JPH10232229A - Cytometer - Google Patents

Abstract

PROBLEM TO BE SOLVED: To provide a cytometer capable of readily recognizing cells positioned on a scanning boundary in a microscope image. SOLUTION: A cytometer two-dimensionally scans laser beams each of a plurality of strips formed by multi-dividing a measurement range on a cell group specimen under control of a scan control circuit 23, fetches optical scanning image data from the cell group specimen into a computer 22, obtain data of each cell in a cell group by an image process with respect to the scanning image data, and displays on a monitor 50 as a statistical graph. In this case, apex coordinates of a plurality of strips are converted into coordinates on a microscope image by the computer 22, and the coordinates-converted strip is displayed on the microscope image of the monitor 50.

Description

DETAILED DESCRIPTION OF THE INVENTION

[0001]

BACKGROUND OF THE INVENTION 1. Field of the Invention The present invention relates to a cytometer that scans a cell population with a laser beam and performs measurement using an image formed from optical data from the cell population.

[0002]

2. Description of the Related Art An individual cell of a biological cell population biochemically labeled with a fluorescent dye is irradiated with a laser spot, and the fluorescence emitted from the individual cell upon excitation is measured at high speed. A flow cytometer is known as a device that analyzes and presents statistical data representing immune characteristics, genetic characteristics, and cell proliferation.

In a conventional flow cytometer, a laser and a spot where the laser beam is focused are fixed, and cells in an isolated suspension state are flown on the spot together with a jet water flow to measure. It is impossible to find cells corresponding to specific data and observe the morphology, and to confirm the measurement data corresponding to individual cells after measurement.

In such a flow cytometer, a cell population on a slide glass is scanned with a spot where a light beam (laser beam) is focused, and fluorescence and scattered light emitted from individual cells of this cell population are detected. For example, as a so-called scanning cytometer, which processes data by using
One disclosed in Japanese Patent No. 55365 is known. Such a scanning cytometer scans a cell population on a slide glass by moving a scanning stage while deflecting a laser beam by a scanner driven according to a scanning waveform. The fluorescence and scattered light emitted from the cells excited by the laser spot by this scanning are converted into electric signals, collected according to the logic of the data input signal output together with the scanning waveform of the scanner, and further formed by scanning. Image processing of the image, the sum of the fluorescence values of individual cells, area, maximum fluorescence value, coordinates on the scanning stage, time from the start of measurement, distance to the nearest cell, outer circumference, number of spots in the cell, etc. Extract measurement data.

[0005] Such a scanning cytometer is
As a feature, by recording each cell coordinate position on the slide glass, and by enabling the reproduction of position coordinates,
That is, it is possible to reproduce and display each cell presenting individual data in the statistical data in the visual field of the microscope and to associate the microscopic observation image with the cell data. In other words, after the measurement, the data of individual cells can be displayed statistically, and the microscope field of view based on the coordinates of the data in a certain subset, such as abnormal peaks of cancer cells and the like on the DNA histogram, is captured by the CCD camera. By reproducing more,
The morphology of each cell can be observed (recall function).

[0006] Contrary to the recall function, by observing the morphology of an individual cell in a microscope image and selecting that cell, the measured data of the corresponding cell is displayed as a numerical value or graph on a computer screen. It can also be displayed (review function).

[0007]

By the way, in such a scanning cytometer, thousands to tens of thousands of cells are usually scanned by scanning a measurement range of several mm to several tens mm square on a slide glass. Need to measure. However, due to the field of view of the microscope objective lens that condenses the laser beam and the capacity of the buffer memory that temporarily stores the scanned image data, the range in which the laser beam can be scanned and the image data collected at one time is several hundred μm square. Is limited to For this reason, in order to measure a larger range, for example, as shown in FIG. 10, a desired measurement range 100 is divided into a plurality of rectangular regions (hereinafter, referred to as strips) 101. , # 1 strip 101 is scanned two-dimensionally, and then the strip 101 # 2 is scanned two-dimensionally so that all the strips 101 are sequentially scanned.

