JP2018108064A - Cell observation system - Google Patents

Abstract

PROBLEM TO BE SOLVED: To simply evaluate the quality of cells.SOLUTION: The present invention provides a cell observation system 1 comprising: a culture observation apparatus 3 which obtains an image in a culture vessel C for culturing cells, with time; a cell-analyzing part which quantitatively analyzes the culture situation of cells cultured in the culture vessel C, based on each image obtained by the culture observation apparatus 3 to perform the statistical analysis of the obtained data; and a monitor 7 which comparably displays the statistical-analysis result in the culture vessel C in a plurality of passage periods obtained by the cell-analyzing part.SELECTED DRAWING: Figure 1

Description

  The present invention relates to a cell observation system.

  Conventionally, for cell culture, each time the cells become confluent, the culture container is removed from the incubator, peeled off from the culture container, diluted, seeded in a new culture container, and cultured. It is. In general, when a cultured cell is repeatedly passaged many times, deterioration such as reduction in proliferation ability occurs, and such a deteriorated cell may affect the test result.

  Therefore, it is desirable that the cells used in the test have stable quality, and it is desirable to have some index for evaluating the quality. For example, the cultured cell evaluation apparatus described in Patent Document 1 classifies cells using the morphological characteristics of cells as an index, and evaluates cell degradation.

International Publication No. 2011/021391

However, the evaluation by the cultured cell evaluation apparatus described in Patent Document 1 requires a complicated algorithm, and there is a problem that work is complicated and time-consuming.
This invention is made | formed in view of the situation mentioned above, Comprising: It aims at providing the cell observation system which can evaluate the quality of a cell easily.

In order to achieve the above object, the present invention provides the following means.
One aspect of the present invention is an image acquisition unit that acquires images in a culture vessel for culturing cells over time, and is cultured in the culture vessel based on the images acquired by the image acquisition unit. An image analysis unit that quantitatively analyzes the culture state of the cells, a statistical analysis unit that statistically analyzes data analyzed by the image analysis unit, and the plurality of passage periods obtained by the statistical analysis unit It is a cell observation system provided with the display part which displays the statistical analysis result in a culture container so that comparison is possible.

  According to this aspect, the image acquisition unit acquires the image in the culture vessel over time, and the image analysis unit quantitatively analyzes the culture state of the cells in the culture vessel based on each image, By statistically analyzing the analyzed data by the statistical analysis unit, the change in the number of cells cultured in the culture vessel can be understood. Here, when the passage is repeated, the cells are deteriorated, and the proliferating ability of the deteriorated cells is lowered.

  In this case, the display unit displays the statistical results in the culture vessel in the plurality of passage cycles obtained by the statistical analysis unit so that they can be compared with each other, without requiring a complicated algorithm. Changes in the cell proliferation ability can be visually recognized. As a result, the quality of the cells can be evaluated with simple work and without any effort.

In the said aspect, the said display part is good also as displaying the growth curve showing the time change of the number of the said cells in each said passage period.
If the cells deteriorate due to repeated passage and the proliferation ability decreases, the ascending curve of the proliferation curve becomes gentle. By comprising in this way, the change of the proliferation ability of the cell of each passage period can be grasped | ascertained at a glance by the inclination of the proliferation curve displayed on a display part.

In the said aspect, the said display part is good also as displaying the said growth curve of the said several subculture period superimposed.
By comprising in this way, it can grasp | ascertain at a glance the difference in the proliferation ability of the cell of the several passage period to which attention is paid.

In the said aspect, it is good also as providing the comparison part which compares the time change of the number of the said cells for every said passage period, and outputs the change of the proliferation rate of the said cell.
By comprising in this way, the comparison part can grasp | ascertain easily the change of the proliferation capability of the cell for every passage period, and can save the effort which a user compares.

In the said aspect, it is good also as providing the quality evaluation part which evaluates the quality of the said cell based on the proliferation rate of the said cell in the said subculture period.
By comprising in this way, the quality evaluation part can be easily divided into the cell of the passage period which has desired quality, and the cell of the passage period which does not have desired quality.

  The cell observation system according to the present invention produces an effect that the quality of the cells can be easily evaluated.

