JPS629311B2 - - Google Patents

Description

[Detailed description of the invention]

The present invention relates to an apparatus that can accurately calculate living cells from a cultured living cell specimen in a very short period of time and without requiring special skills. This calculated value has a smaller coefficient of variation and is more accurate than the conventional artificial cell count calculation value. According to the device of the present invention, it is possible to recognize not only the number of cells but also the shape of the cells. There is an urgent need in various fields to establish the ability to evaluate biological properties such as toxicity and tissue adhesion of biological repair materials and multiphasic biomedical materials, and the tissue culture methods that serve as the basis for this are also rapidly and highly efficient. Accuracy is required. The tissue culture method involves cultivating various types of living cells while testing the harmful effects of the foreign material on living tissues.It is a method to examine how many living cells are killed by the foreign material and what kind of cell behavior occurs when the cells die. However, conventional tissue culture methods have not always been sufficiently rapid and accurate for the following reasons. In other words, the commonly used nuclear number calculation method (NC
In this method, a foreign substance is mixed into cultured living cells and samples are taken out every predetermined number of days to calculate the number of cell nuclei. The number of nuclei per unit volume is read out by spreading the sample on a calculation plate with the cells killed and cell activity fixed.One set of samples is paired with at least three test tubes, and a control sample ( Similarly, a set of 3 (no foreign matter) is used, and since a large number of such sets must be prepared for each period of days, firstly, the experimental system becomes large-scale, and secondly, it is difficult to calculate the nucleus on a calculation plate. However, it is difficult to distinguish between actual nuclei and foreign substances such as dirt, dust, bacteria, etc., and considerable skill is required.There are many steps from staining and fixing the specimen to the calculation plate, which results in the accumulation of human errors. Problems include the fact that the error reaches a level that cannot be ignored as a mass. The present invention has been made in view of the above, and includes (a) simplification of the experimental system, (b) acceleration of calculation speed, (c) elimination of human error, (d) elimination of special skills, and (e) information A preferred embodiment will be described in detail below with reference to the drawings. In the drawings, Fig. 1 is a flowchart of the apparatus of the present invention, and Fig. 2 is an image processing method. A waveform diagram showing the principle of analysis, and FIG. 3 is a diagram explaining the principle of the self-focusing mechanism of the microscope. As shown in FIG.
and an inverted phase contrast microscope 2 that takes a close-up photo of this specimen 1.
A TV camera 3 captures a microscopic image of the living cell specimen 1 through the microscope 2, and the obtained video signal is image scanned to determine the number of living cells based on the optical density of the living cells in the image. This is an automatic calculation device for the number of cultured living cells, which is composed of an image analysis device 4 that calculates , and a computer 6 that processes digital analysis signals from this device 4 . As described above, the apparatus of the present invention attempts to recognize the number of cells and their morphology by scanning the microscopic image of the living cell specimen 1 through the TV camera 3 and performing image analysis to recognize the number of cells and their morphology. The test subject is a live specimen rather than an analysed state; automatic judgment is left to the image analysis device rather than human visual judgment; image formation, statistical processing, and chart creation. It is noteworthy that everything is fully automated. The present invention will be explained in further detail below. (i) Preparation of live cell specimen 1: The cell culture itself is the same as the conventional method, but it should be noted that when calculating the number of cells, the cells are kept alive without being killed and used as a living specimen without staining. be. That is, culture involves culturing normal living cells (primary culture cells and cell lines such as L, Hella, T6L, etc.) in a normal culture solution (MEM culture solution, etc.) for a certain period of time. Thereafter, the cells are transferred to a multi-dish (a dish having a large number of independent holes) as a culture dish, cultured for a period of time, and then subjected to the apparatus of the present invention. (ii) Inverted phase-contrast microscope 2: A known microscope is used that can microscopically view the live specimen being cultured from the bottom of the culture dish (multi-dish) in each hole.The focusing can be done manually, but self-focusing is also possible. It is better to have a scrap mechanism. One of these mechanisms was developed by the present inventor in conjunction with the present invention, and its principle will be outlined based on FIG. The sample stage 21 can be moved up and down by a step motor 22, a timing belt 26, and a pulley 23. This step motor 22 is connected to a microprocessor element 24 built in the image analysis device 4, and the objective lens When the specimen 1 (each hole of the multi-dish) on the specimen stage 21 is taken close-up from below from the cylinder 25, the point where the optical contrast is strongest in the specimen image scanning (the point where the wave height is the steepest in the waveform) A signal is sent from the microprocessor 24 to the step motor 22 to automatically focus and stop the image. The above description is an example in which the sample stage 21 is moved forward and backward relative to the objective lens barrel 25, but the sample stage 21 is fixed using the same elevating means and the objective lens barrel 25 is