However, when the measurement range 100 is divided into a plurality of strips 101 and scanned, if cells cross over any one of the boundaries of each of the strips 101, the cells may not be measured and may not be measured. When the amount of fluorescence from the cell cannot be measured due to discoloration of the cell or variation in the background light near the cell, the measurement may not be performed. Here, cells that have not been measured are referred to as unmeasured cells, whereas cells that have been measured normally are referred to as measured cells. The most frequent cause of unmeasured cells is when the cells are located at any one of the scanning boundaries of the strip 101.

[0009] When the cells are displayed again by the recall function from such a state, the visual field of the microscope is displayed by the image picked up by the CCD camera. Images of unmeasured cells that did not exist are also included. For this reason, from this state, if the operator wants to select a measurement cell by the review function and display the corresponding cell data in order to compare the cells, if the cell image on the screen is a measurement cell that has been measured normally. It is difficult to distinguish between unmeasured cells that have not been measured, and if an unmeasured cell image is selected by mistake, the review function will not operate, and such operations will be repeated, thus deteriorating work throughput. There was a problem of letting it.

In such a case, since the operator cannot understand why there are unmeasured cells in the screen, the operator uses the threshold values for fluorescence values and background processing so that these unmeasured cells can be measured. Cell recognition parameters such as data ranges may be adjusted. With these adjustments, cells that are not recognized due to fading of the cells or variations in the background light near the cells may become recognizable. For cells located near the boundary, even if the cell recognition parameters are adjusted, almost no recognition becomes possible. Even if it is recognized, normal data cannot be obtained because the amount of light is insufficient during scanning. Further, even if the cell recognition parameters are adjusted, the cells located outside the measurement range designated by the operator are not scanned because they are naturally not scanned.

In such a case, repeated useless adjustments by the operator not only increase the burden on the operator, but also cause the laser to scan the cells more than necessary and affect the measurement accuracy such as fading of the cells. Was likely to occur. The present invention has been made in view of the above circumstances, and has as its object to provide a cytometer capable of easily recognizing cells located on a scanning boundary in a microscope image.

[0012]

According to the first aspect of the present invention,
The measurement range on the cell population sample is divided into a plurality of sections to form a plurality of strips, a laser beam is two-dimensionally scanned for each of these strips, optical scan image data from the cell population sample is captured, and an image corresponding to the scanned image data In a cytometer that obtains data of individual cells in a cell population by processing and displays the data as a statistical graph, a display unit that displays a microscope image of the cell population specimen, and vertex coordinates of the plurality of strips are displayed on the microscope image. It is constituted by coordinate conversion means for converting the coordinates into coordinates, and control means for displaying the strip whose coordinates have been converted by the coordinate conversion means on a microscope image on the display means.

According to a second aspect of the present invention, in the first aspect, the control means displays a region on the microscope image, which is outside the strip, in an identifiable manner. According to a third aspect of the present invention, in the second aspect, the display of the region outside the strip on the microscope image is a hatch pattern or a colored display.

As a result, according to the first aspect of the present invention,
Unrecognizable cells located on the scanning boundary of each strip can be presented to the operator in advance. Claim 2
According to the invention described in (3), the operator can easily confirm whether or not the cell to be measured exists in the measurement range.

[0015]

Embodiments of the present invention will be described below with reference to the drawings. (First Embodiment) FIG. 1 shows a schematic configuration of a cytometer to which the present invention is applied.

In this case, a laser beam emitted from the laser light source 1 is appropriately focused by a spot projection lens 18, reflected by a dichroic mirror 2, and further rotated around a rotation axis perpendicular to the plane of the drawing. The light is reflected by the pupil projection lens 4 to form an image on the objective image plane, and then enters the optical path switching mirror 5. Thus, the laser beam is scanned in the vertical direction on the paper at the position of the optical path switching mirror 5 by the presence of the rotating galvanometer mirror 3.

