JP2008237064A - Apparatus for observing cell and method for observing cell - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To provide a small-sized apparatus for observing cells and a method for observing the cells, by which the apparatus is introduced into a usual incubator and most of the culture medium can simultaneously be observed. <P>SOLUTION: The apparatus for observing the cells is equipped with an image observing part 20 thereof arranged under a vessel 40 for culturing the cells installed in the incubator 10. The image observing part 20 is provided with a compound eye image input part for forming a plurality of reduced images of objects on a photodetector with a microlens array in which a plurality of micro-lenses are arranged. The compound eye image input part incorporates ≥50% of the images in the undersurface of the vessel 40. <P>COPYRIGHT: (C)2009,JPO&INPIT

Description

  The present invention relates to a cell observation device and a cell observation method, and more particularly to a cell observation device and a cell observation method in an incubator.

  In recent years, many techniques for culturing human cells in vitro and applying the cultured tissue to patients have been reported. In particular, human skin tissue has reached the level of commercialization in the West. These cultured tissues are also used for proliferating stem cells for various medical uses. Many of these cells are adhesion-dependent cells that stick to the bottom of a culture vessel and grow in two dimensions. However, since three-dimensional growth is difficult, It is necessary to change to a culture vessel with a wider bottom area and further grow.

  As a premise to change the culture vessel as described above, it is necessary to regularly observe the degree of cell proliferation. Until now, various attempts have been made to observe live cells. Observation of cells using an optical microscope is a simple observation technique that has been used for a long time, and is currently most widely used. In this case, a culture apparatus (incubator) is also used, and cells are stored in the culture apparatus except during observation, so that damage caused to the cells by the environment is minimized. However, it is known that in a general observation system in which an optical microscope and a culture apparatus are combined, cells are greatly damaged due to environmental fluctuations when being taken in and out of the culture apparatus. In addition, since the object is attached to and detached from the microscope every time it is observed, it is difficult to reproduce an accurate observation position over a plurality of observations. That is, the observed cells are lost during the next observation, and it is difficult to maintain the identity of the observation target. There is a high possibility that the target cells differ for each observation, and only statistical information can be obtained.

  In order to solve such a problem, the apparatus disclosed in Patent Document 1 discloses a configuration for avoiding the trouble of taking in and out the culture apparatus and avoiding damage to cells due to environmental changes. In this configuration, the growing state is checked while culturing the cells, and the cells can be observed in the culture apparatus as necessary.

Patent Document 2 discloses a culture means for culturing cells, an imaging means for imaging cells contained in the culture means, including at least an infrared light imaging means for imaging cells with infrared rays, and the imaging A cell recognition unit for recognizing a cell from cell image data of an image captured by the unit, a cell parameter measurement unit for measuring a cell parameter indicating a characteristic of the cell recognized by the cell recognition unit based on the cell image data, and a different time point There is disclosed a cell observation device comprising: cell tracking means for discriminating the identity of cells at different time points recognized from the cell image data of the image captured in step 1 based on the cell parameter.
Special table 2001-500744 gazette JP 2006-238802 A JP 2001-61109 A

  However, the devices disclosed in the above two patent documents are special devices, and cannot be put into a normal commercially available incubator, and there is a problem that a special incubator must be used. In particular, the conventional imaging means uses a phase-contrast microscope that observes with transmitted light, and this imaging means is large and the range that can be observed at one time is narrow, and the entire large culture vessel cannot be observed simultaneously. The observation apparatus was not suitable for cell culture.

  The present invention has been made in view of the above points, and an object of the present invention is to provide a cell observation apparatus and a cell observation method that are small in size and can be placed in a normal incubator and can observe most of the medium at the same time. There is.

