JP4731847B2 - Petri dish, chamber apparatus, optical microscope observation method and sample analysis method - Google Patents

Description

  The present invention is suitable for, for example, observation, analysis and processing of living cells, and can be used in combination with fluorescence observation and differential interference observation with a lid attached, a petri dish, a chamber apparatus, an optical microscope observation method, and a sample analysis method The purpose is to provide.

  FIG. 8 is a cross-sectional view showing a conventional general glass bottom petri dish. In the figure, a conventional Petri dish 100 is composed of a lid body 110 and a container body 20 mainly made of transparent styrene resin and the like, and a glass slip 22 is bonded to a through hole 21 formed in the bottom of the container body 20. It was. The upper surface of the lid body 110 and the lower surface of the container body 20 are integrally formed with annular hills 111 and 23, respectively, and the upper surface of the lid body 110 and the lower surface of the container body 20 are damaged. Is preventing. The inner diameter of the hill 111 of the lid body 110 is larger than the outer diameter of the hill 23 of the container body 20 so that a plurality of Petri dishes 100 can be placed on top of each other.

  When processing, observing or analyzing a sample 31 such as cultured cells using such a Petri dish 100, the container 20 is filled with a solution (medium) 32 such as a culture solution, a buffer or physiological saline, and a glass slip. 22 in which the sample 31 is planted. Thereafter, the lid 110 is attached to the container 20 to prevent dust and unnecessary substances from being mixed into the solution 32 and evaporation of the solution 32.

  Next, the case where the sample in the Petri dish is observed with an upright microscope will be described with reference to FIGS. FIG. 9 is a schematic view showing an upright microscope. In the figure, there are two types of illumination light dropping methods for the sample 31 in the upright microscope 200: transmitted illumination and reflected or epi-illumination.

  First, the transmitted illumination will be described. Illumination light emitted from the light source 201 passes through the illumination lens 203 through the mirror base 202, is collected by the condenser lens 204, and then from the lower surface side of the Petri dish 100 to the stage 205. Reach the sample placed on top. Thereafter, the light transmitted through the sample 31 or the light emitted from the sample 31 reaches the objective lens 206 and is observed from the eyepiece lens 208 through the lens barrel 207 or recorded by an imaging device (not shown). In addition, a method in which the lid 110 of the Petri dish 100 is removed and the tip of the objective lens 206 is immersed directly in the solution 32 in the container body 20 for observation is also performed.

  Next, the reflection or epi-illumination will be described. Illumination light emitted from the light source 209 reaches the objective lens 206 through the light projection tube 210. The light condensed on the sample 31 by the objective lens 206 is reflected, scattered, or fluorescently excited by the sample 31 and returns to the objective lens 206 again, passes through the light projection tube 210, passes through the lens barrel 207, and the eyepiece. It is observed from 208 or recorded by the imaging device.

  Next, the case where the sample in the Petri dish is observed with an inverted microscope will be described with reference to FIGS. FIG. 10 is a schematic view showing an inverted microscope. In the same figure, the inverted microscope 300 also employs two types of illumination light for the sample 31: transmission illumination and reflection or epi-illumination, as described above.

  First, transmission illumination will be described. Illumination light emitted from the light source 301 is transmitted through a condenser tube 304 through a light projection tube 303 attached to the transmission illumination support column 302, and in the Petri dish 100 placed on the stage 305. The sample 31 is condensed. The light transmitted through the sample 31 or the light emitted from the sample 31 reaches the objective lens 306 and is observed from the eyepiece lens 308 through the mirror body 307 or recorded by the imaging device.

  Next, the reflection or epi-illumination will be described. Illumination light emitted from the light source 309 reaches the objective lens 306 through the mirror body 307. The light condensed on the sample 31 by the objective lens 306 is reflected, scattered, or fluorescently excited by the sample 31 and returns to the objective lens 306 again, and is observed from the eyepiece lens 308 through the mirror body 307, or Recorded by the imaging device.