The laser beam is transmitted to an optical path switching mirror 5.
After being reflected by the objective lens 6, the light enters the objective lens 6 and is imaged on the specimen 7. In this case, the laser spot formed on the sample 7 scans the laser beam in the left-right direction along the plane of the paper by the optical deflecting means by the rotation of the galvanometer mirror 3, and at the same time, moves the scanning stage 17 in the direction perpendicular to the plane of the paper. By doing so, the laser spot is two-dimensionally scanned on the surface of the sample 7.

In this case, the specimen 7 is composed of a cell population. These cells are biochemically labeled with a fluorescent dye in advance, and emit fluorescence when excited by a laser spot. Fluorescence from the specimen 7 travels in the optical path described above in reverse, passes through the objective lens 6, the optical path switching mirror 5, the pupil projection lens 4, and the galvano mirror 3, passes through the dichroic mirror 2 upward, and further passes through the barrier filter 13. The light is condensed on the light receiving surface of the photomultiplier tube (PMT1) 15 by the condenser lens 14.

On the other hand, the scattered light scattered by the cells on the specimen 7 is condensed by the condenser lens 8 together with the light transmitted through the specimen 7 downward, reflected by the beam splitter 9 and then incident on the ring slit 10. Ring slit 1
Numeral 0 shields the transmitted light from the sample 7, allows only the scattered light to pass, and causes the light to enter the light receiving surface of the photodiode (PD) 11.

That is, the fluorescent light and the scattered light from the specimen 7 two-dimensionally scanned by the laser spot are detected by the photomultiplier tube 15 and the photodiode 11. Although the case where one kind of fluorescent dye is used as the fluorescent dye to be attached to the specimen 7 is described in the drawing, a dichroic mirror 2, a barrier filter 13, and a photomultiplier tube (PMT2, PMT3) 15 are added. By
Fluorescence of different wavelengths from a plurality of fluorescent dyes can be detected simultaneously.

On the other hand, the transmission illumination light source 12 and the epi-illumination illumination light source 1
By using No. 9, it can also be used as a normal microscope. In this case, the optical path switching mirror 5 is provided so as to be insertable into and removable from the optical path of the laser light, and by removing the optical path switching mirror 5 from the optical path, an observation image of the sample 7 is guided to the microscope observation optical system 16. . As a result, the observer
The transmission image and the fluorescence image of the specimen 7 can be observed with the naked eye under a microscope or can be photographed under a microscope with a television camera or a photographing device.

FIG. 2 shows an electric circuit for converting the fluorescence taken in from the photomultiplier tube 15 and the photodiode 11 into an image and processing the image, and a scanning control circuit for two-dimensionally scanning the specimen 7 with a laser spot. ing.

In this case, these circuits are roughly divided into photomultiplier tubes 15a, 15b, 15c (PMTs 1, 2,.
The three signal processing circuits 20a, 20b, which convert the electric signals corresponding to the respective fluorescence intensities obtained from 3) into image data,
20c, a signal processing circuit 20d for converting an electric signal corresponding to the scattered light intensity obtained from the photodiode 11 (PD11) into image data, and these signal processing circuits 20a to 20-2
A computer 22 for processing image data obtained from 0d to create a scanned image and a processed image, a scanning control circuit 23 for two-dimensionally scanning the sample 7 with a laser spot,
It is composed of a main body control unit 24 that controls the operation of each of the signal processing circuits 20a to 20d and the scanning control circuit 23.

The signal processing circuit 20a for the photomultiplier tube PMT1 is connected to the CPU bus 32 of the main body control unit 24. The main body control section 24 has a CPU 33.
The CPU 33 controls the doubling rate by applying a voltage to the cathode of the photomultiplier tube (PMT1) 15a via the CPU bus 32 via a D / A converter inside the signal processing circuit 20a. (PMT1) The offset adjustment voltage of the photoelectric signal detected by 15a is converted to a signal processing circuit 20a.
It is applied to a photoelectric signal via an internal D / A converter.

Other photomultiplier tubes (PMT2, PMT3)
The signal processing circuits 20b and 20c for 15b and 15c are the same as the signal processing circuit 20a for the photomultiplier tube (PMT1) 15a described above, and a description thereof will be omitted.