  In order to solve the above problems, the cell observation device of the present invention includes an image observation unit, a part of which is installed in an incubator, a cell is cultured and installed under a container having a transparent lower surface, and an illumination unit. The image observation unit provided in the incubator has a compound eye image input unit that forms a plurality of reduced object images on a light receiving element by a microlens array in which a plurality of microlenses are arranged. The entire reduced image of the object included 50% or more of the lower surface of the container. Here, the microlens is a lens having a diameter of 2 mm or less. Further, the fact that the entire plurality of reduced object images include images of 50% or more of the lower surface of the container means that 50% of the lower surface of the container is reconstructed by combining the individual object reduced images. All the above appears as an image.

  With such a configuration, the thickness of the image observation unit can be reduced and the focal length can be reduced, so that the cell observation apparatus can be placed in a commercially available incubator with a small capacity and can observe the majority of the medium at the same time. .

  There may be a plurality of compound eye image input units, 50% or more of the lower surface of the container is divided, and each divided image is formed on each compound eye image input unit. .

  The illumination unit may be configured to irradiate the container with light from the side of the container.

  An antireflection treatment may be performed on the inner surface of the upper surface member constituting the upper surface of the container.

  The cell observation method of the present invention is a cell observation method for observing the state of cells in a container having a transparent lower surface in which cells placed in an incubator are cultured, and the above cell observation apparatus is used as a container in an incubator. And a step of observing the culture state of the cells in the incubator with the cell observation device.

  In the present invention, since it has a compound eye image input unit in which a plurality of microlenses are arranged, the thickness of the image observation unit can be reduced, the focal length can be reduced, and more than 50% of the lower surface of the container can be observed simultaneously. .

  Hereinafter, embodiments of the present invention will be described in detail with reference to the drawings. In the following drawings, components having substantially the same function are denoted by the same reference numerals for the sake of brevity.

(Embodiment 1)
FIG. 1 is a diagram schematically showing a cell observation apparatus according to Embodiment 1 installed in an incubator (culture apparatus) 10. The figure shows the interior, in fact there is a door and it is sealed during cell culture. In the incubator 10, there is a shelf 12 on which containers 40 for culturing cells are placed, and an image observation unit 20 of the cell observation device is placed below the shelf.

  When culturing cells or stem cells of human tissue, it is necessary to have the same environment as the inside of the human body, and the temperature inside the incubator 10 is maintained at 37 ° C., humidity 95 RH% or more, and carbon dioxide concentration 5%. In order to keep the carbon dioxide concentration at 5%, a gas cylinder 70 containing carbon dioxide is placed outside the incubator 10, and carbon dioxide is supplied into the incubator 10 through a pipe 71. As shown in FIG. 2, the container 40 for culturing cells has a pentagonal shape like a shogi piece, and the lower surface 42 and side surfaces are transparent so that the culture state inside can be observed. On the other hand, a black matting paint for preventing reflection is applied to the inner surface of the upper surface member constituting the upper surface 41. Although the container 40 is sealed with the screw cap 43, the screw cap 43 is opened when the culture solution or the seed cells are added or when the contents are taken out.

  It is preferable that the shelf 12 is transparent so as not to disturb the observation of cells, or is configured to support the container 40 only with a frame.

  The image observation unit 20 is connected to the image processing unit 60 placed outside the incubator 10 by a connection line 61. The image processing unit 60 processes and analyzes the image data received from the image observation unit 20.

  Further, in the incubator 10, illumination units 50 and 50 are installed on the side of the container 40. The illumination parts 50 and 50 use white LED as a light source. Specifically, a plurality of white LED chips are arranged along the side surface of the container 40 as a light source. By placing the illumination units 50, 50 on the side of the container 40, the reflected light from the lower surface of the container 40 to the image observation unit 20 is eliminated, and the cells can be easily observed. In addition, since the inner side surface of the upper surface of the container 40 is prevented from being reflected, the reflected light from the upper surface to the image observation unit 20 is also eliminated, making it easier to observe the cells.