  In the processing, observation or analysis of the sample 31 using such an upright microscope 200 or an inverted microscope 300, the bottom surface of the container body 20 of the Petri dish 100 is made of a resin such as styrene resin, which has better cell fixability than glass. Although good, glass is often more convenient optically.

  For example, the cell to be the sample 31 is optically close to transparency, and it is difficult to obtain contrast in bright field transmission illumination observation. Therefore, phase difference observation and differential interference observation are performed. In the case of phase difference observation, if the bottom surface of the container body 20 is made of a transparent resin, the angle between the transparent resin and the cell membrane is closer to 90 °. Halation appears due to excessive contrast. On the other hand, in the case of differential interference observation, if the bottom surface of the container body 20 is made of a transparent resin, the amount of shearing that is an element of contrast in differential interference observation is not uniform. Such optical inhomogeneity of the transparent resin produces contrast, and overlaps with the image of the cell, resulting in inconvenience that the target cell cannot be observed.

  Further, in order to support the sample 31 in order to perform the optical microscope observation as described above, it is necessary that the support member has an optically homogeneous property. Further, in order to perform analysis and processing, It is required to be thermally stable. In addition, in the case where the sample 31 is a living body or a cell, it is necessary that the sample 31 has a property that has little influence on the sample 31. Therefore, the glass bottom Petri dish 100 having the above-described configuration is employed, and the highly transparent sample 31 represented by cells can be observed.

Using such a glass bottom Petri dish 100, research papers on the behavior of chromosomes and microtubules in mammalian cell division are listed as the following non-patent documents. On page 379 of this non-patent document, a method and apparatus for observing cells in a 35 mm diameter glass bottom culture dish with an oil immersion objective lens is disclosed.
Tokuko Haraguchi, Toru Kaneda, Yasushi Hiraoka [Dynamics of chromosomes and microtubules visualized by multiple-wavelength fluorescence imaging in living mammalian cell: effects of mitotic inhibitors on cell cycle progression] Genes to cell (1997) 2, 369-380

  When the container 20 of the conventional Petri dish 100 described above is filled with a solution 32 such as a culture solution, the influence of convection due to wave on the liquid surface, concentration change or concentration gradient due to evaporation, and secondary effects of evaporation. There must be little or no. Therefore, it can be said that the lid 110 is indispensable in order to cut off such external influences. On the other hand, when observing a predetermined part (for example, nucleus) of a living cell with fluorescence, observation of the morphology of the whole cell using phase difference observation, Hoffman observation, or differential interference observation is performed. .

  However, if differential interference observation is performed with the cover 110 made of transparent resin attached, polarized light is generated because the optical properties of the transparent resin are inhomogeneous, and the sample 31 passes through the cover 110. However, there is a problem in that differential interference observation cannot be performed unless the lid 110 is removed.

  That is, since differential interference observation is an optical system using polarized light, if a plastic member having non-homogeneous optical properties due to distortion, composition, or the like is interposed in the optical path, the resolution (or appearance) is extremely poor. Become. For this reason, when using the conventional glass bottom Petri dish 100 and using both fluorescence observation and differential interference observation, the lid 110 must be removed.

  However, in observing living cells, removing the lid 110 leads to contamination with various germs, and thus is limited to observation in a short time of about several hours. In addition, since the petri dish 100 that has been observed with the lid 110 opened once may be propagated with various germs, it cannot be expected to observe the subsequent form. Here, there is a method of adding an antibiotic to the solution 32, but this method has been avoided in recent years because of concern about the influence on the cell as a sample. For this reason, when observing a living cell for several days, a predetermined portion of the cell is observed with fluorescence, and the overall appearance of the cell is slightly changed by using the objective lens for fluorescence observation. There was no choice but to observe an image with good contrast or out-of-object phase contrast.