1 is a schematic configuration diagram showing a cell observation system according to a first embodiment of the present invention. It is a longitudinal cross-sectional view which shows the culture observation apparatus of FIG. It is a block diagram of the PC main body of FIG. It is a figure which shows an example of the growth curve of each passage period displayed on the monitor in time series order. It is a figure which shows an example of the growth curve of the number of cells in one passage cycle. It is a longitudinal cross-sectional view which shows the modification of the culture observation apparatus of FIG. It is a longitudinal cross-sectional view which shows the culture observation apparatus of the cell observation system which concerns on 2nd Embodiment of this invention. It is a longitudinal cross-sectional view which shows the culture observation apparatus of the cell observation system which concerns on 3rd Embodiment of this invention. It is a top view which shows the internal structure of the culture observation apparatus of FIG. It is a partial longitudinal cross-sectional view which shows the culture observation apparatus of the cell observation system which concerns on 4th Embodiment of this invention. It is a top view which shows an example of arrangement | positioning of the LED light source in the light source part of the culture observation apparatus of FIG. It is a modification of the culture observation apparatus of FIG. 10, Comprising: It is a partial longitudinal cross-sectional view which shows the case where illumination light is restrict | limited by the light shielding member. It is an example of the light shielding member of FIG. 12, Comprising: (a) When it has a circular single opening, (b) When the radial direction position of an opening differs from (a), (c) Two openings It is a top view which shows the case where each is provided. It is another example of the light-shielding member of FIG. 12, Comprising: It is a top view which shows the case where (a) it has a fan-shaped opening part, and (b) it has a ring-shaped opening part, respectively. It is a partial longitudinal cross-sectional view which shows the other modification of the culture observation apparatus of FIG. It is a partial longitudinal cross-sectional view which shows the other modification of the culture observation apparatus of FIG. It is a partial longitudinal cross-sectional view which shows the other modification of the culture observation apparatus of FIG.

[First Embodiment]
A cell observation system according to a first embodiment of the present invention will be described below with reference to the drawings.
As shown in FIG. 1, the cell observation system 1 according to the present embodiment is acquired by a culture observation device (image acquisition unit) 3 that can culture cells A and acquire images over time, and the culture observation device 3. A PC (Personal Computer) main body 5 for processing the obtained image, and a monitor (display unit) 7 for displaying an image acquired by the culture observation apparatus 3, a processing result obtained by processing the image by the PC main body 5, and the like. I have. Reference numeral 9 denotes an incubator.

  As shown in FIG. 2, the culture observation apparatus 3 includes a base 11 on which a culture vessel C containing cells A to be cultured together with a culture solution B is mounted, a light source unit 13 provided on the base 11, an imaging unit 15, A transmission unit 17 and a control unit 19 are provided.

The culture vessel C is, for example, a cell culture flask and is made of an optically transparent material. Reference sign C1 indicates the bottom surface of the culture vessel C.
As shown in FIG. 2, the base 11 has a mounting surface 11a made of an optically transparent material that closely contacts the lower surface of the culture vessel C, and a culture vessel that is provided upright from the mounting surface 11a and mounted on the mounting surface 11a. And abutting surface 11b that closely contacts one side surface of C. Since the inside of the incubator 9 is in a humid state, the base 11 has a waterproof structure.

  The light source unit 13 is provided with a plurality of LED light sources 13a arranged on the abutting surface 11b, spaced apart from the mounting surface 11a and arranged in a direction parallel to the mounting surface 11a. The optical axis 13b of the illumination light emitted from each LED light source 13a is set to be substantially parallel to the mounting surface 11a.

  The imaging unit 15 includes a condenser lens 15a disposed below the mounting surface 11a inside the base 11, and an imaging element 15b that captures the light collected by the condenser lens 15a and acquires an image. . The imaging element 15b is also disposed on the side opposite to the mounting surface 11a with the condenser lens 15a interposed therebetween, and is disposed on the optical axis 15c of the condenser lens 15a.

The transmission part 17 transmits the image acquired by the image pick-up element 15b to the exterior by radio | wireless.
For example, the control unit 19 includes a timer (not shown), and periodically operates the light source unit 13, the imaging unit 15, and the transmission unit 17.

  As shown in FIG. 3, the PC main body 5 includes a receiving unit 21 that receives an image transmitted from the transmitting unit 17, and cells that are cultured in the culture vessel C based on the image received by the receiving unit 21. A cell analysis unit (image analysis unit, statistical analysis unit) 23 is provided for quantitatively analyzing the culture state of A and statistically analyzing the obtained data.

  The cell analysis unit 23 is configured to count the number of cells A cultured in the culture vessel C by a conventional technique. In addition, the cell analysis unit 23 plots the counted number of cells with respect to the time axis, and creates a growth curve representing the time change of the number of cells A in the culture vessel C in each passage period. ing. The period from the passage work to the next passage work is defined as one passage cycle. The growth curve of the number of cells in each passage period generated by the cell analysis unit 23 is sent to the monitor 7 as information on the number of cells A.

  The monitor 7 displays the time change (statistical analysis results) of the number of cells in the culture vessel C within a plurality of passage periods based on the information on the number of cells A sent from the cell analysis unit 23. It is supposed to be.

  For example, as shown in FIG. 4, the monitor 7 displays a growth curve of the number of cells in each passage period sent from the cell analysis unit 23 in time series. In the example shown in FIG. 4, the growth curve of each passage period obtained by repeating the passage from the first day to the 21st day with one passage period being 3 days is arranged in series along the time axis. ing.