  The cell observation device of this embodiment includes an image observation unit 20, illumination units 50 and 50, and an image processing unit 60, and a cell colony in which light emitted from the illumination units 50 and 50 adheres to the lower surface of the container 40. Then, the light is irregularly reflected and the reflected light is observed by the light receiving element of the image observation unit 20. In the conventional cell observation device, cells are observed with a phase contrast microscope using transmitted light, but in this embodiment, reflected light is observed as it is without using a phase difference formula.

  As shown in FIG. 3, the image observation unit 20 of the present embodiment includes a microlens array 1 in which a plurality of microlenses 1 a are arranged, a partition layer 2 provided under the microlens array 1, and a partition layer. 2 has a compound eye image input unit 30 provided with a light receiving element 3 provided below the light receiving element 3. The structure of the compound eye image input unit 30 is the same as that shown in Patent Document 3. In the drawing, the microlens array 1, the partition wall layer 2, and the light receiving element 3 are drawn apart from each other. However, this is for ease of explanation, and these are actually arranged in an overlapping manner. . The partition layer 2 is an aggregate of a plurality of grid-like partition walls 2a, and the light receiving element 3 is an aggregate of a plurality of light receiving cells 3a.

  One microlens 1a corresponds to one partition 2a, and a plurality of light receiving cells 3a correspond to them. These constitute a signal processing unit U. In the figure, the signal processing unit U is indicated by a rectangular column with a broken line. The partition wall 2a serves to block light from the microlens adjacent to the corresponding microlens 1a, thereby preventing interference of optical signals. A plurality of reduced images of the object on the image observation unit 20 are formed on the light receiving element 3.

  Since the microlens 1a is used in this embodiment, the thickness of the compound eye image input unit 30 itself can be made smaller than that of a normal imaging device. In addition, since the focal length can be reduced, the distance to the target object can be made smaller than that of a normal imaging device. For this reason, the image observation part 20 can be installed in the narrow space in an incubator. In addition, the structure is simple and high-definition image data can be taken out compared to the prior art. In addition, it is possible to simultaneously observe 50% or more of the object having a large area without moving the compound eye image input unit 30.

  The commercially available incubator 10 has a capacity of several tens of liters and the height of the internal space is about 50 to 60 cm. As already described, a commercially available phase contrast microscope cannot be put inside. However, since the image observation unit 20 of the present embodiment can have a height of several mm to several cm and a focal length of several mm to several tens of cm, cells can be observed while the container 40 is placed in the incubator 10. Yes. Thereby, the bad influence on cell culture by taking out container 40 out of incubator 10 can be avoided. As an example of an actual apparatus, the height is 5 mm (about 1 cm including the case), and the focal length is 5 cm.

  Next, a method for observing the cell culture state will be described.

  First, the image observation unit 20 of the cell observation device is installed on the bottom surface inside the incubator 10. Then, a connection line 61 connects to the image processing unit 60.

  Then, the shelf 12 is placed 3 to 4 cm above the image observation unit 20, and the container 40 containing the culture solution and seed cells is placed thereon. Moreover, the illumination parts 50 and 50 are installed on the inner wall of the incubator on both sides of the container 40.

  Next, the door of the incubator 10 is closed and sealed, the temperature, humidity, and carbon dioxide concentration are set to the culture conditions, and cell culture is started.

  Then, every predetermined time, current is supplied to the illumination units 50 and 50 to illuminate the inside of the container 40, and the image observation unit 20 adheres to the lower surface 42 of the container 40 and expands the image data of the cell colonies that are growing. Collected and sent to the image processing unit 60. Image data obtained from each light receiving cell 3a is converted into an image on the lower surface 42 of the container 40 as shown in FIG. 6, for example, and the cell growth rate and larger culture are determined from the number and size of the cell colonies 45, 45, 45. The image processing unit 60 calculates the scheduled transfer date to the container by calculation.

  In the present embodiment, if there is an overlap between the reduced images formed in adjacent signal processing units U, a three-dimensional image can be calculated and extracted.

