appropriate range. If the calculated nuclear density is within the appropriate range (S305: YES), the adjustment unit 21 determines whether the previously performed Z slice corresponds to the last Z position (S306). If the last Z slice is not the last Z position (S306: No), the adjustment unit 21 returns to step S301 described above and adjusts the Z position of the objective lens 126 to the next Z position. do. Then, the adjustment unit 21 causes the microscope device 10 to execute the Z slice again, and the acquisition unit 22 acquires a Z slice image at the set Z position (S302). Then, as described above, the acquisition unit 22 creates brightness contour lines in the acquired Z slice image, discriminates the core portion, and calculates the average value of the brightness of the core portion (S303). Then, the adjustment unit 21 determines the laser intensity I in the next Z slice based on the calculated average value of the luminance of the core portion (S304).

Note that, as shown by the dotted line in FIG. 5, as the Z position of the focal plane moves away from the interface between the cell mass and the well W (the upper surface of the well W), it gradually becomes difficult for the laser light to reach the focal plane. The brightness of the fluorescence decreases accordingly. Therefore, in the acquired Z slice image, the average value of the brightness of the core portion may become small. Therefore, in the process of step S304, if the average value of the brightness of the core part is smaller than the average value of the brightness of the core part in the previous Z stack by a certain percentage or more, the adjustment unit 21 adjusts the brightness of the next Z slice. The laser intensity I in the previous Z slice can be made larger than the laser intensity I in the previous Z slice. Here, the control unit 20 may hold information regarding the correlation between the average brightness of the core portion and the laser intensity I as a table in a memory (not shown). The adjustment unit 21 may refer to a table stored in a memory (not shown) of the control unit 20 and determine the laser intensity I in the next Z slice based on the drop in the average value of the brightness of the core part. . Alternatively, the adjustment unit 21 may determine the laser intensity I in the next Z slice based on the value input by the user through the input unit 40.

Then, as described above, the acquisition unit 22 calculates the nuclear density based on the acquired Z slice image, and determines whether the calculated nuclear density is within an appropriate range (S305). If the calculated nuclear density is within the appropriate range (S305: YES), the acquisition unit 22 determines that the nuclear part has been successfully discriminated in the acquired Z slice image, and performs the above-mentioned process. The process then proceeds to step S306. Note that even if the acquisition unit 22 determines that the calculated nuclear density is within the appropriate range, in steps S303 and S304, the adjustment unit 21 adjusts the laser intensity I in the next Z slice to that in the previous Z slice. In some cases, the laser intensity is determined to be greater than the laser intensity I. In this case, the adjustment unit 21 makes the laser intensity I larger than the laser intensity I in the previous Z slice, and causes the microscope apparatus 10 to execute the next Z slice. Thereby, in the next Z slice, it is possible to increase the average value of the brightness of the core portion in the Z slice image.

In addition, if the average brightness value of the nuclear part in the Z slice image is not sufficient and the nuclear part cannot be discriminated normally, the calculated nuclear density value will be out of the appropriate range and will be an abnormal value. Become. Therefore, when the acquisition unit 22 determines that the calculated nuclear density is not within the appropriate range (S305: No), the adjustment unit 21 adjusts the laser intensity I to the laser that is scheduled to be set in the next Z slice. The intensity is increased to I, and the microscope device 10 is made to perform the Z slice again. Thereby, in the Z slice executed again, it is possible to increase the average value of the brightness of the core portion in the Z slice image.

In the counting device 1 according to the embodiment described above, the adjustment section 21 and the acquisition section 22 can execute the initial value determination process. Thereby, the initial value of the variable parameter Pv can be automatically determined regardless of the difference in user skill level.

The embodiments described above are merely examples and do not limit the scope of the claimed invention. Furthermore, not all of the combinations of features described in the above embodiments are essential. Moreover, various changes can be made to the above embodiment. Modifications of the embodiment will be described below.

In the above description, the reference plane BP was set to pass through a constricted part of the cell mass slightly above the part that contacts the well W. However, the present embodiment is not limited to such an aspect, and the reference plane BP can be set at any Z position as long as a Z slice image in which the nucleus can be discriminated can be obtained.

In the above explanation, the adjustment unit 21 selected the laser intensity I as the variable parameter Pv, but it is also possible to select other setting parameters. For example, when the number of averages is set as the variable parameter Pv, the acquisition unit 22 averages the Z slice images acquired 1, 2, 4, 8, 16, 32 times, etc. in one Z slice. The number of nuclei is counted based on the averaged Z slice images acquired each time (S204, S205). Then, the adjustment unit 21 can obtain the initial value of the variable parameter Pv by obtaining the average number of times at which the count of the number of nuclei becomes constant (S210, S211).