It is also possible to move the sample table 21 forward and backward in the longitudinal direction, and further, it is also possible to fix the sample stage 21 and perform relative movement between the eyepiece lens 27 and/or the objective lens 28 to adjust the focus to a desired position. Such a self-focus mechanism is preferably adopted in order to contribute to full automation of inspection. (iii) TV camera 3: The TV camera 3 captures a microscopic image, and is a commercially available industrial or medical TV camera. (iv) Image analysis device 4: Scans the raw image of the living cell specimen 1 based on the digital video signal from the TV camera 3, determines live cells based on the optical density of the live cells in the image, and calculates the number of live cells. As shown in Figure 1, specimen 1 is displayed on the TV monitor 8 of this device, which has 4 units.
The raw image is displayed, and the content of the analysis given in the raw image is superimposed on the monitor 8. As shown in FIG. 2, the number of cells is determined and calculated as living cells if the waveform of horizontal synchronous scanning has a wave height and a wave width within a certain value. In the same figure, wave height △y and wave width △x
Smaller cells, i.e., ○i and ○ha, are determined to be living cells, and ○ro and ○ni are determined to be living cells (for example, garbage,
It is determined to be caused by bacteria (bacteria). This △y, △x
The value of is appropriately set from (Table 1) described later. If the analysis is based not only on the mere number of cells but also on the area ratio calculation method, a certain area area involved in the calculation is superimposed on the raw image by flashing visible light, making the content of the analysis visible. The output signal from the analyzer 4 is the AD converter 5 in the case of the figure.
is input to the computer 6 as a digital signal. Although it is technically possible to place the AD converter 5 before the analysis device 4, it is better to place it after the analyzer 4 in terms of cost. In order to observe the morphology of living cells in addition to the number of living cells, analysis that determines the shape of living cells from the length of the outer ring line among circular, triangular, polygonal, and amorphous shapes (for example, (For circles, the outer ring wire is the shortest; for polygons, the outer ring wire is the longest.) By attaching the necessary adapter, shape recognition judgment can be performed regardless of size. When some living cells die due to the presence of a foreign substance, or when some kind of abnormal activity continues, the morphology of the cells is determined by finding the cell morphology in the visible image and analyzing it. This is of deep significance. (v) Computer 6: Converts digital analysis signals from analysis device 4 to CRT
A device that processes information including image display,
Examples of such processing include quantitative conversion, graph display, and statistical processing. It goes without saying that the memory means 9, such as an ordinary floppy disk or video memory, can store and save various information. (vi) Others: As shown in Figure 1, by displaying an image of a living specimen taken by a TV camera 3 on a TV monitor 7 (including memory means), the number of cells in the image can be determined and calculated with the naked eye. It is necessary to compare the results with the results of the CRT image of the computer 6 to compare the accuracy of the calculation numbers by the device of the present invention and determine the optimal analysis ability from the example described later. If analysis capability is selected, the TV monitor 7 does not need to be installed. To carry out the apparatus of the present invention based on the above-described configuration, a cultured living cell specimen 1 is prepared, a close-up image of this is taken with an inverted phase contrast microscope 2, and an image of the living specimen is captured with a TV camera 3 to obtain an image. Analyzer 4 scans the image and interprets the cells and calculates the number of cells, and the AD-converted analysis signal is sent to CRT using a computer.
It also performs quantitative conversion, graph display, etc., and furthermore, the memory means 9 automatically records living cell image data over time and performs calculations with various analysis data to perform various displays. It becomes possible to do so. Although we were able to obtain live cell images with a magnification of 4x on the imaging surface and a magnification of 120x on TV monitors 8 and 7 using the microscope 2 and the TV camera 3, improvements in these optical systems may reduce the magnification of the image. should be further improved. Experimental examples and working examples of the apparatus of the present invention are shown below along with comparative examples. Experimental Example In order to discover the analysis ability (waveform processing) that can reduce the error in the cell count calculation value from the live cell sample image in the device of the present invention, we counted the cells visually from the cell image projected on the TV monitor 7. The threshold value of the cell number calculation value according to the present invention when the actual number of cells, the waveform processing for analysis (sensitivity), and the size of the object to clearly distinguish contaminants such as dust and bacteria from cells are varied. The results of the investigation are shown in (Table 1). The culture of living cells in this experimental example was the same as in the example. (Table 1) When the threshold value is close to 1 (least error), the wave height sensitivity is 6.0 and the wave width is 0.4, so the analysis ability (concentration waveform processing) of the analyzer 4 is △y = 6.0 , aimed at △x=0.4.