  In addition, the monitor 7 can superimpose and display a plurality of growth curves of passage periods designated by the operator (user) among the growth curves of the passage periods displayed in time series. Yes. In the example shown in FIG. 4, the growth curve of the passage period a from the 10th day to the 12th day and the growth curve of the passage period b from the 19th day to the 21st day are superimposed and displayed. .

The operation of the cell observation system 1 according to this embodiment configured as described above will be described below.
In order to observe the culture state of the cell A using the cell observation system 1 according to this embodiment, the lower surface of the culture vessel C containing the cell A and the culture solution B to be cultured is placed on the mounting surface 11a of the base 11. The base 11 is mounted so that one side faces the abutting surface 11 b of the base 11.

  In this state, the culture observation apparatus 3 on which the culture vessel C is mounted is accommodated in the incubator 9 and disposed so that the mounting surface 11a is horizontal, so that the temperature and humidity in the incubator 9 are controlled in an environment. The culture of the cell A in the culture vessel C is started. At this time, the timer in the control unit 19 is activated to start timing.

  When the culture is started, the control unit 19 operates the light source unit 13 to turn on the LED light source 13a in accordance with a schedule set in advance according to the timing of the timer, and causes the imaging element 15b to perform imaging.

  The LED light source 13a is provided on the abutting surface 11b that abuts the side surface of the culture vessel C, and makes illumination light enter the culture vessel C from the side surface of the culture vessel C in the direction of the optical axis 13b along the bottom surface C1 of the culture vessel C. . Thereby, like the oblique illumination or the dark field illumination, the cell A that is adhered and growing on the bottom surface C1 of the culture vessel C is illuminated from the side, and a shadow of the cell A is formed.

  A part of the scattered light scattered in the cell A passes through the bottom surface C1 of the culture vessel C and the mounting surface 11a of the base 11, is condensed by the condensing lens 15a in the base 11, and is photographed by the imaging element 15b. . In the imaging unit 15, for example, light from the cell A is captured every several hours by the imaging element 15b, and an image is acquired.

  After each image is acquired, the LED light source 13a is turned off each time. Thus, by intermittently operating the light source unit 13 and the like, the temperature rise of the apparatus can be suppressed and the influence of heat on the cells A can be reduced.

  Then, the images acquired over time by the image sensor 15 b are respectively sent from the control unit 19 to the transmission unit 17 and sequentially transmitted to the outside by the transmission unit 17. Outside the incubator 9, images received from the transmitting unit 17 are sequentially received by the receiving unit 21 of the PC main body 5 and displayed on the monitor 7, whereby the culture vessel C is taken out from the incubator 9 and observed. Furthermore, the culture state of the cells A in the culture vessel C can be confirmed outside the incubator 9 without opening the door of the incubator 9. That is, there is an advantage that the troublesome confirmation work at the time of cell culture can be greatly reduced. Moreover, since it is not necessary to take out the culture container C out of the incubator 9, environmental changes (changes in temperature, pH, etc.) with respect to the cells A can be eliminated.

  The images received by the receiving unit 21 are sequentially sent to the cell analyzing unit 23, and the number of cells A cultured in the culture vessel C is sequentially counted by the cell analyzing unit 23 based on the respective images. And the cell analysis part 23 plots the counted cell number with respect to a time axis, and produces the growth curve of the cell number in the culture container C in one passage period. The growth curve of the number of cells generated by the cell analysis unit 23 is displayed on the monitor 7.

  When the grown cell A reaches a state (confluent) that spreads over one surface of the culture vessel C, the operator once takes out the culture observation device 3 from the incubator 9, dilutes the number of cells, and passes it to a new culture vessel C. Perform substitute work.

  The new culture container C after the subculture work is mounted on the base 11 and the culture observation apparatus 3 is accommodated in the incubator 9 again. Then, similarly to the cells A in the previous culture container C, the cells A are cultured in an environment in which the temperature and humidity in the incubator 9 are controlled, and an image thereof is acquired and displayed on the monitor 7. Further, the number of cells is counted by the cell analysis unit 23 using the image, and a growth curve of the number of cells in the culture container C within one passage period is created and displayed on the monitor 7. Repeat this for multiple passage cycles.

  On the monitor 7, as shown in FIG. 4, the growth curves of the number of cells in the culture vessel C within a plurality of passage periods are displayed in series along the time axis. By displaying in this way, the time change of the number of cells in the culture vessel C in each passage period can be easily compared. In addition, when cell A deteriorates due to repeated passages and the proliferation ability decreases, the growth curve rises gradually. The change in the proliferation ability of the cell A can be grasped at a glance.