The signal processing circuit 20d for the photodiode 11 is substantially the same as the other signal processing circuits 20a to 20c. However, the cathode voltage is not applied to the photodiode 11, so that the signal processing circuit 20d is connected to the signal processing circuit 20d. Is not provided.

The main body control unit 24 includes a GPIB interface control circuit (GPIB) for performing various communications between the CPU 33 and the computer 22 with respect to the CPU bus 32.
(I / F) 36, an RCU 37 as a group of movement control switches for the scanning stage, and an input / output circuit 38. The scanning stage 17 of the scanning control circuit 23 is connected to the CPU bus 32 via driving circuits 41 and 42 in the X-axis and Y-axis directions.
Two stepping motors 3 for moving in the axial direction
9, 40, and a waveform generating circuit 45 for forming a waveform for driving the galvanometer mirror 3 are connected. The galvanomirror 3 is connected to the waveform generation circuit 45 via a D / A converter 46, and the waveform generated by the waveform generation circuit 45 is applied to the galvanomirror 3 via the D / A converter 46. The galvanomirror 3 is driven according to the applied voltage. Thereby, the main body control unit 24
Controls the operation of the scanning stage 17 and the galvanomirror 3 via the scanning control circuit 23, and allows the laser spot to arbitrarily and two-dimensionally scan the specimen 7.

The RCU 37, which is a group of control switches for moving the scanning stage, has four direction (up, down, left, and right) keys.
The contents are notified to U33, and the CPU 33 gives an instruction to the drive circuits 41, 42 of the stepping motors 39, 40 in accordance with the notified contents to move the scanning stage 17. When the movement of the scanning stage 17 is completed, the contents are notified to the computer 22 via the GPIB interface control circuit 36. As a result, the operator can control the scanning stage 1 by the RCU 37.
7 can be operated freely, and the computer 22 can know that the operation of the scanning stage 17 by the operator has been completed.

On the other hand, the computer 22 is provided with a GPIB board 47 for performing GPIB control.
It is possible to communicate with the CPU 33 of the main body control unit 24 via the GPIB interface control circuit 36 on the fourth side. Thus, the stage 17 and the galvanomirror 3 are transmitted from the computer 22 through the main body control unit 24.
Control such as start and end of the operation. Further, as described above, when the scanning stage 17 is moved by the RCU 37, a notification can be received from the main body control unit 24.

The computer 22 includes a CPU 22
1, two memory boards 48a and 48b as expansion slots, and one GPIB board 4 described above.
7, one video board 49 is mounted. Memory circuits for two channels are mounted on the memory boards 48a and 48b. As shown in the figure, the transfer data from the signal processing circuit 20a is input to the channel 1 memory circuit of the one memory board 48a, and the transfer data from the signal processing circuit 20b is input to the channel 2 memory circuit. The transfer data from the signal processing circuit 20c is input to the channel 3 memory circuit of the other memory board 48b, and the transfer data from the signal processing circuit 20d is input to the channel 4 memory circuit.

Further, one of the memory boards 48a, 48b
One channel is composed of two bank memories, and while the signal processing circuit 20a is transferring data to one bank, the other bank can be accessed by the CPU 221.

The video board 49 has a CCD camera 16
a is connected. This CCD camera 16a
For capturing a transmission image or a fluorescence image of the microscope, and is attached to the microscope as described above. A video signal captured by the CCD camera 16a is input to a video board 49,
The image is displayed on the monitor 50 by being combined with a graphic image in accordance with an instruction from the CPU 221.

The microscope image synthesized here can be displayed in real time, and a computer graphic can be overlaid on the microscope image. Also, C
The microscope image displayed according to the instruction of the PU 221 is decomposed into pixels of a maximum of 640 × 480 dots, and what is μm when one pixel on the screen is converted into the moving coordinate system of the stage is measured in advance as a coordinate conversion value. deep. in this case,
Since the position coordinate data of each cell is in the coordinate system on the scanning stage 17, it can be converted into coordinate values on the microscope image by the coordinate conversion values. As a method of measuring the coordinate conversion value, a target image is displayed on a microscope screen, the target image is selected, coordinates on the screen are obtained, and a range in which the target value is known and moves within the screen. Then, the stage is moved by the moving amount, and by selecting the same target image again, the ratio is calculated and obtained from the moving amount on the screen and the actual moving amount of the scanning stage.