  In addition, when a high-magnification objective lens is used, the working distance (WD) of the objective lens is short. For example, the WD of a 100 × objective lens is about 0.15 mm. The WD of the condenser lens corresponding to this is about 0.7 mm. For this reason, if the lid 110 of the Petri dish 100 is formed of a transparent resin having a thickness of about 0.5 to 1 mm, there is a problem that differential interference observation can no longer be performed.

  When fluorescence observation and phase contrast observation or Hoffman observation are combined, when switching from fluorescence observation assuming the use of an oil objective to phase difference observation or Hoffman observation assuming the use of a dry type objective lens In addition, there has been a problem that a region that has been observed until now is shifted, or that an imaging failure occurs during phase difference observation or Hoffman observation due to adhesion of residual oil during fluorescence observation. In addition, if the fluorescence observation is performed with the same objective lens as the phase difference observation or Hoffman observation in order to eliminate the replacement of the objective lens, a light amount loss due to a modulator such as a slit, a phase film or a knife edge in the objective lens occurs. There is a problem.

  The present invention has been made in view of the above problems, and can be used in combination with fluorescence observation and differential interference observation with the lid attached, and the air between the condenser lens and the objective lens in the differential interference observation. It is an object of the present invention to provide a petri dish, a chamber apparatus, an optical microscope observation method, and a sample analysis method that can eliminate the layer and can be used in combination with high resolution and high magnification differential interference observation and fluorescence observation.

In order to achieve the above object, a first Petri dish of the present invention includes a container body for storing a sample, a lid body having a through hole covering the container body, and being pushed through the through hole, At least two pipes for supplying and discharging the solution to and from the body, and a sealing member for sealing the through hole and holding the pipe in close contact with each other, facing both the lid and the container Each of the through-holes is formed, and each of the through-holes is closed by a plate member capable of transmitting light, which is glass that suppresses autofluorescence and is optically homogenized. A configuration further provided with another pipe that is pushed through and supplies an additive substance other than the solution into the container, or a configuration in which the plate member is made of quartz glass, or a through hole of the lid, From the through hole of the container body And listen to the configuration.

  In order to achieve the above object, the chamber apparatus of the present invention is configured such that at least two pipes for supplying and discharging the solution are passed through the lid body in the Petri dish, and are put into the container body through these pipes. The solution is circulated, and preferably, the lid is made to pass through another pipe for supplying an additive substance other than the solution into the container.

  In order to achieve the above object, the optical microscope observation method of the present invention irradiates illumination light into the container body from above the plate member of the lid body in the Petri dish or chamber apparatus, and the plate of the container body The sample is observed by the objective lens from below the member. Preferably, oil or liquid is interposed between the condenser lens for condensing the illumination light on the sample and the plate member of the lid, and the observation of the sample is a combination of fluorescence observation and differential interference observation. To do. In order to achieve the above object, the sample analysis method of the present invention analyzes the sample based on the observation image obtained by the optical microscope observation method.

  According to the Petri dish, the chamber apparatus, the optical microscope observation method, and the sample analysis method of the present invention, it is possible to irradiate the sample with unpolarized illumination light through the plate member of the lid in the Petri dish or the chamber apparatus. This makes it possible to perform fluorescence observation and differential interference observation of living cells using a general oil objective lens without removing the lid. Further, since the lid is not removed, there is an effect that the cells are not deteriorated even when fluorescence observation and differential interference observation of living cells are performed for a long time.

  Furthermore, when a through-hole part protruding toward the inside of the container body is formed on the lid in the Petri dish or chamber apparatus, and the lower end opening of the through-hole part is closed by a plate member capable of transmitting light, The plate member of the lid body is immersed in the solution in the container body, the air layer existing between the plate member and the sample can be eliminated, and by using an oil condenser lens to collect the illumination light, High-resolution, high-magnification differential interference observation that cannot be used in combination with fluorescence observation of living cells is possible.

