JP2009229339A - Sample preparation method for microscopic observation, and kit used for the same - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To identify a cell to be observed using an electron microscope among floating cells in a liquid, by using an optical microscope, and observe the identified cell in detail by using the electron microscope. <P>SOLUTION: A sample preparation method includes: a first process for preparing a mixed liquid of the floating cells and a mesh polymer at a temperature that the mesh polymer is not converted into a gel; a second process for forming a layer, containing the floating cells and the mesh polymer from the mixed liquid on a substrate, and converting the layer into the gel; a third process for observing the gel layer by using the optical microscope, and identifying the floating cell to be observed for the electron microscope; and a fourth process for preparing an ultrathin slice as a sample for the electron microscope so as to contain a portion of the cell identified in the third process, after the cell has been fixed and embedded in the gel layer. <P>COPYRIGHT: (C)2010,JPO&INPIT

Description

  The present invention relates to a method for preparing a sample for observing floating cells floating in a liquid with a microscope, and a kit for preparing the sample.

  Conventionally, an electron microscope has been widely used as a means for observing the ultrastructure inside a cell. In order to perform observation with a resolution of the order of several nanometers with an electron microscope, it is necessary to solidify a cell sample to be observed, and then make an ultrathin slice with a thickness of 100 nanometers or less including a round slice of the cell sample. . For this reason, it is extremely difficult to reconstruct a sample for observation with an electron microscope, and cell samples that were not included in the sample are destroyed and discarded when the sample is prepared. Care must be taken to ensure that it is included.

  A sample for observation with an electron microscope is obtained by fixing a cell sample to be observed with a chemical fixing agent, embedding (embedding) it in a resin, and slicing the solidified sample into an ultrathin section with a microtome (for example, Patent Documents 1 to 4 and Non-Patent Document 1).

  Among cells, adherent cells that can be attached to the surface of a glass plate or the like can easily locate target cells by displaying position coordinates on the surface. It becomes possible to observe. That is, after searching and confirming the target cell with an optical microscope, fixation and embedding can be performed by the above-described method, the same cell can be searched with reference to the position coordinates, and an electron microscope observation sample including this can be prepared . As a result, it is possible to select specific cells that are carrying out the reaction intended for research and observe them in detail with an electron microscope.

  However, floating cells such as Paramecium and blood cells are not stationary in the liquid and move freely. Therefore, it is difficult to specify the position of the cell in both the vertical and horizontal directions, and even if the target cell is specified by observation with an optical microscope, it is necessary to fix and embed the floating cell. The position moved, and it was extremely difficult to observe the same cell as observed with an optical microscope with an electron microscope.

  In particular, the influence of drugs on blood cells has become a social problem in recent years, and specific cells affected by drugs are selected from a group of blood cells floating in a liquid, and this is used as an optical microscope. Observing with both electron microscopes was an urgently important issue.

In addition, since surviving floating cells freely move around in the liquid, it was difficult to observe one living cell over time with an optical microscope. For this reason, it has been desired to develop a method that can fix floating cells in a alive state and observe them with an optical microscope by a simple technique.
JP 2007-309872 A Japanese Patent Laid-Open No. 5-306979 JP 2006-177692 A JP 2005-174657 A Usukura Jiro, "Electron microscope technology for structural cell biology", "1. Ultrathin section method as basic technology" and "2. Immunocytochemistry", [online], 2007, Hitachi High-Tech, [2008] February 12, 2011], Internet <URL: http://www.hitachi-hitec.com/em/emworld/technique/index.html>

  In view of the present situation described above, the present invention specifies a cell to be observed with an electron microscope from floating cells suspended in a liquid using an optical microscope, and the identified cell is detected with an electron microscope. An object of the present invention is to provide an electron microscope observation sample preparation method that enables detailed observation.

  It is another object of the present invention to provide an optical microscope observation sample preparation method that enables suspension or movement of floating cells to be observed with an optical microscope.

  As a result of intensive studies to solve the above problems, the present inventors mixed floating cells with a sol of a network polymer such as agarose, and applied the obtained mixture to a centrifuge or applied pressure to the mixture from above. After adding the floating cells in layers by adding them, the floating cells can be quiesced by making them into a gel by the action of the above-mentioned network polymer. It has been found that the sample can be observed and that a sample for observation with an electron microscope can be prepared by performing normal cell fixation or embedding in a resin with respect to the layer obtained above. The present invention has been completed.