  Further, when the operator designates a growth curve for any of a plurality of passage cycles, the designated growth curves for those passage cycles are superimposed on the monitor 7 and displayed. Thereby, it is possible to grasp at a glance the difference in proliferation ability of the cells A of a plurality of passage periods of interest.

In addition, the operator can evaluate the degree of deterioration of the cell A using the change in the growth curve of each passage cycle as an index. For example, the slope of each growth curve for each passage period may be calculated and compared quantitatively using that as an index. In this case, for example, as shown in FIG. 5, the number of cells at the beginning of the passage cycle (time t ₀ ) is N _0, and the number of cells at the end of the passage cycle (time t) is N _t . Then, the slope a of the growth curve can be calculated by a = (N _t −N ₀ ) / (t−t ₀ ).

Alternatively, the slopes of two arbitrary passage periods may be calculated and used as an index for quantitative comparison. Assuming that the number of cells at an arbitrary time point (time t ₁ ) is N ₁ and the number of cells at another time point (time t ₂ ) is N ₂ , the slope a of the growth curve is a = (N ₂ −N ₁ ) / (t ₂ -t ₁ ).

Alternatively, the growth rate may be calculated and compared quantitatively using it as an index. In this case, the growth rate α can be calculated by (N _t −N ₀ ) / N ₀ . The growth rate α is (N ₂ −N ₁ ) / N, where N ₁ is the number of cells at any time (time t ₁ ) and N ₂ is the number of cells at other time (time t ₂ ). ₁ can be calculated.

Moreover, the time (doubling time) required until the number of cells doubles for the proliferation curve of each passage cycle may be calculated and used as an index for quantitative comparison. In this case, the doubling time T can be calculated by (t−t ₀ ) log ₁₀ 2 / log ₁₀ (N _t / N ₀ ) or (t−t ₀ ) / log ₂ (N _t / N ₀ ). .

Alternatively, the doubling time at any two points in the passage period may be calculated and compared quantitatively using it as an index. The number of cells at an arbitrary time point (time t ₁ ) is N _1, and the cell number at another time point (time t ₂ ) is N ₂ , and the doubling time T is (t ₂ −t ₁ ) log ₁₀ It can be calculated by 2 / log ₁₀ (N ₂ / N ₁ ) or (t ₂ -t ₁ ) / log ₂ (N ₂ / N ₁ ). Here, t ₁ and t ₂ are preferably two arbitrary points in the logarithmic growth phase.

  As described above, according to the cell observation system 1 according to the present embodiment, the monitor 7 displays the change in time of the number of cells in the culture vessel C within a plurality of passage periods in a comparative manner. A change in the proliferation ability of the cell A during each passage period can be visually recognized without requiring a simple algorithm. Thereby, the quality of the cell A can be evaluated by a simple operation without taking time and effort.

  Further, since the imaging unit 15 captures the scattered light transmitted through the bottom surface C1 of the culture container C among the scattered light in the cultured cells A, the culture container C is used when the culture is performed in a high temperature and humidity environment. A clear image can be obtained without being affected by water droplets formed by dew condensation on the upper surface.

  Moreover, in this embodiment, since the LED light source 13a is used as the light source part 13, there exists an advantage that heat_generation | fever can be suppressed, the influence on the cell A can be reduced and power consumption can also be suppressed.

  In the present embodiment, a cell growth curve may be created by calculating a cell density (the number of cells per unit area) instead of the number of cells and plotting it with respect to the time axis. Alternatively, the cell growth curve may be created by calculating the area occupied by the cells A in the culture vessel C instead of the number of cells, and plotting it with respect to the time axis.

  In the present embodiment, for example, the cell analysis unit 23 may function as a comparison unit that compares changes in the number of cells for each passage period and outputs changes in the proliferation rate of the cells A. . By doing in this way, the operator can grasp | ascertain easily the change of the proliferation ability of the cell A for every passage period, and can save the effort which an operator himself compares.

  In the present embodiment, for example, the cell analysis unit 23 may function as a quality evaluation unit that evaluates the quality of the cell A based on the proliferation rate of the cell A in each passage cycle. By doing in this way, the cell analysis part 23 can evaluate easily the cell A which has desired quality.

  In this case, for example, when the growth rate of the cells A is larger than a predetermined threshold at the end of the culture process, the cells A in the culture container C may be determined to have good quality. In addition, when the cell A deteriorates and the proliferation ability decreases, the doubling time of the number of cells becomes longer. Therefore, when the doubling time until the predetermined number of cells is reached is shorter than a predetermined threshold value, The cell A may be determined to have good quality.