  In addition, according to the chamber apparatus, the optical microscope observation method, and the sample analysis method of the present invention, the solution in the container can be kept at a constant component, concentration, and temperature, and the solution in the container can be kept fresh. . In addition, by supplying an additive substance having a preset time, concentration, and amount from the pipe, it is possible to perform an experiment for stimulating a living cell as a sample or knowing a reaction to an environmental change.

  Hereinafter, a petri dish, a chamber apparatus, an optical microscope observation method, and a sample analysis method according to an embodiment of the present invention will be described with reference to the drawings. First, a petri dish according to the first embodiment of the present invention will be described with reference to FIG. FIG. 1 is a sectional view showing a petri dish according to the first embodiment of the present invention. In addition, about the location similar to the prior art mentioned above, the same code | symbol is attached | subjected and detailed description is abbreviate | omitted.

  In the figure, a petri dish 1 of this embodiment is composed of a lid body 10 and a container body 20 made of transparent styrene resin or the like. A circular through hole 11 is formed in the approximate center of the lid 10. The through-hole 11 has a positional relationship facing the through-hole 21 formed in the bottom of the container body 20, and a plate member 12 capable of transmitting light is bonded from the back side (the container body 20 side). It is blocked by.

  The plate member 12 is preferably one that suppresses autofluorescence and is optically homogenized in order to reduce optical resolution reduction factors such as polarization and birefringence. For example, in order to suppress autofluorescence, a very thin plate glass, a plate glass formed of a non-fluorescent material, or the like can be used. Further, as an optical homogenization treatment, an antireflection film is applied to reduce the wavelength selectivity of the plate member 12, a distortion is eliminated, a composition that easily homogenizes optical properties, an internal stress is reduced. For example, it can be made uniform. From such a viewpoint, the material of the plate member 12 is preferably a synthetic quartz glass that is chemically synthesized and has few impurities. However, sufficient resolution can be obtained even with a glass slip similar to the plate member 22 of the container body 20. Note that the plate member 12 of the lid body 10 and the plate member 22 of the container body 20 may be made of the same material or different materials depending on the observation method.

  On the other hand, the adhesive for fixing the plate member 12 to the through-hole 11 is preferably one that does not have both swellability and autofluorescence, or one that does not have only one depending on the purpose. Further, a configuration in which all the joint surfaces of the plate member 12 and the plate member 12 are filled with the adhesive, or a configuration in which airtightness and watertightness can be secured with a small amount of adhesive may be employed. Note that the bonding of the lid 10 and the plate member 12 is not limited to bonding with an adhesive, but may be performed by other bonding means such as thermal bonding.

Moreover, the shape of the through-hole 11 and the plate member 12 is not limited to a circle as in the present embodiment, and may be, for example, a rectangle or an ellipse. However, it is necessary to make the shape and size such that a part of the illumination light incident from the through hole 11 is not blocked in the middle of reaching the sample 31 in the container body 20. Therefore, it is desirable that the diameter L _{1 of the} through hole 11 of the lid 10 is the same as or larger than the diameter L ₂ of the through hole 21 of the container body 20. Here, FIG. 2 is a cross-sectional view showing a modified example of the Petri dish 1, and the plate member 12 of the lid 10 may be bonded or joined from the front side of the through-hole 11 as shown in FIG.

  Next, the optical microscope observation method according to the first embodiment of the present invention will be described with reference to FIG. FIG. 3 is a schematic view for explaining an optical microscope observation method according to the first embodiment of the present invention.

  In this embodiment, fluorescence observation and differential interference observation of the sample 31 are performed using the Petri dish 1 shown in FIG. 2 and the inverted microscope 300 shown in FIG. First, the Petri dish 1 is placed on the stage 305 of the inverted microscope 300 shown in FIG. Next, as shown in FIG. 3, the illumination light S is irradiated into the container body 20 from above the plate member 12 of the lid body 10 in the present Petri dish 1, and the objective lens from below the plate member 22 of the container body 20. By 306, fluorescence observation and differential interference observation of the sample 31 are performed simultaneously or alternately. The space between the plate member 22 and the objective lens 306 (interval = about 0.1 mm) is filled with oil 306a. Thereafter, the sample 31 is analyzed based on observation images obtained by such fluorescence observation and differential interference observation.