  That is, the first aspect of the present invention is a first step of preparing a mixed solution of floating cells and a network polymer sol at a temperature at which the network polymer does not gel, and from the mixed solution, the floating cells and A layer containing the network polymer is formed on a substrate, the second step of making the layer into a gel state, and the gelled layer is observed with an optical microscope to be a target for electron microscope observation A third step for identifying cells, and after fixing and embedding the gelled layer, an ultrathin section, which is an electron microscope observation sample, is a part of the cells identified in the third step. And a fourth step of manufacturing the sample for electron microscope observation.

  The second aspect of the present invention is a first step of preparing a mixed solution of floating cells and a network polymer sol at a temperature at which the network polymer does not gel, and from the mixed solution, the floating cells and the The present invention relates to a method for producing a sample for optical microscope observation, including a second step of forming a layer containing a network polymer on a substrate and making the layer into a gel.

  The third aspect of the present invention is a kit for preparing a sample capable of observing floating cells with an optical microscope and a sample capable of observing the floating cells with an electron microscope, and is used for specifying a position of a mesh polymer and unevenness. And a substrate having the following indication.

  According to the first aspect of the present invention, a cell to be observed by an electron microscope is searched for and identified from among floating cells suspended in a liquid by an optical microscope. The structure can be observed with an electron microscope.

  According to the second aspect of the present invention, it becomes possible to observe with an optical microscope by stopping floating or movement of floating cells suspended in a liquid by a very simple technique.

  According to the third aspect of the present invention, the sample preparation method for microscopic observation according to the first aspect of the present invention and the second aspect of the present invention can be easily implemented.

  First, a method for producing an electron microscope observation sample according to the first aspect of the present invention will be described in detail. Since each step in the method for producing an optical microscope observation sample according to the second invention is the same as the first step and the second step in the first invention, the description thereof is omitted.

  In the first step, a mixed solution of floating cells and a network polymer sol is prepared at a temperature at which the network polymer does not gel.

  In the present invention, a floating cell means a cell that does not adhere to other cells or tissues and survives in a state where it can freely move in a liquid. Specific examples include, for example, blood cells such as red blood cells and white blood cells, and self-propelled unicellular organisms such as Paramecium and Tetrahymena that move by moving cilia growing on the cell surface. In the first aspect of the present invention, the floating cells may be pre-fixed using a glutaraldehyde solution, a formaldehyde solution, or a mixed solution of both, or a living cell may be used. Good. In any case, by observing floating cells with an optical microscope in the third step, cells having characteristics that are desired to be observed with an electron microscope from among floating cells, or a stage where observation with an electron microscope is desired Confirm the cells in and identify them as target cells.

  The suspended cells may be used as they are in the culture solution, but it is preferable to collect and use the concentrated suspension cells by centrifuging the culture solution.

  A network polymer means a polymer having a three-dimensional network network formed by covalent bonds, and when dispersed in an aqueous medium, it becomes a sol state (showing fluidity) at a high temperature. Then, it is in a gel state (not showing fluidity). Specific materials include natural polymers such as agarose and synthetic polymers such as polyacrylamide. From the viewpoint of cytotoxicity, natural polymers such as agarose are preferable. Further, as the network polymer, it is desired to select a polymer that can maintain a certain degree of transparency when gelled so as not to interfere with microscopic observation.

  The network polymer sol used in the first step is obtained by dispersing the network polymer in an aqueous medium such as water and heating it to a gelling temperature or higher. For example, when the gelation temperature is about 30 ° C., the network polymer sol can be easily obtained by heating to about 60 ° C. Since the gelation temperature varies depending on the concentration of the network polymer, the temperature during heating is determined in consideration of this point.

  In the first step, a mixed solution of floating cells and a reticular polymer sol is prepared, and the temperature at the time of adjustment is a temperature at which the reticular polymer does not gel. That is, the suspended cells and the reticulated polymer are sufficiently mixed in a state where the mixed solution maintains fluidity, and the suspended cells and the reticulated polymer are uniformly dispersed in the mixed solution.

  When cells that have been pre-fixed as floating cells are used, the suspension cells and the sol of the reticulated polymer are mixed at a temperature that does not destroy the ultrastructure of the floating cells.

