JP2018026197A - Microscopic observation method and microscopic observation auxiliary device - Google Patents

Abstract

PROBLEM TO BE SOLVED: To provide a method capable of providing microscopic observation at high resolution by stabilizing the position of an organic sample, such as a biological sample, in a solution, in microscopic observation, and a microscopic observation auxiliary device therefor.SOLUTION: A solution containing a sample is dropped on a stage, an elastic film is placed so as to cover the solution on the stage, and the sample is pressed to the stage by discharging the solution while deaerating between the stage and the film.SELECTED DRAWING: Figure 3

Description

  The present invention relates to a method for observing a sample in a solution on the stage of a microscope and an auxiliary device thereof, and more particularly to a method for stabilizing the position of a sample and providing a microscope observation with high resolution and a microscope observation auxiliary device therefor.

  An inverted optical microscope is known for the upright type. This is because the objective lens is mounted upward on the lower side of the stage. For example, even if an organic sample floats in a solution such as a biological sample, the sample is placed in a petri dish placed on the stage. Observable from below. At this time, not only a thin glass layer with a petri dish bottom but also a solution exists between the objective lens and the sample, so the distance between the objective lens and the sample cannot be reduced, and the resolution does not come out. Therefore, high-resolution observation is also performed by allowing only a sample to settle and adhere to the bottom of the petri dish by some method.

  By the way, an inverted scanning electron microscope (SEM) that emits an electron beam from below to above has also been proposed. However, the path of the electron beam from the electron gun needs to be maintained in a vacuum state higher than a certain level, and the structure becomes complicated compared to an optical microscope.

  For example, Non-Patent Document 1 describes an atmospheric pressure SEM having an open sample chamber as such an inverted SEM. The upper optical microscope section and the lower SEM section are arranged coaxially with the sample in between, so that the optical microscope observation and the SEM observation can be given simultaneously in the same field of view. The partition wall that separates the optical microscope portion and the SEM portion is provided with a through hole through which an electron beam passes, and this is blocked by a very thin film that allows the electron beam to pass therethrough. This thin film constitutes a sample stage on which the sample is placed. That is, since the sample is disposed on the optical microscope unit side and is under atmospheric pressure, the SEM unit side can be evacuated regardless of the sample, and the electron beam stability is excellent, so high-resolution SEM observation is possible.

  On the other hand, in the above-described inverted atmospheric pressure SEM, the sample is irradiated with an electron beam through the electron beam transmission film that separates the optical microscope unit side and the SEM unit side. It is necessary to form thinly so as to increase the thickness. On the other hand, it is also necessary to have a mechanical strength that can withstand the pressure difference between both sides of the membrane.

  Therefore, for example, Patent Document 1 discloses a dish using a SiN film that can withstand such a pressure difference even if the mechanical strength is high and thin. According to this, the biological sample etc. on a dish can be observed by SEM with high resolution using an inverted SEM.

JP 2008-267889 A

Komine et al .; Development of underwater observation method using atmospheric pressure electron microscope ASEM; Synthesiology, Vol. 3, pages 116-126

  When observing an organic sample in a solution such as a biological sample in an inverted atmospheric pressure SEM as described above, if the sample is lifted from the electron beam transmission film of the dish, the electron beam is rapidly attenuated to lower the resolution. I will let you. Therefore, it is necessary to stably observe the position of the sample on the electron beam permeable film.

  The present invention has been made in view of the situation as described above, and the object of the present invention is to stabilize the position of an organic sample in a solution such as a biological sample in high-resolution observation under a microscope. The present invention relates to a method capable of providing microscopic observation and a microscope observation auxiliary device.

  An observation method according to the present invention is a method of observing a sample in a solution on a stage of a microscope, wherein the solution containing the sample is dropped on the stage, and an elastic film is applied so as to cover the sample on the stage. It is characterized in that the sample is pressed against the stage by discharging the solution while degassing between the stage and the film.

  According to this invention, it is possible to give a high-resolution microscope observation by pressing while discharging the solution so that the position of the sample in the solution is stabilized on a stage such as an electron beam permeable film.

  In the above-described invention, the microscope may be a scanning electron microscope, and the scanning electron microscope may be an inverted type. Further, the elastic film may have a light transmission property through which the sample can be observed with an optical microscope. According to such an invention, observation with an optical microscope can be performed together, and observation assistance by SEM can be provided.