  According to such a Petri dish and an optical microscope observation method of this embodiment, the plate member 12 of the lid 10 is optically homogenized, so that illumination light having no polarization is transmitted through the plate member 12 to the sample 31. Can be irradiated. This makes it possible to perform fluorescence observation and differential interference observation of living cells using a general oil objective lens without removing the lid 10. Further, since the lid 10 is not removed, there is an effect that the cells are not deteriorated even when fluorescence observation and differential interference observation of living cells are performed for a long time.

  In addition, since a good differential interference observation, or an observation image of fluorescence observation and differential interference observation can be obtained by the illumination light transmitted through the optically uniform plate member 12, the sample 31 can be analyzed based on the observation image. It will be possible to do well.

  Next, a petri dish according to the second embodiment of the present invention will be described with reference to FIG. FIG. 4 is a sectional view showing a petri dish according to the second embodiment of the present invention. In addition, about the location similar to embodiment mentioned above, the same code | symbol is attached | subjected and detailed description is abbreviate | omitted.

  In the figure, the Petri dish 2 of the present embodiment forms a cylindrical through-hole portion 41 projecting toward the inside of the container body 20 in the lid body 40, and the lower end opening 41 a of the through-hole portion 41 is light-transmitted. It is set as the structure obstruct | occluded by the plate member 42 which can permeate | transmit. With such a configuration, the plate member 42 of the lid body 40 is immersed in the solution 32 in the container body 20, and the air layer existing between the plate member 42 and the sample 31 is eliminated. The distance between the plate member 42 and the sample 31 is set to about 1.5 mm.

As in the first embodiment, the plate member 42 is made by suppressing autofluorescence and optically homogenizing in order to reduce optical resolution reduction factors such as polarization and birefringence. preferable. Further, it is desirable that the diameter L _{1 of the} through-hole portion 41 of the lid body 40 is the same as or larger than the diameter L ₂ of the through-hole 21 of the container body 20 in order to prevent the incident loss of illumination light.

  Next, an optical microscope observation method according to the second embodiment of the present invention will be described with reference to FIG. FIG. 5 is a schematic view for explaining an optical microscope observation method according to the second embodiment of the present invention.

  As in the first embodiment, fluorescence observation and differential interference observation of the sample 31 are performed using the Petri dish 2 shown in FIG. 4 and the inverted microscope 300 shown in FIG. 10. In this embodiment, the condenser lens 304 is used. And an oil condenser lens in which the space between the plate members 42 of the lid 40 (interval = about 0.1 mm) is filled with oil 304a is used.

  First, the Petri dish 2 is placed on the stage 305 of the inverted microscope 300 shown in FIG. Next, as shown in FIG. 5, the condenser lens 304 is inserted into the through-hole portion 41 of the lid body 40 in the Petri dish 2, and the illumination light S is irradiated into the container body 20 through the oil 304a and the plate member 42. . On the other hand, fluorescence observation and differential interference observation of the sample 31 are simultaneously or alternately performed from below the plate member 22 of the container body 20 by the objective lens 306. Thereafter, the sample 31 is analyzed based on observation images obtained by such fluorescence observation and differential interference observation.

  According to the Petri dish and the optical microscope observation method of this embodiment, the plate member 42 of the lid 40 in the Petri dish 2 is immersed in the solution 32 in the container body 20, and the plate member 42 and the sample 31 are interposed between the plate member 42 and the sample 31. The existing air layer can be eliminated, and coupled with the use of an oil condenser lens, high-resolution differential interference observation that cannot be used together with fluorescence observation of living cells so far becomes possible.