  On the other hand, when living cells are used as floating cells and they are subject to observation with an optical microscope in a living state, the mixture of the floating cells and the reticular polymer sol is mixed with the temperature at which the floating cells can survive. To do. As described above, when heating to about 60 ° C. for sol formation, the temperature of the sol is lowered to a temperature at which floating cells can survive, and then mixed with the floating cells. For example, when a unicellular organism (for example, Paramecium or Tetrahymena) that is active under normal outside air temperature is used as the floating cell, the temperature during mixing is preferably about 10 to 30 ° C. When blood cells are used as floating cells, it is preferable that the temperature during mixing be around 36 ° C.

  For this reason, the network polymer used preferably has a property of not gelling (solidifying) even at such a low temperature. From this viewpoint, when the network polymer is agarose, commercially available “low melting point agarose”, for example, SeaPlaque (registered trademark) Agarose manufactured by Cambrex, can be preferably used.

  The amount and concentration of the reticulated polymer are adjusted so that the reticulated polymer gels at an appropriate temperature, and the floating cells can be entangled with the network of reticulated polymer and become stationary. .

  In the second step, a layer containing the floating cells and the network polymer is formed on the substrate from the mixed solution of the floating cells obtained in the first step and the network polymer sol, and the layer is gelled. Shape. Since the layer containing floating cells is gelled by the action of the network polymer, the floating cells become entangled with the network of the network polymer, and the movement of the floating cells can be stopped. This makes it possible to observe stationary floating cells. Further, even when a living cell is used as a floating cell, the cell can survive in the gelled layer, so that the living floating cell can be made stationary and observed.

  According to an embodiment of the present invention, the formation of the layer can be achieved by subjecting the mixed solution to a centrifugal operation to form a layer in which the floating cells are concentrated. When the mixed solution is put in a container and subjected to a centrifugal operation, floating cells are concentrated in the lowermost part of the mixed liquid. The lowermost part corresponds to the aforementioned layer, and the bottom wall of the container corresponds to the aforementioned substrate. . In this lowermost part, the floating cells do not overlap each other in the vertical direction, and the floating cells are arranged in almost one layer, so that it can be suitably used as a sample for optical microscope observation. The number of rotations and time during the centrifugal operation may be adjusted so that the lowermost part is suitable for observation with an optical microscope (cell arrangement, layer thickness and cell concentration). It is about 1 minute at several 3000 rpm.

  The lowermost part contains not only floating cells but also a network polymer at a concentration sufficient for gelation. During and after the centrifugal operation, the temperature of the mixture decreases, and the network polymer gels, and the entire mixture including the bottom solidifies (the fluidity of the mixture is lost). ). The lowermost part of the liquid mixture thus solidified is set as an observation object by an optical microscope.

  As a container for storing the liquid mixture during the centrifugal operation, it is preferable that the inner surface of the bottom wall has a position specifying display by unevenness. Thereby, when the target cell is specified by the optical microscope observation in the third step, the position can be easily recorded. Therefore, when preparing an ultrathin section in the fourth step, it becomes easy to include a part of the identified target cell in the ultrathin section. As such a container, a commercially available dish with an address can be used.

  According to another embodiment, the formation of the layer in the second step is performed by applying pressure from above to the mixed solution of the floating cells and the network polymer sol obtained in the first step (stretching) the substrate. This can be achieved by thinning the film. Specifically, the mixed solution may be placed on a substrate such as the above-mentioned addressed dish or glass plate, and the prepared mixed solution may be stretched thinly on, for example, a preparation. After stretching, the preparation can be removed. According to this embodiment, the floating cells do not overlap each other when viewed from the vertical direction, and are arranged in almost one layer, so that a sample for observation with an optical microscope can be suitably used. Moreover, in this form, since floating cells can be pressed down strongly, the movement of the self-propelled cells such as Tetrahymena that are actively swimming can be stopped relatively easily.

  The thickness of the thin film to be formed should be determined in consideration of the size of the floating cells. For example, when Tetrahymena having a size of about 50 μm in length and width and about 30 μm in height is used, the thickness is about 100 μm. A thin film may be formed. By thinning the thickness of the thin film in this way, observation with an optical microscope becomes easy.

  Further, as described above, it is convenient in a later step to use a substrate having a surface-specific indication by unevenness as described above. During and after the pressure application, the temperature of the thin film decreases, whereby the network polymer is gelled and the thin film is solidified (the fluidity of the thin film is lost). The thin film thus solidified is an object to be observed with an optical microscope.

  In the third step, the gelated layer in the second step is observed with an optical microscope, and from these, floating cells to be observed with an electron microscope are searched and selected. The cell selected here is a specific cell for which it is desired to observe its ultrastructure with an electron microscope, or a cell in a specific stage for which similar observation is desired. Note that observation with an optical microscope can be performed with a resolution of micrometer order.

