JP4048265B2 - Single-cell long-term observation device - Google Patents

Description

[0001]
BACKGROUND OF THE INVENTION
The invention of this application relates to a one-cell long-term observation apparatus. More specifically, the invention of this application relates to a single cell that can be cultured in units of one cell in the state of a specific cell in a biotechnology research field using microorganisms or cells, and can be continuously observed and measured. The present invention relates to an observation apparatus.
[0002]
[Prior art]
In the conventional fields of biology, medicine, and pharmacy, to observe changes in cell status or cell response to drugs, the average value of the cell population is as if it were a single cell characteristic. I have observed it. In reality, however, cells rarely have a synchronized cell cycle in the population, and each cell expresses a protein in a different cycle. In order to solve these problems, techniques such as synchronized culture have been devised, but since the origin of the cultured cells is not from the same single cell, the difference in the genes of each of the derived cells before culture is a protein. Differences in expression may occur, and when analyzing the results of responses to stimuli, whether the fluctuations are derived from the response fluctuations universally possessed by the cell response mechanism itself ( In other words, it was difficult to clarify whether the fluctuation originated from the difference in genetic information. For the same reason, cell lines are generally not completely cultured from a single cell, so it has been difficult to clarify whether the reproducibility of responses to stimuli fluctuates due to differences in the genes of each cell. Further, there are two types of stimulation (signal) for cells: those given by the amount of signal substances, nutrients and dissolved gas contained in the solution around the cells, and those by physical contact with other cells. Conventionally, when observing cells in a biotechnology research field, a part of a group of cells cultured in a large incubator has been temporarily removed from the incubator and set in a microscope for observation. Or the whole microscope was enclosed with the plastic container, temperature was controlled, and the microscope observation was performed, managing a carbon dioxide concentration and humidity using another small container in it. At this time, there are many inventions as a method of making the solution conditions constant by exchanging an old culture solution and a fresh culture solution while culturing cells. For example, in the method disclosed in Japanese Patent Laid-Open No. 10-191961, the circulating pump raises / lowers the level of the medium with respect to the substrate surface between a level higher than the upper edge height of the substrate and a level lower than the lower edge height. When the level is lowered to the low level, the medium is supplied. When the level is raised to the high level, the nutrient state is kept constant by a mechanism for discharging the medium. Further, in Japanese Patent Laid-Open No. 8-172956, an introduction tube for introducing a new medium into the culture vessel, a discharge tube for discharging the culture medium in the culture vessel to the outside, a gas portion of the culture vessel, and a pump are provided in the culture vessel. Each end of the trachea communicating with each other is inserted, and a filter is provided in each of the introduction tube, the discharge tube, and the trachea to prevent the invasion of bacteria into the culture container, and the nutrient state of the culture tank is kept constant. It is configured. However, in any of the inventions, there is no known example of culturing while controlling the solution environment of cultured cells and the physical contact between the cells. Therefore, the inventors of this application have solved these problems, newly selected only one specific cell, cultured the cell as a cell line, and a cell solution when observing the cell. A technology for controlling the environmental conditions and controlling the cell concentration in the container to be constant, and a technology for culturing and observing the cells while identifying interacting cells were invented, and the application was filed as Japanese Patent Application No. 2000-356827.
[0003]
[Problems to be solved by the invention]
However, the invention filed by the above-mentioned inventors made great progress toward solving the problems of the prior art, but means for continuously observing and measuring a large number of microchambers built on a substrate. There has not yet been sufficient investigation. For example, it is not a problem if only one microchamber is continuously observed, but when a large number of microchamber arrays constructed on a substrate are optically observed continuously, the focal range is Since the observation position must be moved while performing observation at a narrow high magnification, a number of problems arise. For example, (i) If the stage cannot be completely aligned and fixed in the X-axis and Y-axis directions, the microchamber array may be lost due to the stage movement, and (ii) the substrate may be distorted slightly. The micro that should have been in the focal plane of the microscope when the position to be observed was moved by moving the stage due to the minute change in the height of the substrate surface caused by the minute tilt of the substrate fixed on the microscope stage The chamber position may be out of focus and the focus may be blurred depending on the observation position, and (iii) biological samples will be damaged if white light is applied for a long time for observation. .
[0004]
Therefore, the invention of this application solves the above-mentioned problems, and can continuously observe and measure a plurality of microchamber arrays arranged in an arbitrary direction on the microchamber array substrate, It is an object of the present invention to provide an improved new one-cell long-term observation apparatus that does not cause damage.
[0005]
[Means for Solving the Problems]
In order to solve the above-mentioned problems, the invention of this application firstly, a plurality of micro chambers arranged in an arbitrary direction on the micro chamber array substrate are moved continuously in accordance with the arrangement direction and continuously. Means for measuring two axis directions of the XY stage for fixing the substrate and an angle formed by the microchamber array on the substrate, and means for moving the two axes of the XY stage in accordance with this angle in order to measure And a one-cell long-term observation device having a means for tilting a camera for observation according to this angle.