In the above description, the illumination unit 100 was equipped with three CW lasers 102 with wavelengths of 405 nm, 488 nm, and 561 nm, but the number of CW lasers and wavelengths are not limited to the example of the above embodiment, and can be set arbitrarily. . Further, although the microscope device 10 is a confocal laser microscope equipped with a laser light source as a light source, the present embodiment is not limited to such a configuration. For example, it may be a multiphoton excitation microscope, and a thin sheet It may also be a light sheet microscope that irradiates excitation light shaped into a shape from the side of the sample. Alternatively, for example, OCT (Optical Coherence Tomography) may be used.

In the above description, in the Z stack execution process, the acquisition unit 22 calculates the nuclear density based on the acquired Z slice image, and determines whether the calculated nuclear density is within an appropriate range. . However, it is not always necessary to make the determination in step S305 based on the nuclear density. For example, the acquisition unit 22 may calculate the distribution of the circumference of the nucleus and determine whether the distribution exceeds an expected range and becomes an abnormal distribution. Alternatively, the ratio between the brightness of the core portion and the brightness of the portion other than the nucleus may be calculated, and it may be determined whether the brightness ratio is equal to or greater than a certain value.
Further, in the above description, the nucleus of the cell has been used as an example of the cell structure, but the cell structure is not limited to the nucleus. For example, cell organelles such as lysosomes, Golgi bodies, and mitochondria, proteins, second messengers, mRNAs, genes, etc. may be used.

In the above description, the setting parameters of the microscope device 100 are adjusted at different positions in the Z-axis direction of the cell mass, but the present invention is not limited thereto. For example, the setting parameters of the microscope device 100 may be adjusted at different positions in the X-axis direction of the cell mass. Further, for example, the setting parameters of the microscope device 100 at different positions in the Y-axis direction of the cell mass may be adjusted. Of course, you can adjust the setting parameters for each of these X-axis, Y-axis, and Z-axis directions individually, or you can combine the X-axis, Y-axis, and Z-axis directions as appropriate. I do not care. For example, the setting parameters at a predetermined position on the XY plane at the same Z position of the cell mass may be used to set the setting parameters at a position different from the predetermined position.

In the above explanation, the sample S is a mass of cells (see FIG. 1) in which the cells are uniformly distributed. However, in this embodiment, the sample to be measured is It is not limited to clusters of cells distributed in For example, as shown in FIG. 7, the sample S may be a mass of cells containing blood vessels inside. Even in this case, in the Z stack execution process, the acquisition unit 22 does not necessarily need to make the determination in step S305 based on the nuclear density. This is because the nuclear density is not constant depending on the distribution of blood vessels. Therefore, the acquisition unit 22 may calculate the distribution of the circumference of the nucleus, as described above, and determine whether the distribution exceeds the expected range and becomes an abnormal distribution. Alternatively, the ratio between the brightness of the core portion and the brightness of the portion other than the nucleus may be calculated, and it may be determined whether the brightness ratio is equal to or greater than a certain value.

Furthermore, in the above description, the internal structure of a cell was exemplified as the cell structure, but the structure is not limited to this. For example, an example of cell information is the number of dendritic spines, which are cell structures on the cell surface, in a neuron, as shown in FIG. The counting device 1 according to the present embodiment can also be applied to counting the number of dendritic spines using a sample S of nerve cells including a plurality of dendritic spines as shown in FIG. I can do it. Note that the dendritic spines are stained in advance with an appropriate dye. In this case, in the Z-stack execution process, the acquisition unit 22 does not necessarily need to make the determination in step S305 based on the density of dendritic spines. This is because the density of dendritic spines is not necessarily constant. Therefore, as in the case described above, the acquisition unit 22 calculates the distribution of the circumference of dendritic spines and determines whether the distribution exceeds the expected range and becomes an abnormal distribution. Good too. Alternatively, the ratio of the brightness of the dendritic spine to the brightness of the other parts may be calculated, and it may be determined whether the brightness ratio is equal to or higher than a certain value.

Note that the purpose of measuring images is not limited to measuring the number of cells in a cell cluster. The purpose may also be to obtain a clear image of a predetermined position within a cell mass. For example, by analyzing an image of a predetermined cell in a cell cluster, when examining the state of the cells in the cell cluster, the state of the cell cluster can be appropriately evaluated. For example, it is possible to accurately evaluate the effect of a drug injected into a cell mass on the cells inside the cell mass.