  Although the layer obtained in the second step is gelled, it retains transparency to the extent that it can be observed with an optical microscope. When this is observed with an optical microscope, the floating cells can be observed in a stationary state. This is the same even if the floating cell is a living cell. However, it is not necessary for observation with an optical microscope that all cells in the floating cells are stationary, and it is only necessary that some cells are stationary. The suspension of floating cells by the method of the present invention is considered to be caused by the floating cells becoming entangled with the network of gelled network polymer. In particular, when floating cells are self-propelled cells such as Tetrahymena, the one in which two cells are joined in the middle of cell division tends to get entangled in the network of reticulated macromolecules and capture the stationary state. Cheap.

  As the optical microscope used in the present invention, a fluorescence microscope can be used. In this case, the floating cells may be stained with a fluorescent reagent in advance before the first step. This makes it possible to search for a cell that is performing a specific reaction intended for observation with an electron microscope in a molecule-specific manner. When searching for a cell using a fluorescence microscope, for example, a reaction in the cell nucleus may be searched using the shape or size of the fluorescence image as a clue. Such a fluorescent reagent is not particularly limited as long as it can stain the cell nucleus. For example, if Hoechst 33342 or DAPI is used, the DNA portion can be stained.

  According to the present invention, when the floating cell is a living cell, the intracellular activity of the floating cell can be observed over time by an optical microscope. Since floating cells, especially free-running floating cells, were very difficult to observe the intracellular activity of quiescent live cells over time, the present invention made it possible to observe over time. Significance is enormous. In addition, by observing living cells over time, we have identified cells that are in an academically important stage, such as the moment of cell division where changes are most noticeable from a structural or functional point of view. When fixation or embedding is performed, the ultrastructure of the cells at such a stage can be observed with an electron microscope.

  As described above, in the case where the substrate has a display for specifying the position by unevenness on the surface, when the target cell is specified, the display in the vicinity thereof may be recorded. When this recorded display is used as a mark in the fourth step, it becomes easy to prepare an ultrathin section including the specified cell.

  In the fourth step, after the target cells are identified in the third step, the gelled layer is subjected to cell fixation and embedding, and then an ultrathin section, which is an electron microscope observation sample, is obtained. It is prepared so as to include a part of the target cell specified in the process. Observation with an electron microscope enables observation with a resolution of nanometer order.

  The above-mentioned cell fixation aims to maintain the structure and physical properties of living cells as much as possible, while embedding means that cells can be prepared so that ultrathin sections including cell round slices can be prepared. The purpose is to allow the resin to penetrate inside and cure. Cell fixation and embedding are generally known as those used when preparing an ordinary electron microscope observation sample, and the drugs and conditions used are not particularly limited.

  When a transmission electron microscope is used, the cells and the network polymer can be chemically fixed by immersing the sample after selecting the specific cells in the third step in an osmium acid solution. (However, when living cells are used as floating cells, they are pre-fixed by dipping in a glutaraldehyde solution, formaldehyde solution, or a mixture of both before dipping in an osmic acid solution.) After dehydrating the sample using ethanol, the sample is sufficiently infiltrated with a curable resin such as an epoxy resin. Next, the curable resin is cured by heating or ultraviolet irradiation. As a result, the sample is solidified after the network polymer and the cells and the resin are fused. When the centrifugation operation is used in the second step, the whole mixed solution containing the layer is solidified in such a state that the layer containing the floating cells contained in the network polymer is substantially in contact with the surface of the substrate. In addition, when pressure is applied in the second step, a thin film made of suspended cells and a network polymer is solidified.

  Thereafter, the solidified material is peeled off from the substrate such as the bottom wall of the dish. When the surface of the substrate has a display for specifying the position by the unevenness, the display in which the unevenness is inverted is engraved on the surface of the solidified product that has been peeled off, which is in contact with the substrate. Even after cell fixation and embedding, the position of floating cells that have been quiesced by the reticulated polymer has not changed, so use the display stamped on the sample surface as a mark to identify in the third step. Target cells can be easily found.

  Next, while confirming the display engraved on the surface of the sample with a stereomicroscope, trimming is performed using a file or razor in order to cut out the site containing the specific cells selected in the third step from the separated solidified product. After trimming, the machined part is sliced with a microtome, and an ultrathin section as an electron microscope observation sample is cut out. Thereby, an ultrathin section including a part of the cells (slice of cells) identified in the third step can be prepared. However, the portion including the unevenness imprinted on the sample surface is discarded so that it is not included in the ultrathin slice as the sample for electron microscope observation.