(Embodiment 2)
The second embodiment is different from the first embodiment in the compound eye image input unit, and the other points are the same as those in the first embodiment, so the compound eye image input unit will be described.

  As shown in FIG. 4, the compound eye image input unit 31 of the present embodiment is configured to have nine divided compound eye image input units 33, 33,. Each divided compound eye image input unit 33, 33,... Has nine signal processing units U. .. Are arranged along the shape of the lower surface 42 of the container 40 so that images of 50% or more of the lower surface 42 can be observed. In this embodiment, 90% or more of images can be observed. Each of the divided compound eye image input units 33, 33,... Forms the divided image of the lower surface 42 of the container 40 on the light receiving element.

  As shown in FIG. 5, the divided compound eye image input unit 33 is configured such that a partition layer 37 is placed on an image sensor (light receiving element) 38, a microlens array 36 is placed thereon, and a diaphragm 35 is placed thereon. Is placed. Then, a spacer 34 is placed on the diaphragm 35, and these stacked parts are fixed by a frame. The aperture 35 is a hole having a smaller diameter than the microlens formed in the plate, and the spacer 34 has a large opening at the center so as not to interfere with cell observation.

  In the present embodiment, since a plurality of divided compound eye image input units 33 are configured using a plurality of small light receiving elements, they can be manufactured at low cost. Also, the resolution goes up. In addition, the same effects as those of the first embodiment are obtained.

(Other embodiments)
The above embodiments are merely examples, and the present invention is not limited to these examples. For example, in the second embodiment, the number of divided compound eye image input units 33 may be more or less than nine. Further, the number of signal processing units U constituting one divided compound eye image input unit 33 may be larger or smaller than nine.

  The shape and size of the container are not particularly limited. A filter may be attached to the compound eye image input unit and the divided compound eye image input unit. The antireflection treatment on the upper surface of the container may be performed by sandblasting or changing the material itself to a nonreflective one.

  As described above, the cell observation apparatus according to the present invention is useful as an apparatus for culturing and observing stem cells because the image observation section is thin and can be installed in an incubator.

Schematic diagram of the cell observation apparatus according to Embodiment 1 installed in the incubator Perspective view of vessel for culturing cells Schematic exploded perspective view of compound eye image input unit Plan view of compound eye image input unit according to Embodiment 2 Exploded perspective view of split compound eye image input unit Diagram showing colonies of cultured cells

Explanation of symbols

DESCRIPTION OF SYMBOLS 1 Micro lens array 1a Micro lens 10 Incubator 20 Image observation part 30,31 Compound eye image input part 40 Container 41 Container upper surface 42 Container lower surface 50 Illumination part 60 Image processing part 61 Connection line

Claims (5)

A cell observation device partially installed in an incubator,
An incubator is provided with an image observation unit that is placed under a container in which cells are cultured and has a transparent lower surface, and an illumination unit,
The image observation unit includes a compound eye image input unit that forms a plurality of reduced object images on a light receiving element by a microlens array in which a plurality of microlenses are arranged.
The whole of the plurality of reduced object images includes a cell observation device including an image of 50% or more of the lower surface of the container. There are a plurality of compound eye image input units,
The cell observation device according to claim 1, wherein an image of 50% or more of the lower surface of the container is divided, and each divided image is formed on each compound eye image input unit.   The cell observation apparatus according to claim 1, wherein the illumination unit irradiates light to the container from a side of the container.   The cell observation apparatus according to any one of claims 1 to 3, wherein an inner surface of an upper surface member constituting the upper surface of the container is subjected to an antireflection treatment. A cell observation method for observing the state of cells in a container having a transparent lower surface in which cells placed in an incubator are cultured,
Installing the cell observation device according to any one of claims 1 to 4 below a container in an incubator;
And observing the culture state of the cells in the incubator with the cell observation device.

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