In the embodiment described above, the structure, arrangement, etc. of each component of the microscope apparatus 10 (illumination section 100, optical system 120, light receiving section 140, movable stage 160) can be changed as appropriate. For example, in the above embodiment, the optical system 120 has been explained while showing specific optical elements, but the present teachings are not limited to such a form, and the number, type, and arrangement of the optical elements constituting the optical system 120 are explained. etc. may be changed as appropriate. Further, in the embodiment described above, the counting device 1 includes the microscope device 10, the adjustment section 21 and the input section 40 that function as a measurement condition setting device for setting measurement conditions of the microscope device 10. Therefore, in the embodiment described above, the adjustment section 21 and the input section 40 functioned as a measurement condition setting device that sets the measurement conditions of the microscope device 10 included in the same counting device 1. Not limited. For example, the adjustment section 21 and the input section 40 may be configured to set measurement conditions for another independently provided microscope device.

Although preferred embodiments and modified examples of the present invention have been described above with reference to the accompanying drawings, it goes without saying that the present invention is not limited to these examples. The various shapes and combinations of the constituent members shown in the above example are merely examples, and can be variously changed based on design requirements and the like without departing from the gist of the present invention. The components of the embodiments described above can be combined as appropriate. Furthermore, some components may not be used. In addition, to the extent permitted by law, all public announcements and US patent disclosures regarding each of the above-described embodiments and modifications are incorporated herein by reference. All other embodiments, operational techniques, etc. made by those skilled in the art based on the embodiment described above are included in the scope of the present embodiment.

1 Counting device 10 Microscope device 20 Control section 30 Display section 40 Input section 100 Illumination section 120 Optical system 140 Light receiving section 160 Movable stage

Claims (12)

an illumination optical system that includes an objective lens and scans and irradiates the cell mass with illumination light from a light source in a plane intersecting the optical axis direction of the objective lens through a scanning unit ;
a detection optical system that focuses light from the cell mass onto a light receiving unit via the scanning unit;
comprising a control unit;
When the scanning unit two-dimensionally scans the illumination light at each of the plurality of different positions along the optical axis direction to obtain a two-dimensional image, the control unit controls the control unit to control the illumination light at each of the plurality of positions as a pre-process. In the process of acquiring the two-dimensional image, the density of the cell structure is determined based on the two-dimensional image acquired at the first position of the cell mass without performing image acquisition to determine the image acquisition conditions of , information on the perimeter of the cell structure, and ratio information between the brightness of the cell structure and the brightness of a portion other than the cell structure, and The microscope determines image acquisition conditions at a second position, which is different from the first position, along the optical axis direction based on the information obtained. The microscope according to claim 1, wherein the control unit changes the image acquisition conditions at the second position until the specified information falls within an appropriate range. 3. The microscope according to claim 1, wherein the cell structure is an internal structure of the cell or a surface structure of the cell. 4. The microscope according to claim 3, wherein the internal structure of the cell is the nucleus of the cell. 4. The microscope according to claim 3, wherein the structure on the surface of the cell is a surface protrusion. The image acquisition conditions include at least one of the intensity of the illumination light, the scanning speed of the scanning unit, the voltage applied to the light receiving unit, and the number of times the two-dimensional image is averaged at the second position. The microscope according to any one of claims 1 to 5. Any one of claims 1 to 6, wherein the control unit changes the image acquisition conditions so that the density of the cell structures included in the two-dimensional image acquired at the first position falls within an appropriate range. The microscope described in section. The control unit changes the image acquisition conditions so that a distribution of perimeters of the cell structures included in the two-dimensional image acquired at the first position falls within an appropriate range. The microscope according to any one of the items. The cell mass is placed on a holding member,
The microscope according to any one of claims 1 to 8, wherein the first position is set at a different position along the optical axis direction from a position where the cell mass and the holding member are in contact with each other. The microscope according to claim 9, wherein the first position is set within 10 μm along the optical axis direction from a position where the cell mass and the holding member are in contact with each other. The microscope according to any one of claims 1 to 10, wherein the cell mass includes a tissue section. an illumination optical system that includes an objective lens and scans and irradiates the cell mass with illumination light from a light source in a plane intersecting the optical axis direction of the objective lens through a scanning unit;
a detection optical system that focuses light from the cell mass onto a light receiving unit via the scanning unit;
In a microscope equipped with
When acquiring a two-dimensional image by two-dimensionally scanning the illumination light with the scanning unit at each of a plurality of different positions along the optical axis direction, as a pre-process, image acquisition conditions for each of the plurality of positions are determined. In the process of acquiring the two-dimensional image, information on the density of the cell structure, information on the density of the cell structure, based on the two-dimensional image acquired at the first position of the cell mass, without image acquisition for determining identifying at least one of information on the perimeter of the structure of the cell, and information on a ratio between the brightness of the cell structure and the brightness of a portion other than the cell structure;
A method for setting a microscope, comprising: determining image acquisition conditions at a second position different from the first position along the optical axis direction based on the specified information.

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