  In the present embodiment, the illumination light from the light source unit 13 is incident in the culture vessel C in the horizontal direction along the optical axis 13b parallel to the bottom surface C1 of the culture vessel C. The illumination light from the light source unit 13 may enter the culture vessel C at an angle of ± 30 ° or less with respect to the horizontal direction. Even if such an angle is adopted, a shadow similar to that of dark field illumination or oblique illumination can be formed on the cell A, and a three-dimensional image can be taken.

  Further, in the present embodiment, as shown in FIG. 6, a partial image of the bottom surface C1 is acquired by making the optical axis 15c of the condenser lens 15a orthogonal to the bottom surface C1 of the culture vessel C. Good. In this case, for example, the optical axis 15c of the condenser lens 15a may be bent by one or more mirrors 15d. Although the certainty of the determination of the culture state is lower than when photographing the entire bottom surface C1 of the culture vessel C or a relatively wide part of the bottom surface C1, the culture state may be estimated from the image of this partial region. .

In the present embodiment, the control unit 19 includes a timer and periodically activates the light source unit 13 and the like. Instead, a receiving unit (not shown) is connected to the control unit 19. Then, a command signal from the outside of the incubator 9 may be received, and the control unit 19 may drive the light source unit 13 or the like according to the command signal. The light source unit 13 may be turned on / off or photographed by remote operation according to instructions input in advance by the operator, or the light source unit 13 may be turned on / off or photographed by remote operation at an arbitrary timing.
Transmission and reception of image signals and command signals may be performed wirelessly or may be performed by wire.

Moreover, in this embodiment, although the aspect which displays a cell growth curve was shown, you may display as a straight line by plotting with respect to a logarithmic axis. By doing in this way, the part which corresponds to the logarithmic growth phase in the passage period is displayed as a straight line, and the cell proliferation ability can be grasped and compared more intuitively.
Further, instead of the cell growth curve, the change in the number of cells may be displayed as a bar graph or may be displayed as discontinuous dots (points).

[Second Embodiment]
Next, a cell observation system according to a second embodiment of the present invention will be described below with reference to the drawings.
As shown in FIG. 7, the cell observation system 31 according to the present embodiment is different from the cell observation system 1 according to the first embodiment in the imaging unit 33.
In the description of the present embodiment, portions having the same configuration as those of the cell observation system 1 according to the first embodiment described above are denoted by the same reference numerals and description thereof is omitted.

  The imaging unit 33 is disposed below the mounting surface 11a of the base 11 and a microlens array 35 including a plurality of microlenses 35a arranged on a plane substantially parallel to the mounting surface 11a, and further below the microlens array 35. The image pickup device 35b is provided. The microlens 35a of the microlens array 35 is arranged corresponding to each pixel of the image sensor 35b.

  The focal length of each microlens 35a is set larger than the thickness dimension obtained by adding the thickness dimension of the transparent member constituting the mounting surface 11a and the thickness dimension of the bottom surface C1 of the culture vessel C mounted on the mounting surface 11a. Has been. Each microlens 35a is arranged below the mounting surface 11a with the focal position aligned with the cell A adhered to the bottom surface C1 of the culture vessel C, thereby projecting an image of the cell A onto the imaging surface of the image sensor 35b. Can be done.

  It is not always necessary for the imaging device 35b to capture an image of the entire bottom surface C1 of the culture vessel C. An image is acquired for an arbitrary partial region such as a central portion where the cell A is highly likely to exist. Based on this, the culture state may be estimated.

[Third Embodiment]
Next, a cell observation system according to a third embodiment of the present invention will be described below with reference to the drawings.
As shown in FIGS. 8 and 9, the cell observation system 41 according to the present embodiment includes a focus adjustment mechanism 43 that moves the condenser lens 15 a of the imaging unit 15 in a direction along the optical axis 15 c, and the imaging unit 15. This is different from the first embodiment in that it includes a moving mechanism 45 that moves two-dimensionally in a direction along the mounting surface 11a.
In the description of the present embodiment, portions having the same configuration as those of the cell observation system 1 according to the first embodiment described above are denoted by the same reference numerals and description thereof is omitted.

  The focus adjustment mechanism 43 is a linear motion mechanism including a motor 43a and a ball screw 43b, for example. The focus adjustment mechanism 43 is fixed to the nut 43c by rotating the ball screw 43b by the operation of the motor 43a and linearly moving the nut 43c engaged with the ball screw 43b along the optical axis 15c of the condenser lens 15a. The condenser lens 15a can be moved along the optical axis 15c.

  As shown in FIG. 9, the moving mechanism 45 includes two linear motion mechanisms 47 and 49 arranged orthogonally. The first linear motion mechanism 47 includes a guide rail 47a fixed to the base 11, a slider 47b supported so as to be movable in the first horizontal direction X along the guide rail 47a, and a drive mechanism 47c for moving the slider 47b. It has. The drive mechanism 47c includes a motor 47d and a ball screw 47e.