  In the above-described invention, the elastic film may be silicon rubber. The elastic film may be a balloon body, and the interior of the cup provided on the stage may be decompressed so as to cover the balloon body after the balloon body is placed on the solution. Good. According to this invention, the solution can be efficiently discharged without damaging the sample, and the position of the sample can be stabilized on a stage such as an electron beam permeable film, so that high-resolution microscope observation can be provided.

  In the above-described invention, the balloon body may have a conduit that communicates with the outside of the cup, and the internal pressure thereof may be controlled. According to this invention, the solution can be discharged more efficiently, the position of the sample can be stabilized on a stage such as an electron beam permeable film, and a higher resolution microscopic observation can be provided.

  An observation auxiliary device according to the present invention is an auxiliary device for observing a sample in a solution on a stage of a microscope, and gives the solution containing the sample dripped on the stage so as to cover the stage. And a deaeration means for deaerating between the stage and the film, and the sample can be pressed against the stage by discharging the solution.

  According to this invention, it is possible to give a high-resolution microscope observation by pressing while discharging the solution so that the position of the sample in the solution is stabilized on a stage such as an electron beam permeable film.

  In the above-described invention, the microscope may be a scanning electron microscope, and the scanning electron microscope may be an inverted type. Further, the elastic film may have a light transmission property through which the sample can be observed with an optical microscope. According to such an invention, observation with an optical microscope can be performed together, and observation assistance by SEM can be provided.

  In the above-described invention, the elastic film may be silicon rubber. Further, the elastic film is a balloon body, the cup body is placed on the solution, and the cup is placed on the stage so as to cover the balloon body. Means. According to this invention, the solution can be efficiently discharged without damaging the sample, and the position of the sample can be stabilized on a stage such as an electron beam permeable film, so that high-resolution microscope observation can be provided.

  In the above-described invention, the balloon body may have a conduit that communicates with the outside of the cup, and the internal pressure thereof may be controlled. According to this invention, the solution can be discharged more efficiently, the position of the sample can be stabilized on a stage such as an electron beam permeable film, and a higher resolution microscopic observation can be provided.

  In the above-described invention, the cup may be a suction cup that is attracted to the stage. According to this invention, the solution can be discharged more efficiently, the position of the sample can be stabilized on a stage such as an electron beam permeable film, and a higher resolution microscopic observation can be provided.

It is a figure which shows the structure of an inverted microscope. It is a figure which shows the principle of operation of this invention. It is sectional drawing of the principal part of FIG. It is a figure which shows the Example of this invention. It is a scanning electron micrograph of the biological sample as a comparative example. FIG. 6 is a scanning electron micrograph of a biological sample similar to FIG. 5 observed using the apparatus of FIG.

  Hereinafter, a method for observing a sample in a solution, particularly an organic sample in a solution such as a biological sample, on the stage of an inverted scanning electron microscope according to one embodiment of the present invention will be described with reference to FIGS. However, it will be explained.

  As shown in FIG. 1, the microscope 1 is also referred to as an atmospheric pressure scanning electron microscope (ASEM) and opens a sample chamber to a low vacuum or atmospheric pressure. Therefore, it is suitable for observing a sample that requires pretreatment to be placed in a vacuum, for example, an organic sample in a solution such as a biological sample.

  The microscope 1 includes an upper optical microscope section A and a scanning electron microscope section B including a lower vacuum chamber 21.

  A movable stage 10 is attached to the upper surface of the vacuum chamber 21 (not shown), and a dish 12 is disposed on the movable stage 10 so as to be movable in a horizontal plane. A window 15 is provided at the center of the dish 12, and the electron beam permeable film 14 closes the window 15. The electron beam permeable film 14 is a thin film body that is partially or wholly thin enough to transmit an electron beam, and is a thin film that has a strength that does not break even by negative pressure due to evacuation inside the vacuum chamber 21. is there. As such a thin film, an inorganic material thin film, such as a thin film made of SiN, is known.

  In the optical microscope section A, the sample 2 is arranged on the electron beam transmission film 14 of the dish 12, and from above, the objective lens 3 moves up and down and right and left to give an optical microscope observation. Note that the eyepiece and the like are not shown.

  In the scanning electron microscope section B, the inside of the vacuum chamber 21 can be evacuated by a vacuum means 21a including a vacuum pump, and an electron beam is directed upward from the electron gun 22 based on control from the control section 22a. Fired. An electron beam from the electron gun 22 passes through the electron beam transmissive film 14 and is irradiated to the sample 2. The reflected electron beam or the like from the sample 2 is detected by the electron detector 23, and is appropriately observed by a scanning electron microscope by the image display means 23a.