【table】

[Table] Example (a) Preparation of living cell specimen: L cells were added with 5% calf serum.
Preliminary culture was performed in MEM culture solution at 7-day intervals, and after 4 days of main culture, the cells were divided into 24-well multi-dishes, and after 2, 4, and 7 days of culture, they were applied to the apparatus of the present invention.
Note that the number of samples was 30. (b) Analysis ability: △y = 6.0, △x = 0.4 (c) Magnification: 120x on TV monitor (d) Results: Examine the rate of variation (that is, the variation in error) from the results obtained under the above conditions. The results are shown in (Table 3). Comparative example A specimen exactly the same as in the above example was prepared, and the conventional
The cell numbers obtained according to the NC method are shown in (Table 2).

【table】

[Table] As is clear from Tables 2 and 3, in the case of the NC method, the coefficient of variation was 29.3 within 2 hours from the start of the experiment.
%, 29.3% after 2 days and 12.2% after 4 days, whereas according to the device of the present invention, the coefficient of variation was 5.56% 2 hours after the start of the experiment, 2.7% after 2 days, and 2.25 after 4 days. %, which is much lower than that of the NC method, and it can be seen that the accuracy has been dramatically improved. As described above, the device of the present invention performs image analysis while displaying the microscopic image of a cultured living cell specimen taken with a TV camera to determine and calculate the number of living cells, so the calculation speed is faster. It can be done quickly, there is no human error, the experimental system can be small-scale and the calculation costs can be kept low, the processing capacity can be increased, and the degree of cell proliferation can be continuously and regularly recorded. It has excellent effects that can eliminate the problems of conventional methods, such as being able to store data, making it possible to make various comparisons and judgments based on old and new data, and being able to recognize the morphology of living cells. It is something.

[Brief explanation of the drawing]

FIG. 1 is a flowchart of the apparatus of the present invention, FIG. 2 is a waveform diagram showing the principle of image analysis, and FIG. 3 is a diagram explaining the principle of the self-focusing mechanism of the microscope. (Explanation of symbols), 1... Living cell specimen, 2... Inverted phase contrast microscope, 3... TV camera, 4... Image analysis device, 5... AD converter, 6... Computer, 7, 8... ...TV monitor, 9...memory means.

Claims (1)

[Claims] 1. A living cell specimen 1 in culture, an inverted phase contrast microscope 2 for taking a close-up photo of the specimen 1, a TV camera 3 for taking a microscopic image of the living cell specimen 1 through the microscope 2, and a TV camera 3 for taking a microscopic image of the living cell specimen 1 through the microscope 2; an image analysis device 4 that scans the image signal of the image, determines the number of living cells based on the optical density of the living cells in the image, and calculates the number of living cells; and a computer 6 that processes the digital analysis signal from this device 4. An automatic counting device for the number of viable cells in culture. 2. The apparatus according to claim 1, wherein the determination calculation of living cells by the image analysis apparatus 4 is performed based on the wave height and wave width of the concentration waveform of the horizontal scan of the image. 3 The video signal of the TV camera 3 is displayed on the TV monitor 7, and the analysis image of the image analysis device 4 is displayed as a unique image.
2. The device according to claim 1, wherein the analog analysis signal of the device 4, which is imaged on a TV monitor 8, is inputted to the computer 6 via the AD converter 5. 4. The apparatus according to claim 3, wherein the digital analysis signal from the AD converter 5 is stored and stored in the memory means 9. 5. The apparatus according to claim 1, wherein the inverted phase contrast microscope 2 includes a self-focusing mechanism.