  The second linear motion mechanism 49 includes a guide rail 49a fixed to the slider 47b of the first linear motion mechanism 47, a slider 49b supported so as to be movable in the second horizontal direction Y along the guide rail 49a, And a drive mechanism 49c for moving the slider 49b. The drive mechanism 49c includes a motor 49d and a ball screw 49e.

  The operator confirms whether or not the focus of the condenser lens 15a properly matches the cell A based on the image acquired by the image sensor 15b. A command signal for operating in this direction is input, and the focus adjustment mechanism 43 is operated by the control unit 19.

  When the ball screw 43b is rotated by the rotation of the motor 43a, the nut 43c moves in one of the horizontal directions according to the rotation direction of the ball screw 43b, and the condenser lens 15a fixed to the nut 43c moves in the horizontal direction. As a result of the movement, the focal position of the condenser lens 15a is moved in the vertical direction.

  That is, when the condenser lens 15a is moved in the direction approaching the mirror 15d, the focal position is raised, and when the condenser lens 15a is moved away from the mirror 15d, the focal position is lowered. . Thereby, it is possible to obtain a clear image by adjusting the focal position to an appropriate position.

  When the operator wants to observe a different position, the operator inputs a command signal for moving the moving mechanism 45 in either direction, and the control unit 19 activates the moving mechanism 45. By moving the imaging unit 15 along the second linear movement mechanism 49, the observation position is moved in the horizontal direction Y, and the second linear movement mechanism 49 and the imaging unit 15 are moved along the first linear movement mechanism 47. As a result, the observation position is moved in the horizontal direction X. Thereby, the observation position can be adjusted two-dimensionally.

  You may arrange | position the light source part 13 of this embodiment in the position illuminated from above the culture container C. FIG. Moreover, you may arrange | position the light source part 13 in the position illuminated from the side surface of the culture container C. FIG. Further, the light source unit 13 may be disposed at a position where the light source unit 13 is illuminated from the bottom surface of the culture vessel C, and the light source unit 13 may be moved together with the imaging unit 15 by the moving mechanism 45.

[Fourth Embodiment]
Although the said 1st Embodiment and 2nd Embodiment showed the aspect which irradiates illumination light from the side with respect to the cell A, the aspect which irradiates illumination light from the downward direction of the cell A is demonstrated below.
In the cell observation system 51 according to the present embodiment, as shown in FIG. 10, the culture observation apparatus 3 is disposed below a stage 53 on which a culture vessel C containing cells A is placed, and below the stage 53. An objective lens 55 that collects light transmitted from above 53, a photographing optical system 57 that captures light transmitted through the cell A, and a radially outer side of the objective lens 55, and passes through the stage 53. And a light source unit 59 for emitting illumination light upward.

An optically transparent material, for example, a glass plate 53a is disposed on the stage 53 so as to cover the objective lens 55 and the light source unit 59, and the culture vessel C is placed on the upper surface of the glass plate 53a. It has become.
The bottom surface of the culture vessel C is indicated by reference symbol C1, and the top plate of the culture vessel C is indicated by reference symbol C2.

  As shown in FIGS. 10 and 11, the light source unit 59 includes a plurality of LED light sources (light sources) 61 arranged around the objective lens 55 at intervals in the circumferential direction and the radial direction, and the LED light sources 61. Correspondingly arranged, a plurality of collimating lenses 63 for making the illumination light generated in each LED light source 61 substantially parallel light, and a diffusion plate 65 for diffusing the illumination light made substantially parallel light by the collimating lens 63 are provided. Yes.

The light source unit 59 can turn on specific LED light sources 61 independently (FIGS. 10 and 11 show the LED light sources 61 that are turned on by hatching).
That is, by turning on only the LED light sources 61 at different positions in the radial direction of the objective lens 55, the glass plate 53a and the bottom surface C1 of the culture vessel C are transmitted from the bottom to the top as shown by the solid line in FIG. After that, the angle of the illumination light reflected on the inner surface of the top plate C2 of the culture vessel C and transmitted through the cell A, the bottom surface C1 of the culture vessel C and the glass plate 53a from obliquely above and incident on the objective lens 55 is indicated by a broken line It can be switched as shown in.

  In addition, by lighting only the LED light source 61 at a specific position in the circumferential direction of the objective lens 55, the cell A can be illuminated only from a specific direction in the circumferential direction. Further, as shown in FIG. 11, by turning on the LED light source 61 arranged in two or more directions in the circumferential direction of the objective lens 55, in particular, in an axially symmetric direction with respect to the optical axis S of the objective lens 55. The cell A can be irradiated with illumination light with reduced illumination unevenness.