The positional relationship between the scanning stage 17 and the microscope image is determined by aligning the optical axis of the objective lens 6 shown in FIG. 1 with the center of the microscope image.
Although the magnification can be changed by exchanging according to the specimen 7, since the coordinate conversion value differs depending on the magnification of the objective lens 6 to be used, the value of each objective lens 6 is stored in a memory (not shown) of the computer 22. Keep it.

The basic software used in the computer 22 is Microsoft Windows.
Is used to statistically display cell data in a graph in a display area called a window, or to use a CCD camera 16a.
To display real-time images. Also,
These windows can be scaled up and down on the screen of the monitor 50 and can be changed as needed. The software of the computer 22 that controls this apparatus can be started on such Windows.

Next, a case where a cell is actually measured and the recall function and the review function are used will be described with reference to FIG. First, when the operator instructs the computer 22 to start measurement, in step 301, cell data collection is started. In this case, for data collection, the galvanometer mirror 3 and the scanning stage 17 are moved to two-dimensionally scan the sample 7 with a laser spot, and fluorescence and scattered light from the sample 7 are detected by the photomultiplier tube 15 and the photodiode 11. To do this automatically. Each cell data thus measured is stored in the memory of the computer 22 together with the mark display information, and at the same time, the position coordinates of the recognized cell are also stored. The position coordinates are calculated by adding the coordinates of the collected cell data having the origin at the upper left of the strip 101 to the stage coordinates at the upper left point of the strip 101 described above. At this time, the stained cells may be discolored or the amount of fluorescence from the cells may not be measured due to the influence of background light near the cells. Becomes unmeasured cells without being recorded. In this case, the mark display information is not stored.

The range that can be scanned by the galvanometer mirror 3 is about several hundred μm, and the size of the horizontal strip 101 is limited as shown in FIG. Further, the size of the scanned image data that can be collected at one time is limited by the memory capacity of the bank memory. Therefore, in order to measure a large amount of cells, as shown in FIG.
00 is divided into a plurality of strips 101. And
In one of the strips 101, scanning is performed by rotating the galvanometer mirror 3 in the horizontal direction, and scanning is performed by moving the scanning stage 17 in the vertical direction. When the strip 101 of # 1 ends, the strip 101 of # 2 is moved to the strip of # 2. Then, all the strips 101 are sequentially scanned, but the cells present at the vertical boundary between the adjacent strips 101 and the strips 101 are not recognized as one cell due to insufficient light quantity at the time of scanning. In some cases, the cell data is not stored, but becomes unmeasured cells. Also in this case, the mark display information is not stored.

When the data collection is completed, the data is displayed in step 302. In this case, each measured cell data is processed by the computer 22 as statistical data. For example, the horizontal axis indicates the total amount of fluorescence of individual cells, the vertical axis indicates the area of the cell, or the horizontal axis indicates the total amount of fluorescence of individual cells, and the vertical axis indicates a DNA histogram. The graph is displayed on the monitor 50 by.

FIG. 4 shows a display example of the data displayed on the monitor 50. In this example, the cell data graph 40 is shown.
1 and a microscope image 402 are displayed. These displays are displayed in a display range called a window, and the scale of the display range and the display position can be arbitrarily changed.
Further, the microscope image 402 can display a real-time image from the CCD camera 16a or overlay a computer graphic on the microscope image 402. The mouse pointer 403 can be freely moved on the displayed cell data graph 401 or microscope image 402 by the designation of the operator, and the position on the display screen can be taken into the computer 22.

Next, in step 303, confirmation by the operator is performed. This confirmation is an operation for the operator to verify the cell data. Here, the operator moves the mouse pointer 403 to a desired plot 405 on the cell data graph 401 and takes the position into the computer 22 to select data for which the cell morphology is to be observed.