  The ultrathin section prepared in this way contains some of the target cells identified in the third step, so after passing through the necessary treatment, it was identified with an optical microscope in the third step by observing with an electron microscope. The ultrastructure of the target cell can be observed.

  A kit for preparing a microscope observation sample according to the third aspect of the present invention will be described. This kit is for preparing both a sample in which floating cells can be observed with an optical microscope and a sample in which the floating cells can be observed with an electron microscope. The first and second aspects of the present invention are implemented. Can be used to

  The kit includes a reticulated polymer and a substrate having an indication for position determination by unevenness, and these are described above. When the network polymer is dispersed in an aqueous medium, it becomes a sol state at a high temperature and becomes a gel state at a low temperature, but the gelation temperature is within a temperature range where the floating cells can survive. It is preferable to select. As a result, the first step and the second step of the first invention and the second invention can be carried out in a temperature range where the floating cells can survive. Is possible. The substrate having a display for specifying the position by unevenness may be a container having the display on the inner surface of the bottom wall, or a glass plate having the display.

  The kit according to the present invention includes reagents and instruments necessary for preparing a sample for optical microscope observation or a sample for electron microscope observation (for example, a reagent used for cell fixation or embedding) in addition to the network polymer and the substrate. May be provided.

  The kit can be used based on the contents already described above, but for the convenience of the user, depending on the type and properties of the network polymer, the concentration of the polymer when suspended in an aqueous medium, It is advisable to attach specifications regarding temperature control for sol-formation or gelation, suspension cell pretreatment method, centrifuge rotation speed, time, and the like. This makes it possible to produce a sample with good reproducibility. These conditions can be appropriately determined by those skilled in the art based on the above description and the description of the following examples.

  The present invention will be described in more detail with reference to the following examples, but the present invention is not limited to these examples.

Example 1
<Electron microscope observation procedure when pre-fixed cells are embedded in agarose>
Tetrahymena cells were subjected to centrifugation to precipitate, and the supernatant was removed. A fixing solution (4 mM paraformaldehyde and 2.5% glutaraldehyde in 10 mM Tris-HCl (pH 7.5)) was added to the precipitated cells, and the mixture was allowed to stand at 4 ° C. for 2 hours for prefixation. As paraformaldehyde, special reagent paraformaldehyde powder for electron microscope (Nacalai Tesque) is used, and as glutaraldehyde, special reagent glutaraldehyde (25% aqueous solution) for electron microscope (Nacalai Tesque) It was used.

  Prefixed cells were stretched and collected, and the precipitated cells were washed with a phosphate buffer. In order to observe the morphology of the cell nucleus with a fluorescence microscope, the cell nucleus was stained with DAPI (4 ′, 6-diamidino-2-phenylindole).

  Agarose (SeaPlaque (registered trademark) Agarose, manufactured by Cambrex) was added to the phosphate buffer so as to be 0.5%. This was heated in a microwave oven to completely dissolve and cooled to 37 ° C.

  The cell suspension and 0.5% agarose solution were mixed at a volume ratio of 1: 4 to obtain a mixture. 10 L of the mixture was placed on an addressed dish (35 mm Glass Base Dish with Grid, Grid Size: 175 μm, IW AKI), and the dish was immediately centrifuged (centrifugation condition: centrifuge LC-21 (TOMY)), Rotation speed 3000 rpm, centrifugation time 1 minute). At this time, the cells settled to the bottom of the dish, and the agarose was cooled and solidified.

  To prevent the cells and agarose from drying, 100 ml of a phosphate buffer was added, and the cell morphology and intracellular structure (particularly cell nuclei) were observed with a fluorescence microscope (IX70 manufactured by OLYNPUS). The whole image of the cell and the address of the dish were observed with transmitted light, and the cell nucleus was stained with DAPI, so it was observed with fluorescence.

The phosphate buffer was removed, and a phosphate buffer containing 1% OsO ₄ was added to the cells, followed by chemical fixation at room temperature for 1 hour. After removing OsO ₄ and washing the cells with ultrapure water, ethanol was added and dehydrated.

  Epoxy resin (EPON812 RESIN EMBEDDING KIT manufactured by TAAB EMBEDDING MATERIALS) was infiltrated into the cells. The resin was cured by placing in an incubator (60 ° C.) and allowing to stand for 16 hours. At this time, a capsule containing a resin was placed on the sample.