An observation method using the cell observation system 51 according to the present embodiment configured as described above will be described below.
In order to observe the transparent cell A using the cell observation system 51 according to the present embodiment, as shown in FIG. 10, the culture vessel C is adhered to the bottom surface C1 of the culture vessel C as shown in FIG. Is placed on the glass plate 53a of the stage 53 so that its bottom surface C1 is on the lower side.

  In this state, any one of the LED light sources 61 of the light source unit 59 is operated to generate illumination light. The illumination light generated in the LED light source 61 is made into substantially parallel light by the collimating lens 63 arranged corresponding to the LED light source 61 and diffused by the diffusion plate 65, and the bottom surface of the glass plate 53a and the culture vessel C. The light passes through C1 from the bottom to the top, is reflected from the inner surface of the top plate C2 of the culture vessel C, and is irradiated obliquely from above to the cell A.

  Of the illumination light irradiated to the cell A, the transmitted light of the illumination light that has passed through the cell A passes through the bottom surface C1 of the culture vessel C and the glass plate 53a from the top to the bottom, and enters the objective lens 55. At this time, the illumination light is refracted and scattered by the shape and refractive index of the cell A, or is attenuated by the transmittance of the cell A to become transmitted light on which information on the cell A is placed. The light is condensed and photographed by an image sensor (not shown).

  As described above, according to the cell observation system 51 according to the present embodiment, the imaging optical system 57 including the light source unit 59 and the objective lens 55 is disposed below the cell A. Compared with the transmitted light observation device in which the light source unit and the photographing optical system are arranged in the light source unit 59, the light source unit 59 and the photographing optical system 57 are concentrated only on one side of the cell A, and the device can be thinned. There is. In addition, the thinned cell observation system 51 has an advantage that a subject such as a cell can be observed without labeling by photographing the transmitted light.

  The illumination light from the light source unit 59 is emitted from the outside in the radial direction of the objective lens 55 and reflected on the inner surface of the top plate C2 of the culture vessel C, so that the cell A is irradiated obliquely from above and the objective lens. Since the light is condensed by 55, by appropriately setting the incident angle to the cell A, light and darkness can be formed in the image of the cell A, and an easy-to-see image can be obtained even for a transparent subject such as a cell. There is an advantage that you can.

  Further, in the present embodiment, the light source unit 59 includes a plurality of LED light sources 61 that are arranged in the radial direction around the objective lens 55 and can be turned on independently. Moreover, the irradiation angle of the illumination light incident on the cell A can be changed by changing the radial position of the LED light source 61 to be lit. Accordingly, when the incident angle is smaller than the capture angle of the objective lens 55, bright field illumination with less illumination unevenness is obtained, and when the incident angle is greater than the capture angle of the objective lens 55, dark field illumination in which the fine structure is emphasized. In addition, in the case of an incident angle equivalent to the capture angle of the objective lens 55, oblique illumination in which the cell A can be viewed three-dimensionally can be obtained.

  In the present embodiment, the light source unit 59 includes a plurality of LED light sources 61 that are arranged in the circumferential direction around the objective lens 55 and can be turned on independently. By changing the position, the irradiation direction of the illumination light incident on the cell A can be changed. Thereby, the direction of the shadow of the image of the formed cell A can be changed, and the appearance can be changed.

  Further, as shown in FIG. 11, lighting unevenness is achieved by simultaneously lighting a plurality of LED light sources 61 at different positions in the circumferential direction, in particular, simultaneously lighting a plurality of LED light sources 61 arranged in an axial symmetry. There is an advantage that an image of the cell A with less unevenness can be acquired.

  Further, in the present embodiment, since the diffusion plate 65 is provided corresponding to each LED light source 61, the illumination light emitted from the LED light source 61 is uniformly diffused and illumination with uniform intensity with little illumination unevenness. The cell A can be irradiated with light.

  In the present embodiment, the plurality of LED light sources 61 are arranged in an array and are turned on independently to switch the illumination angle, illumination direction, and the like of the illumination light. 12 to FIG. 14, the light source unit 59 includes a light source 67 disposed around the objective lens 55, and a light shielding member 69 disposed above the light source 67 and shielding illumination light from the light source 67. You may decide to prepare.

  That is, the light-shielding member 69 has an opening 69a that opens in a part in the circumferential direction or a part in the radial direction, and a transmission that transmits the light that is reflected by the inner surface of the top plate C2 of the culture vessel C and transmitted through the cell A The hole 69b is provided, and by replacing the light shielding member 69, the position of the opening 69a can be adjusted to change the irradiation angle and irradiation direction of the illumination light.

  The light source unit 59 may include an LED light source 61, a collimating lens 63, and a diffusion plate 65 arranged in an array like the above, but does not require a function of switching the light emission position of the illumination light, and has an opening 69a. Any light source that can emit illumination light from a wider range may be used.