When the recall function is designated in step 304, a cell image corresponding to the data selected by the confirmation in step 303 is displayed on the microscope image 402. In this case, the position coordinates of the selected data are read from the memory of the computer 22, and the scanning stage 17 is moved based on the position coordinates, so that the real-time captured image of the CCD camera 16a at this time becomes the microscope image 402. Will be displayed.

In this case, as shown in FIG. 5, the microscope image 402 contains the cell image 40 corresponding to the selected image data.
4a together with another cell image 40 present in the microscope field of view.
4b is also displayed, and a confirmation cursor 406 surrounding the cell image 404a in a square is displayed on the cell image 404a corresponding to the selected data among them,
Also, a cell image 404a corresponding to the selected cell data
Regarding and the other cell images 404b, the mark 407 is overlaid and displayed by computer graphics for all cells that are recognized as measurement cells at the time of measurement and to which mark display information is added. In this case, the mark 407 has a circular shape surrounding the cell images 404a and 404b, makes the cell images easy to see, and furthermore, the cell images 404a and 404b.
Since the background occupying most of the screen is dark black, the mark 407 is made to be conspicuous by using a high-luminance color, for example, white. In contrast, unmeasured cells 404c
Does not display anything.

The mark 407 is displayed after being converted to the coordinate system on the microscope screen by the above-described coordinate conversion values because the position coordinate data of the measurement cells is stored in the coordinate system on the scanning stage 17. .

The verification of the cell data by the operator is performed until it is determined in step 305 that the confirmation is completed. Then, when it is determined in step 305 that the confirmation has been completed, the process proceeds to step 306, where the verification of the cell image is performed as confirmation by the operator.

The verification of the cell image is performed by checking the confirmation cursor 406.
Is performed by comparing the form of the cell image 404a displayed as an overlay with the form of the other cell image 404b. To do this, the operator moves the mouse pointer 403 onto the corresponding cell image to select another cell image in the microscope image 402, and takes the position coordinates into the computer 22. In this case, the mark 4
There are cell images 404a and 404b in which 07 is displayed and cell image 404c in which mark 407 is not displayed, but cell image 404c in which mark 407 is not displayed.
Since it is possible to easily confirm that the cell is an unmeasured cell that is not recognized as a measurement cell during measurement, the operator can select a desired cell from the cell image 404b on which the mark 407 is displayed without hesitation.

Then, in step 307, cell data corresponding to the position coordinates of the cell image 404b selected by the operator is retrieved from the data group stored in the memory of the computer 22. Here, in step 308,
If it is determined that there is data, the corresponding data is highlighted in step 309. In this case, if there is data, the corresponding data is highlighted such as changing the display color of the cell data plot on the cell data graph display 401 shown in FIG. In addition, characteristic amounts such as cell area, sum of fluorescence values, and peak values can be displayed.

On the other hand, if it is determined in step 308 that there is no data, an error or the like is displayed as to whether or not to do anything. Thereafter, in step 310, the operator confirms whether or not to end the processing, and upon confirming the end, ends the processing.

In this way, the operator can display the cell images 404 a and 404 displayed on the microscope image 402.
The operator can select only the cells recognized as the measurement cells at the time of measurement simply by indicating the one where the mark 407 is displayed from b and 404c. Therefore, the operator uses the review function to compare the cells with each other. Even if you want to select the measurement cells and display the corresponding cell data, you can eliminate the possibility of selecting an unmeasured cell image by mistake.
Work throughput can be greatly improved.

On the other hand, as shown in FIG. 10 described above, when the measurement range 100 is divided into a plurality of strips 101 and scanned, if a cell extends over any one of the boundaries of each of the strips 101, the measurement target is determined. However, in such a case, in the present invention, the computer 22
By converting the vertex coordinates of each of the divided strips 101 into the coordinates of the microscope image 402, a rectangle connecting the vertex coordinates of each strip 101 can be overlaid on the microscope image 402 by computer graphics.

In this case, since the vertex coordinate data of the strip 101 obtained by dividing the measurement range 100 shown in FIG. 10 is stored in the coordinate system on the scanning stage 17, the rectangle created by these vertex coordinate data is The coordinates are converted into the coordinate system of the microscope image 402 by the above-described coordinate conversion and displayed.