  After confirming that the resin was cured, it was peeled off from the dish. At this time, since the cells were embedded in the resin, they were detached together.

  The exfoliated material was set in a microtome (EM UC6 manufactured by Leica) with the surface in which the cells were embedded facing up. After scraping off other than the target site with a razor, an ultrathin section was prepared with a diamond knife.

  The sections were placed on a grid. The entire grid was set on a sample stage of an electron microscope (JEM-2000EX II manufactured by JEOL) and observed with an electron microscope.

  The photograph obtained by the above fluorescence microscope observation and electron microscope observation is shown in FIG. In FIG. 1, A is an observation image by bright field, B is an observation image of cell nuclei by fluorescence, C is an image in which A and B are superimposed, and D is a portion surrounded by a white square in C. Is an image observed with an electron microscope.

Example 2
<Electron microscope observation procedure when living cells are embedded in agarose>
The same procedure as in Example 1 was performed except the points described below. The agarose solution having a concentration of 3% was cooled to about 30 to 33 ° C. and then mixed with the cell suspension.

  A mixture of cells and agarose was placed on an addressed dish, and after agarose was hardened, a cell culture solution was added. At this time, the agarose was thinly solidified on the dish so that the cells in the agarose can easily breathe. As a thinning method, the same centrifugal treatment as in Example 1 or a method of pressing agarose with a cover glass or the like was used.

  Similarly, it observed with the fluorescence microscope. As a result, the living floating cells were allowed to rest and observed over time.

Next, the cells in agarose were fixed using the fixing solution used in Example 1. Then washed with ultrapure water, fixed after in OsO ₄ the same manner as in Example 1, to create a super-thin sections, it is possible to perform electron microscopy.

  According to the present invention, it is possible to observe floating cells with an optical microscope, specify target cells, and then further observe the ultrastructure with an electron microscope. Therefore, the internal structure of floating cells such as blood cells can be obtained. In order to elucidate the functions and their functions, electron microscope observation can be carried out efficiently and reliably. Therefore, it is possible to accelerate the elucidation of cell functions such as gene delivery, and it can be expected to greatly improve the efficiency of the determination of the medicinal effects on cells, which is indispensable for the drug discovery business in the pharmaceutical and agricultural fields.

  Furthermore, according to the present invention, it is expected that a new inspection function will be given to an inspection apparatus for blood cells, etc., and electron microscope observations in the life science field where dangerous and important issues such as environment and aging are piled up. Can provide a very efficient means for the preparation of medical samples.

Fluorescence micrograph and electron micrograph obtained in Example 1

Claims (9)

A first step of preparing a mixture of floating cells and a reticular polymer sol at a temperature at which the network polymer does not gel;
A second step of forming a layer containing the floating cells and the network polymer on the substrate from the mixed solution, and forming the layer into a gel;
A third step of observing the gelled layer with an optical microscope to identify floating cells to be observed with an electron microscope;
Fourth step of preparing an ultrathin section, which is a sample for electron microscope observation, so as to include a part of the cells specified in the third step after cell fixation and embedding on the gelled layer. Including,
Preparation method of sample for electron microscope observation.   The preparation method according to claim 1, wherein the floating cell is a living cell, and the mixed liquid preparation in the first step is performed at a temperature at which the floating cell can survive.   The production method according to claim 1 or 2, wherein the network polymer is agarose.   The manufacturing method according to any one of claims 1 to 3, wherein the substrate has a display for position identification by unevenness on the surface thereof.   The layer formation in the second step is achieved by subjecting the mixed solution to a centrifugal operation to form a layer in which the floating cells are concentrated.   The layer formation in a 2nd process is a manufacturing method in any one of Claims 1-4 achieved by applying a pressure from above to the said liquid mixture located on the said board | substrate, and developing the said liquid mixture.   The production method according to claim 1, wherein the floating cells used in the first step are stained with a fluorescent reagent, and the optical microscope is a fluorescent microscope. A first step of preparing a mixture of floating cells and a reticular polymer sol at a temperature at which the network polymer does not gel;
Forming a layer containing the floating cells and the network polymer from the mixed solution on a substrate, and making the layer into a gel;
Preparation method of sample for optical microscope observation. A kit for preparing a sample capable of observing floating cells with an optical microscope and a sample capable of observing the floating cells with an electron microscope,
A network polymer,
And a substrate having a display for position identification by unevenness.