[0006]
In addition, the invention of this application is, secondly, in order to correct the focal position shift caused by the unevenness of the substrate, means for microscopic measurement of the microchamber using phase difference method or differential interference method, and measurement Means for measuring the luminance distribution of the shape of the microchamber and means for moving the focal position of the objective lens to a position where the inclination of the luminance distribution is steepest. A single-cell long-term observation device, and thirdly, means for limiting the wavelength of the light source for optical observation to a specific wavelength band in order to prevent the biological sample from being damaged by the observation light, Provided is a one-cell long-term observation apparatus having means for irradiating light only for a minimum time necessary for measurement.
[0007]
DETAILED DESCRIPTION OF THE INVENTION
The invention of this application has the features as described above, and an embodiment thereof will be described below.
[0008]
First, FIG. 1 schematically shows an example of the system configuration of the one-cell long-term observation apparatus of the invention of this application. Reference numeral 101 in the figure denotes a light source of a phase contrast microscope or a differential interference microscope, and a halogen lamp is generally used. Reference numeral 102 denotes a band-pass filter that transmits only light having a specific wavelength from light from a light source for observation of a stereoscopic microscope such as a phase difference. For example, when a sample such as a cell is used, damage to the sample can be prevented by using a narrow band near a wavelength of 700 nm. Reference numeral 103 denotes a shutter, which has a function of blocking light irradiation while image measurement is not being performed, such as when the XY stage 105 is moved. A condenser lens 104 introduces a phase difference ring for phase difference observation, and introduces a polarizer for differential interference observation. A substrate 100 on which a microchamber array is processed is placed on the XY stage 105, and the XY stage is moved by a driving device 119 to observe and measure different microchambers on the substrate. To be able to. The presence state of the cells in the micro chamber is observed with the objective lens 106. The focal position of the objective lens 106 can be moved by the driving device 107. For fluorescence observation, control is performed such that light from the light source 108 is transmitted through only the excitation light wavelength by the band-pass filter 109 and the sample is irradiated with the excitation light only when observed by the shutter 110. The excitation light that has passed through the shutter 110 is irradiated onto the sample by the dichroic mirror 111. At this time, the objective lens observes a phase difference image or differential interference image of the sample by the light transmitted from the light source 101 and a fluorescent image emitted from the sample by the excitation light from the light source 108. Only the phase contrast microscope image or the differential interference microscope image in the flow path is observed by the camera 115 by the dichroic mirror 112 and the band pass filter 114 that reflect light having the same wavelength as that transmitted through the band pass filter 102. The light receiving surface of this camera can be rotated according to the inclination of the microchamber fixed on the XY stage, and can be made to coincide with the inclination of the microchamber. On the other hand, the fluorescent image can be observed with the camera 117 by selectively transmitting only the wavelength band of the fluorescence observation by the mirror 113 and the band pass filter 116 of the light passing through the objective lens. Similarly to the camera 115, the light receiving surface of the camera 117 can be rotated in accordance with the inclination of the microchamber to match the inclination of the microchamber. The phase difference image or differential interference image taken by the camera 115 is analyzed by the image processing unit 118, and for example, detection of the tilt of the microchamber based on pattern matching, control of the amount of movement of the XY stage, or the light receiving surfaces of the cameras 115 and 117 The rotation amount of the lens, the height control of the objective lens 106, the focusing, etc. can be performed.
[0009]
FIG. 2 is a diagram showing an example of the arrangement of a microchamber array that can actually be used. In the example of FIG. 2, micro chambers 202 are periodically arranged on a substrate 201. In actual observation, the inside of the cell culture container 203 in each microchamber is observed at a high magnification. Here, when using 100 times (numerical aperture 1.35) which is the maximum magnification of the optical microscope, it is generally desirable that the thickness of the substrate in contact with the objective lens is 0.2 mm or less.
[0010]
FIG. 3 is a diagram for explaining an example of a microchamber observation procedure. In general, when a substrate is fixed on an XY stage, it is difficult to completely control the position and angle because a thin substrate is fixed even if a jig is devised. Therefore, a procedure for sequentially measuring the microchamber array on the substrate that is substantially aligned with the position and angle will be described. The microchamber is formed at an angle θ formed between the two movement axes of the XY stage, the X axis 311 and the Y axis 312. First, the observation region 301 recorded on the light-receiving surface of the camera by the high-magnification objective lens is rotated by θ so that all microchambers having a size that makes the best use of the visual field can be viewed. Can be observed inside. After observing the field 301 of the microchamber, it is moved to the field 302 of the adjacent microchamber by the movement amount 305. At this time, the movement amount L is a value used in advance as the arrangement period of the microchamber array at the time of manufacture. Then, the movement amount X1 of the stage in the X direction and the movement amount Y1 in the Y direction are respectively expressed by the following equations:
X1 = Lcosθ
Y1 = Lsinθ
The movements in the X-axis and Y-axis directions are interlocked while maintaining this relationship.
[0011]