  Here, as shown in FIG. 2A, the sample 2 is in the solution 2a and is on the electron beam transmission film 14 as a droplet Sp. At this time, since the sample 2 can float in the solution 2a, its position is not stable and cannot be observed with an electron microscope with high resolution. Therefore, it is necessary to stabilize the position of the sample 2 by pressing it onto the electron beam transmission film 14 serving as an observation stage.

  As shown in FIG. 2B, a cup 30 containing a balloon 32 made of an elastic film such as silicon rubber slightly inflated with air or the like is placed on the droplet Sp, and the inside of the cup 30 is not shown. Deaerate and depressurize by decompression means. The balloon body 32 is inflated by the internal pressure, the shape is regulated along the inner periphery of the cup 30, and the sample 2 floating in the droplet Sp is pressed against the electron beam permeable film 14. At this time, it is preferable to control the internal pressure of the balloon body 32 and the deaeration / depressurization state of the cup 30 so as not to destroy the sample 2.

  Needless to say, the deaeration and decompression in the cup 30 by the decompression means are completely different from the decompression as in the vacuum chamber 21 of the scanning electron microscope section B. In addition, it is preferable that the space in the cup 30 is reduced by the state in which the balloon body 32 is expanded, and only the solution 2 a of the droplet Sp is discharged to the outside of the cup 30.

  Further, FIG. 3 shows in more detail how the sample 2 in the droplet Sp is pressed against the electron beam permeable film 14.

  As shown in FIG. 3A, before the degassing in the cup 30 or in the initial stage of degassing, the elastic film of the balloon body 32 surrounds the droplet Sp lightly on the electron beam permeable membrane 14, that is, It is preferable that it exists so as to “paste” it. For this purpose, the flexibility and the internal pressure of an elastic film such as silicon rubber are adjusted.

  As shown in FIG. 3B, when the inside of the cup 30 is deaerated, the solution 2a is discharged outward, and the sample 2 is pressed against the electron beam permeable membrane 14. At this time, by wrapping the sample 2 with the elastic film of the balloon body 32, the position of the sample 2 on the electron beam permeable film 14 becomes more stable, and the solution 2a is formed between the sample 2 and the electron beam permeable film 14. It does not intervene, and makes the transmission of the electron beam better. That is, it enables scanning electron microscope observation with higher resolution.

  Further, if the elastic film of the balloon body 32 is a material having optical transparency, the sample 2 can be observed with an optical microscope from above through this, and the observation assistance for the observation with the scanning electron microscope is given.

  According to the embodiment described above, the solution 2a is discharged and pressed so as to stabilize the position of the sample 2 in the solution 2a on the stage such as the electron beam permeable film 14, and the high resolution by the scanning electron microscope is obtained. It can give observation.

  Next, the state of observation with a scanning electron microscope according to another embodiment of the above-described embodiment will be described.

  As shown in FIG. 4A, the microscope observation auxiliary device 50 includes a cup 40 having a bellows-like suction cup 40b that is attached to a stage including the electron beam permeable membrane 14, and a balloon 32 made of silicon rubber inside the cup 40. Consists of.

  For the suction cup 40b of the cup 40, a PVC suction pad for FSGA22 packaging machine made by Schmalz, Germany was used. The silicon tube 40a was connected to this, and it connected to the pump (not shown). The balloon body 32 is a balloon B type # 3204 for posterior nasal cavity manufactured by Koken Co., Ltd., and a silicon tube 32a is connected to the balloon body 32 through a hole in the middle through the inside of the silicon tube 40a of the cup 40. Pulled out. The periphery of the hole was sealed with an adhesive for silicon so that air did not leak.

  First, a droplet Sp provided with sample 2 (the lung is taken out from the mouse into a sliced slice. The lung tissue contains metastasized breast cancer cells) is dropped onto the electron beam permeable membrane 14 in water 2a. And the suction cup 40b was covered so that the droplet Sp might be covered. When the silicon tube 32a of the balloon body 32 is closed and the inside of the suction cup 40b is degassed and depressurized from the silicon tube 40a, the height of the suction cup 40b is increased while pressing the balloon body 32 against the electron beam permeable membrane 14. It will be reduced. At this time, the elastic film of the balloon body 32 swells along the inner surface of the suction cup 40b due to internal pressure, and stops in balance with the contraction of the suction cup 40b. In addition, the sample 2 is pressed onto the electron beam permeable membrane 14 while discharging the water 2a to the outside so that the tip 40b 'of the suction cup 40b is turned up.