  More specifically, as shown in FIGS. 1 and 2, the interface between the plate member 12 and the air and the interface between the air and the solution 32 have a large difference in refractive index, that is, a reflectance and a refractive index. A large reflectance means that the amount of light reaching the sample 31 is reduced, and a large refractive index means that the aberration is large and the Airy disk at the focal point becomes large and the spatial resolution is lowered.

  If the plate member 42 of the lid body 40 is immersed in the solution 32 in the container body 20 as in the Petri dish 2 of the present embodiment, two interfaces having a high reflectance and a refractive index can be eliminated, and the reflectance and the refractive index can be eliminated. Only the interface between the plate member 42 and the solution 32 having a small rate remains. The influence of the interface can be ignored by setting the refractive indexes of the plate member 42 and the solution 32 to the same or very close values.

  In addition, the oil condenser lens can illuminate with a high NA (aperture angle), which enables illumination suitable for single-cell observation. Further, no air layer is interposed between the condenser lens 304 and the objective lens 306. As a result, illumination suitable for high-magnification observation and observation that detects weak fluorescence are possible.

  In the above embodiment, oil is interposed between the condenser lens 304 and the plate member 42 of the lid body 40. However, a recess is formed on the surface of the plate member 42 to hold oil and other liquids. You may do it. In addition, considering the switching of the condenser lens and the working distance (WD), the diameter of the through-hole portion 41 in the lid portion 40 is increased and the depth thereof is reduced, so that the focal position of the condenser lens is raised and lowered. It can be exchanged or switched without moving.

  Next, a chamber apparatus according to an embodiment of the present invention will be described with reference to FIG. FIG. 6 is a cross-sectional view showing a chamber apparatus according to an embodiment of the present invention. In addition, about the location similar to embodiment mentioned above, the same code | symbol is attached | subjected and detailed description is abbreviate | omitted.

  In the figure, the chamber apparatus 3 of the present embodiment has two through holes 14 and 15 formed in the lid 10 of the petri dish 1 shown in FIG. 2 and a pipe 51 in each of the through holes 14 and 15. 52, and the through holes 14 and 15 are sealed with seal members 61 and 62, respectively.

  The pipe 51 is for supplying the solution 32 into the container body 20, and the pipe 52 is for discharging the solution 32 in the container body 20. These pipes 51 and 52 are made of glass, metal, or plastic, and are connected to a pump of a solution circulating device (not shown) so that the solution 32 in the container body 20 circulates.

  FIG. 7 is a cross-sectional view showing a modified example of the chamber device 3. As shown in the figure, a pipe 53 for supplying an additive substance into the container body 20 is added to the supply-side through-hole 14 in addition to the pipe 51 for supplying the solution 32. The through hole 14 is sealed by a two-hole seal member 63.

  In addition, as the sealing members 61, 62, 63, for example, a material having a good sealing property such as silicon rubber or fluorine rubber is used, and these sealing members 61, 62 are elastically closed before the pipes 51, 52 are inserted. In this state, a hole capable of tightly holding the press-fitted pipes 51 and 52 is provided. By such seal members 61, 62, 63, the tips of the pipes 51, 52, 53 can be held at a predetermined height. For example, the liquid level of the solution 32 can be kept constant by holding the discharge pipe 52 at a predetermined height.

  According to the chamber apparatus 3 of this embodiment, the solution 32 in the container body 20 can be kept at a constant component, concentration, and temperature, and the solution 32 in the container body 20 can be kept fresh. In addition, by supplying an additive substance having a preset time, concentration, and amount from the pipe 53, it is possible to perform an experiment for stimulating a living cell as the sample 31 or knowing a reaction to an environmental change.

For example, the solution 32 kept at a predetermined temperature is supplied from the pipe 51 and the carbon dioxide gas is supplied from the pipe 53 to keep the solution 32 in the container body 20 at a temperature of less than 37 ° C. and a CO ₂ concentration of 5%. In addition, by maintaining the humidity of the environment in which the chamber apparatus 3 is placed at 90%, the activity of living cells as the sample 31 can be maintained for a long period of time.