On the other hand, in order to change the observation direction by 90 degrees and move from the visual field 303 to the visual field 304 in the direction of 306 and observe the microchamber in the next lower row, the amount of movement X2 of the stage in the X direction and the Y axis direction The movement amount Y2 of the movement moves in conjunction with each other while maintaining the relationship of the following equation.
[0012]
X2 = Lsinθ
Y2 = Lcosθ
FIG. 4 (1) shows a phase contrast microscopic image of the microchamber, (2) its AA cross-sectional view, and (3) a luminance distribution diagram on the AA cross-section. In the phase contrast micrograph of the micro chamber 401 in FIG. 4A, a schematic diagram of the AA cross section is as shown in FIG. 4B, and the structure 403 is in the shape of the micro chamber on the substrate 402. Has been added. Here, when observing the shape in the structure in water, using a stereomicroscope may be difficult to observe depending on the thickness of the structure and the difference in absorption from water. This is because the absorption of light depends on the thickness of the object through which the light passes, and is sufficient for light having a thickness of about 1 micrometer, such as a microfabricated product. This is because the contrast cannot be obtained. In addition, substances with high water content such as gelatin and agarose have almost the same refractive index and absorption as water, and it is difficult to obtain sufficient contrast. Therefore, it is difficult to use a conventional image focusing mechanism that obtains contrast based on the differential processing of the entity image. As can be seen from the luminance distribution diagram of the AA cross section of the phase contrast microscope image of FIG. 4 (3), the luminance changes greatly at the boundary surface of the structure, and the luminance 408 on the structure and the substrate (single) It can be seen that the average value of the position luminances 409 is substantially the same. However. Here, line 404 is the luminance distribution of the AA cross section, line 405 is the maximum luminance, 406 is the minimum luminance, and 407 is the luminance at the position of the midpoint. The slope of the luminance distribution is the steepest distribution when the focus is best, and becomes a gentle slope as the focus is shifted.
[0013]
FIG. 5 is a diagram showing the luminance distribution on the substrate in more detail. The spatial distribution of luminance is as shown by a line 501, and the width 505 or 506 of the luminance distribution takes the minimum value when the microscope image is in focus at the middle line 504 of the highest luminance 503 and the lowest luminance 502. For example, by measuring the widths 505 and 506 by image processing and selecting a place where the value that changes with the movement of the objective lens becomes a minimum, it is possible to focus on the steepest position of the luminance distribution described above. . In this embodiment, a method of focusing by focusing on the width of the luminance middle line of the luminance distribution of the phase contrast microscope image has been shown. However, the differential interference image can be similarly focused. It is also possible to adjust the focus position by directly measuring the slope of the luminance curve.
[0014]
【The invention's effect】
As described in detail above, the invention of this application makes it possible to continuously observe a plurality of microchambers on a substrate on which minute microchambers are arranged.
[Brief description of the drawings]
FIG. 1 is a schematic diagram showing an example of the system configuration of a one-cell long-term observation apparatus of the invention of this application.
FIG. 2 is a diagram showing an example of an arrangement of microchamber arrays that can actually be used in the invention of this application.
FIG. 3 is a diagram illustrating an example of a microchamber observation procedure according to the invention of this application.
FIGS. 4A and 4B are (1) a phase contrast microscopic image of a microchamber, (2) a sectional view taken along the line AA, and (3) a luminance distribution diagram taken along the section AA.
FIG. 5 is a diagram illustrating a luminance distribution on a substrate in more detail.
[Explanation of symbols]
100, 201, 402 Substrate 101, 108 Light source 102, 109, 114, 116 Band pass filter 103, 110 Shutter 104 Condenser lens 105 XY stage 106 Objective lens 107, 119 Drive device 111, 112 Dichroic mirror 113 Mirror 115, 117 Camera 118 Image processing analysis unit 202, 401 Micro chamber 203 Cell culture vessel 301, 302, 303, 304 Micro chamber observation region 305, 306 XY stage movement amount 311, 312 XY stage movement axis 403 Structure 404, 501 A-A Cross-sectional luminance distribution curves 405 and 503 Maximum luminance 406 and 502 Minimum luminance 407 and 504 Intermediate value between maximum luminance and minimum luminance 408 Structure luminance 409 Substrate luminance 505 and 506 Maximum luminance and minimum luminance Width of intermediate value of degree

Claims (3)

In order to continuously measure a plurality of microchambers arranged in an arbitrary direction on a microchamber array substrate in accordance with the arrangement direction, two axial directions of an XY stage for fixing the substrate and on the substrate Characterized in that it has means for measuring the angle formed by the microchamber array, means for moving the two axes of the XY stage in conjunction with this angle, and means for tilting the camera for observation in accordance with this angle. One-cell long-term observation device. In order to correct the deviation of the focal position caused by the unevenness of the substrate, means for microscopically measuring the microchamber using the phase difference method or differential interference method, means for measuring the luminance distribution of the shape of the measured microchamber, 2. The one-cell long-term observation apparatus according to claim 1, further comprising means for moving the focal position of the objective lens to a position where the gradient of the luminance distribution is steepest. In order to prevent damage to the biological sample due to the observation light, it has a means to limit the wavelength of the light source for optical observation to a specific wavelength band, and irradiates light only for the minimum time necessary for measurement The single-cell long-term observation apparatus according to claim 1 or 2, further comprising:

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