  FIGS. 13A to 13C are examples having a circular opening 69a, and show examples in which the radial direction and the number of openings 69a are different. 14A shows a case where the opening 69a has a fan shape, and FIG. 14B shows a case where the opening 69a has an annular shape. Any size, position and shape of the opening 69a can be adopted.

  In the present embodiment, the cells A are accommodated in the culture vessel C having the top plate C2 such as a cell culture flask, and the illumination light is reflected on the inner surface of the top plate C2 of the culture vessel C. It is not limited to this. For example, when the cell A is accommodated in a container 71 having no top plate such as a petri dish (without a lid) as a culture container C, as shown in FIG. 15, the mirror is placed at a position where the upper opening of the petri dish is closed. The reflective member 73 as described above may be disposed, and the reflective light 73 may reflect the illumination light transmitted through the bottom surface 71b of the container 71 from the bottom to the top. The reflection member 73 may be provided so as to be insertable / removable at an upper position of the cell A by direct movement or swinging.

Further, in the present embodiment, as shown in FIG. 16, a light shielding member 75 made of a material that shields light may be provided above the top plate C <b> 2 of the culture vessel C.
By doing in this way, since the external light from the outside is shielded by the light shielding member 75, it is possible to suppress the external light from entering the culture vessel C from the top plate C2 and perform observation efficiently. .

In the present embodiment, as the light source unit 59, the LED light source 61, the collimating lens 63, and the diffusion plate 65 are arranged substantially horizontally along the glass plate 53a. However, instead of this, FIG. As shown in FIG. 4, the LED light source 61, the collimating lens 63, and the diffusion plate 65 may be disposed inclined toward the optical axis S.
By doing in this way, the loss of the illumination light emitted from the LED light source 61 can be suppressed, and illumination light can be efficiently irradiated to the cell A.

In the present embodiment, the light source unit 59 is illustrated as including the diffusion plate 65, but the diffusion plate 65 may not be included.
In the present embodiment, the light source unit 59 includes the collimator lens 63, but the collimator lens 63 may not be included.
In the present embodiment, the light source unit 59 includes the collimator lens 63 and the diffusion plate 65, but the arrangement of the collimator lens 63 and the diffusion plate 65 may be reversed. The collimating lens 63 and the diffusion plate 65 may not be provided.

Further, in the present embodiment, the configuration in which the light source unit 59 is disposed below the culture vessel C has been described as an example. However, for example, the light source unit 59 is disposed above the culture vessel C to Illumination light may be irradiated from above.
In the present embodiment, the photographing optical system 57 may be moved in the X and Y directions by a drive mechanism. In this case, the light source unit 59 may be moved together with the photographing optical system 57 by a driving mechanism.

  In the present embodiment, the light source unit 59 has been described by exemplifying a configuration in which a plurality of LED light sources (light sources) 61 are arranged around the objective lens 55 at intervals in the circumferential direction and the radial direction. A plurality of LED light sources (light sources) may be arranged at intervals only in the circumferential direction. For example, four LED light sources (light sources) may be arranged at intervals of 90 degrees in the circumferential direction. One LED light source (light source) may be arranged.

  The embodiment of the present invention has been described in detail with reference to the drawings. However, the specific configuration is not limited to this embodiment, and includes design changes and the like within a scope not departing from the gist of the present invention. For example, the present invention is not limited to those applied to the above-described embodiments and modifications, but may be applied to embodiments in which these embodiments and modifications are appropriately combined, and is not particularly limited. . Further, transmission / reception between the transmission unit 17 and the reception unit 21 may be wired or wireless.

1, 31, 41, 51 Cell observation system 3 Culture observation device (image acquisition unit)
7 Monitor (display section)
23 Cell analysis part (image analysis part, statistical analysis part, comparison part, quality evaluation part)
A cell C culture vessel

Claims (5)

An image acquisition unit for acquiring images in a culture vessel for culturing cells over time;
Based on each of the images acquired by the image acquisition unit, an image analysis unit that quantitatively analyzes a culture state of the cells cultured in the culture container;
A statistical analysis unit that statistically analyzes the data analyzed by the image analysis unit;
A cell observation system comprising: a display unit configured to display the statistical analysis results in the culture vessel in a plurality of passage periods obtained by the statistical analysis unit in a comparable manner.   The cell observation system according to claim 1, wherein the display unit displays a growth curve representing a temporal change in the number of the cells within each passage period.   The cell observation system according to claim 2, wherein the display unit displays the growth curves of the passage periods in a superimposed manner.   The cell observation system according to any one of claims 1 to 3, further comprising a comparison unit that compares a time change of the number of cells for each passage period and outputs a change in the proliferation rate of the cells. The cell observation system according to any one of claims 1 to 4, further comprising a quality evaluation unit that evaluates the quality of the cells based on the proliferation rate of the cells in the passage period.

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