FIG. 6 shows the boundary of the strip 101 as the white line 6.
It is the figure displayed with 01. In this case, the white line 601 is an overlap display of the boundary between strips on the microscope image 402, and an unmeasured cell image 404c is displayed near the white line 601.

Next, each of the divided strips 101
The timing at which the vertex coordinates are converted to the coordinates of the microscope image 402 will be described with reference to FIG. This coordinate conversion is executed when the movement end notification of the scanning stage 17 from the main body control unit 24 arrives. In this case, the coordinates of the strips 101 obtained by dividing the measurement range 100 and the number of the strips 101 are calculated in advance when the operator specifies the measurement range 100.

In FIG. 7, when a movement end notification of the scanning stage 17 is received from the main body control unit 24, the strip counter I is cleared in step 701. next,
In step 702, it is determined whether or not the counter I is smaller than the number of strips. If the value of the counter I is larger than the number of strips, the process is terminated. If the value of the counter I is smaller than the number of strips, the process proceeds to step 703. Count up I. Then, step 704
Then, the coordinates of the strip I corresponding to the counted-up I are converted into the coordinates on the microscope image 402.

Thereafter, in step 705, it is determined whether or not the transformed coordinates fall within the range of the currently displayed microscope image 402. If so, in step 706, the microscope image 4 is displayed.
02 is overlaid with strip I.

Then, the flow returns to step 702 again, and the above-mentioned processing is repeated. Thereby, the operator can check the scanning boundary of each strip 101 on the microscope image 402, and when the target cell image is located near the scanning boundary, the operator can easily reduce the possibility of becoming an unmeasured cell. I can judge. In such a case, by changing the measurement start position and moving the scanning boundary of the strip 101 to remove the target cell from the boundary, the target cell is changed to a recognizable position. This eliminates the need to perform meaningless scanning, saving labor.
Cell fading can be prevented by reducing the number of scans. (Second Embodiment) The configurations of an optical system and an electric circuit according to the second embodiment are the same as those shown in FIGS. 1 and 2, so that the drawings will be referred to and the description here will be omitted. Omitted.

In this case, as shown in FIG. 8, the area outside the measurement range 801 on the slide glass 800 is an area obtained by removing the area of the measurement range 801 from the coordinate space of the scanning stage 17, and is divided into rectangular areas. Out of measurement range area 8
02, outside the measurement range area 803, outside the measurement range area 804,
It is divided into a region 805 outside the measurement range.

The coordinates of the vertices of the plurality of divided out-of-measurement areas 802 to 805 are obtained from the coordinates on the scanning stage 17 from the microscope image 40 in the same manner as in the previous embodiment.
The coordinates are converted to the coordinates on the microscope image 2 and are overlapped on the microscope image 402 by computer graphics.

In this case, the areas 802 to 805 outside the measurement range
In FIG. 9, a boundary line where regions outside the measurement range are in contact with each other is transparent, and a hatch pattern is displayed as an overlay. In the figure, reference numeral 901 denotes the inside of the measurement range, and 902 denotes the outside of the measurement range.

In this case, the same effects as those of the first embodiment can be expected, and unrecognizable cells located clearly on the scanning boundary of each strip can be presented to the operator. Erroneous measurement will be prevented while the target cell is out of the measurement range, and a decrease in work throughput can be prevented.

In the second embodiment, the inside of the measurement range 901 and the outside of the measurement range 90 are determined by using a hatch pattern.
Although the two regions are distinguished, they can be distinguished by changing the color tone and gradation of the region 902 outside the measurement range. In addition, in the above description, the hatching pattern or the coloring is applied to the area 902 outside the measurement range, but the same can be naturally applied to the area 901 inside the measurement range. (Third Embodiment) When printing a photograph or a hard copy of a microscope image, an operator sometimes wants to erase a display of a measurement range, a strip, a mark of a measurement cell, and the like. Further, when the measurement range is wide, a slight time lag occurs in a search process for searching for an area to be displayed in an overlapped manner.