  The chamber apparatus 3 can be used for microscopic observation similar to the Petri dish 1 according to the above-described embodiment. In this case, the lid body 10 and the container body 20 are sandwiched and fixed, or a clamp or a clemmel is used. It is good to hold down.

  The petri dish, chamber apparatus, optical microscope observation method, and sample analysis method of the present invention are not limited to the above-described embodiments. For example, the Petri dish of the present invention can be applied to a photographic apparatus in addition to an upright microscope, an inverted microscope, a stereomicroscope, or a microscope that is observed from above and below the Petri dish. It can also be applied to analyzers that use optics, magnetism, and radiation. Moreover, the petri dish and chamber apparatus of this invention are applicable not only to an optical microscope observation method and a sample analysis method like the said embodiment but to the various processing methods in a sample preparation stage.

It is sectional drawing which shows the petri dish which concerns on 1st Embodiment of this invention. It is sectional drawing which shows the example of a change of the said Petri dish. It is the schematic for demonstrating the optical microscope observation method which concerns on 1st Embodiment of invention. It is sectional drawing which shows the petri dish which concerns on 2nd Embodiment of this invention. It is the schematic for demonstrating the optical microscope observation method which concerns on 2nd Embodiment of this invention. It is sectional drawing which shows the chamber apparatus which concerns on one Embodiment of this invention. It is sectional drawing which shows the example of a change of the said chamber apparatus. It is sectional drawing which shows the conventional common glass bottom Petri dish. It is the schematic which shows an erecting microscope. It is the schematic which shows an inverted microscope.

Explanation of symbols

DESCRIPTION OF SYMBOLS 1, 2 Petri dish 10 Cover body 11 Through-hole 12 Plate member 13 Elevation 14,15 Through-hole 20 Container body 21 Through-hole 22 Plate member (glass slip)
23 Plateau 31 Sample 32 Solution 40 Lid 41 Through-hole 42 Plate member 43 Plateau 3 Chamber device 51, 52, 53 Pipe 61, 62, 63 Packing

Claims (10)

A container for containing a sample ;
A lid body with a through hole covering the container body;
At least two pipes that are pushed through the through-holes to supply and discharge the solution to and from the container body;
A sealing member that seals the through hole and holds the pipe tightly;
With
Forming through-holes facing each other in both the lid and the container,
A petri dish characterized in that these through-holes are respectively closed by a plate member capable of transmitting light, which is glass that is optically homogenized and suppresses autofluorescence . Another pipe that is pushed through the through-hole and supplies an additive substance other than the solution into the container.
The petri dish according to claim 1, further comprising: Claim 1 or 2 petri dish according to any one, characterized in that said plate member is a quartz glass. The petri dish according to any one of claims 1 to 3 , wherein the through hole of the lid body is made larger than the through hole of the container body. In the claims 1-4 petri dish according to any one, the lid is penetrated at least two pipes for conducting exhaust the supply of the solution was circulated the solution was placed in the container body through these pipes The chamber apparatus characterized by the above-mentioned. Wherein the additive material other than the solution is passed through the other pipe to be supplied to the container body, according to claim 5, characterized in that the solution was put into the container body through the pipe was circulated through the lid Chamber equipment. The petri dish according to any one of claims 1 to 4 or the chamber apparatus according to claim 5 or 6 , wherein the container body is irradiated with illumination light from above the plate member of the lid, and the plate member of the container body An optical microscope observation method characterized by observing a sample from below with an objective lens. 8. The optical microscope observation method according to claim 7 , wherein oil or liquid is interposed between a condenser lens for condensing the illumination light on a sample and a plate member of the lid. The optical microscope observation method according to claim 7 or 8 , wherein the observation of the sample is a combination of fluorescence observation and differential interference observation. A sample analysis method, wherein the sample is analyzed based on an observation image obtained by the optical microscope observation method according to any one of claims 7 to 9 .

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