  Note that the pressure of the sample 2 on the electron beam permeable film 14 can be adjusted by taking air in and out of the silicon tube 32 a of the balloon body 32.

  FIGS. 5 and 6 show photographs in the scanning electron microscope observation when the above-described microscope observation auxiliary device 50 is not used and when it is used. In FIG. 6, the entire field of view is in focus and observation is possible over a wide range. The small nuclei indicated by white arrows in FIG. 6 (b) are the nuclei of normal alveolar cells, and the white arrows in FIG. 6 (c) represent the large nuclei of breast cancer cells that have metastasized. On the other hand, in FIG. 5, it is dark except for a part of the lower left, and the range of just focus is very narrow. This is because the sample 2 is floating in the water 2 a and the sample 2 cannot be pressed onto the electron beam permeable film 14.

  As described above, by using the microscope observation auxiliary device 50, high-resolution scanning electron microscope observation can be easily performed. In addition, even if the microscope observation auxiliary device 50 is a case where the sample 2 is pressed onto the electron beam permeable film 14 from below, it can be dealt with by placing the sample 2 facing upward.

  Here, for example, not only a scanning electron microscope but also various microscopes such as a high-resolution optical microscope and a super-resolution optical microscope are applicable as long as they form an image from fluorescence that warps through a thin film glass. Can be observed. The apparatus could also be used in a high resolution optical microscope where the optical system is upside down and upright as a whole.

  As mentioned above, although the Example by this invention and the modification based on this were demonstrated, this invention is not necessarily limited to this, A person skilled in the art will deviate from the main point of this invention, or the attached claim. Various alternative embodiments and modifications could be found without doing so.

DESCRIPTION OF SYMBOLS 1 Microscope 2 Sample 2a Solution 3 Objective lens 10 Movable stage 12 Dish 14 Electron beam permeable film 15 Window 21 Vacuum chamber 22 Electron gun 23 Electron detector 23a Image display means 30, 40 Cup 32 Balloon body 40b Suction cup 50 Microscope observation auxiliary device A Optical microscope part B Scanning electron microscope part

Claims (15)

A method of observing a sample in a solution on a microscope stage,
The solution containing the sample is dropped on the stage, an elastic film is placed so as to cover it on the stage, the solution is discharged while degassing between the stage and the film, and the sample is removed. A microscope observation method characterized by being pressed against a stage.   The microscope observation method according to claim 1, wherein the microscope is a scanning electron microscope.   The microscope observation method according to claim 2, wherein the scanning electron microscope is an inverted type.   3. The microscope observation method according to claim 2, wherein the elastic film has a light transmission property through which the sample can be observed with an optical microscope.   The microscope observation method according to claim 1, wherein the elastic film is silicon rubber.   The elastic film is a balloon body, wherein the inside of a cup provided on the stage is decompressed so as to cover the balloon body after the balloon body is placed on the solution. 5. The microscopic observation method according to 5.   The microscope observation method according to claim 6, wherein the balloon body has a pipe line communicating with the outside of the cup, and the internal pressure thereof is controlled. An auxiliary device for observing a sample in a solution on a microscope stage,
An elastic film provided so as to cover the solution containing the sample dropped on the stage on the stage, and degassing means for degassing the stage and the film, A microscope observation auxiliary device, wherein the sample can be ejected and the sample can be pressed against the stage.   9. The microscope observation auxiliary device according to claim 8, wherein the microscope is a scanning electron microscope.   The microscope observation auxiliary device according to claim 9, wherein the scanning electron microscope is an inverted type.   10. The microscope auxiliary device according to claim 9, wherein the elastic film has a light transmission property through which the sample can be observed with an optical microscope.   12. The microscope auxiliary apparatus according to claim 11, wherein the elastic film is made of silicon rubber.   The elastic film is a balloon body, and a cup is placed on the stage so as to cover the balloon body after the balloon body is placed on the solution, and a decompression means for decompressing the inside of the cup. The microscope observation auxiliary device according to claim 12, comprising:   14. The microscope observation assisting apparatus according to claim 13, wherein the balloon body has a conduit communicating with the outside of the cup, and the internal pressure thereof can be controlled. The microscope auxiliary device according to claim 13 or 14, wherein the cup is a suction cup that is adsorbed to the stage.

Images