Therefore, in the third embodiment, the operator can designate the display / non-display of the measurement range, the strip, and the mark of the measurement cell. The designation method in this case is designated by selecting a switch on software operating on Windows. Then, these display / non-display settings are saved in the software. When the microscope image is redisplayed, the display / non-display setting contents are referred to. When the display is set to display, the strip is connected to the microscope as described in the first and second embodiments. Display overlap on the image. If not displayed, no overlap display is performed.

[0062]

As described above, according to the present invention, unrecognizable cells located on the scanning boundary of each strip can be presented to the operator in advance. In such a case, the measurement start position is changed and the scanning boundary is changed. To move the target cell off the boundary and change it to a recognizable position.Therefore, there is no need to perform meaningless scanning, labor is saved, and the number of scans is reduced by reducing the number of scans. Fading can be prevented.

Further, since unrecognizable cells located on the scanning boundary line of each strip can be presented to the operator in advance, there is no possibility of erroneous measurement while the target cells are out of the measurement range. Drop can be prevented.

[Brief description of the drawings]

FIG. 1 is a diagram showing a schematic configuration of a cytometer according to a first embodiment of the present invention.

FIG. 2 is a diagram illustrating a schematic configuration of an electric circuit and a scanning circuit according to the first embodiment.

FIG. 3 is a flowchart for explaining the operation of the first embodiment;

FIG. 4 is a view showing an example of a screen display on a monitor according to the first embodiment;

FIG. 5 is a diagram illustrating a display example of a microscope image according to the first embodiment.

FIG. 6 is a diagram showing a display example of a microscope image according to the first embodiment.

FIG. 7 is a flowchart for explaining the operation of the first embodiment;

FIG. 8 is a diagram illustrating a region outside a measurement range according to a second embodiment of the present invention.

FIG. 9 is a diagram illustrating a display example of a microscope image according to the second embodiment.

FIG. 10 is a diagram for explaining a method of dividing a measurement range into a plurality of strips and scanning.

[Explanation of symbols]

 DESCRIPTION OF SYMBOLS 1 ... Laser light source 2 ... Dichroic mirror 3 ... Galvano mirror 4 ... Pupil projection lens 5 ... Optical path switching mirror 6 ... Objective lens 7 ... Sample 8 ... Condenser lens 9 ... Beam splitter 10 ... Ring slit 11 ... Photodiode 12 ... Transmission illumination light source DESCRIPTION OF SYMBOLS 13 ... Barrier filter 14 ... Condenser lens 15, 15a-15c ... Photomultiplier tube (PMT) 16 ... Microscope observation optical system 16a ... CCD camera 17 ... Scanning stage 18 ... Spot projection lens 19 ... Epi-illumination light source 20a-20d ... Signal Processing circuit 22 Computer 22a CPU 23 Scan control circuit 24 Body control unit 32 CPU bus 33 CPU 36 GPIB interface control circuit 37 RCU 38 Input / output circuit 39, 40 Stepping motors 41, 42 Stepping Motor drive Moving circuit 45 Waveform generating circuit 46 D / A converter 47 GPIB board 48a, 48b Memory board 49 Video board 50 Monitor

Claims (3)

[Claims] 1. A measurement range on a cell population sample is divided into a plurality of strips to form a plurality of strips, a laser beam is two-dimensionally scanned for each of these strips, and optically scanned image data from the cell population sample is captured. Obtaining data of individual cells in the cell population by image processing on the scanned image data;
A cytometer for displaying as a statistical graph, display means for displaying a microscope image of the cell population specimen, coordinate conversion means for converting vertex coordinates of the plurality of strips to coordinates on the microscope image, and the coordinate conversion means Controlling means for displaying the strip subjected to the coordinate conversion on the microscope image of the display means. 2. The cytometer according to claim 1, wherein the control means displays a region on the microscope image that is outside the strip so as to be identifiable. 3. The cytometer according to claim 2, wherein the display of the area outside the strip on the microscope image is a hatch pattern or a colored display.