JP3053731B2 - Biological sample preparation method for scanning electron microscope and biological sample observation method - Google Patents

Description

DETAILED DESCRIPTION OF THE INVENTION

[0001]

BACKGROUND OF THE INVENTION 1. Field of the Invention The present invention relates to a method for preparing a biological sample for a scanning electron microscope for observing a biological sample containing water with a scanning electron microscope, and a method for observing a biological sample.

[0002]

2. Description of the Related Art When observing a biological sample containing water with a scanning electron microscope, it is necessary to maintain the sample as close as possible to a living body. However, biological samples
If the sample is taken out and inserted into a scanning electron microscope as it is after extraction, the moisture is vaporized and the shrinkage due to drying often occurs due to the vacuum inside the apparatus.

Therefore, at present, a biological sample is processed before it is observed with a scanning electron microscope. FIG.
(A) is shown for explaining the procedure of the conventional biological sample preparation, and the conventional procedure of the biological sample preparation is as follows.

[0004] A sample is extracted. Extraction of the sample needs to be subdivided quickly so that the sample does not dry out.

[0005] The extracted sample is washed. The reason for washing is that mucus and intracellular granules are adhered to the surface of the sample as it is, and there is a possibility that the site to be observed of the sample may be obscured when observed with a scanning electron microscope. Normally, the end of the extracted sample is pinched with tweezers and shaken as it is in a buffer solution (this is 2-3 times).
Times). The time required for this cleaning is about 30 minutes.

[0006] The fixed sample is subjected to a fixing process. Fixing is to cure the sample by applying a chemical change. When fixing a biological sample, it is usually 1 to 2.5.
% Glutaraldehyde fixative, 1% osmium fixative, etc. are used. The fixation methods are "single fixation", which fixes only one fixative, and "two fixatives," which use two kinds of fixatives sequentially. There is "fixed heavy". Glutaraldehyde fixative mainly fixes proteins only, whereas osmate fixative is fixed relatively hard because proteins and lipids are fixed. In "single fixation", a biological sample is left in a glutaraldehyde fixative for 1 to 2 hours or more and in an osmium fixative for 1 to 2 hours or less. In the "double fixation", a biological sample is left in a glutaraldehyde fixative for 1 to 2 hours or more.

[0007] The biological sample is washed with a buffer to remove the fixative that has not chemically reacted with the sample. If excess fixative remains, crystals such as osmic acid precipitate on the sample surface when the sample is dried, which hinders observation. The time required for this cleaning is about 30 minutes to 2 hours.

[0008] The water in the biological sample is converted to ethyl alcohol,
A dehydration treatment is performed to replace and remove the organic solvent such as acetone. This is because the organic solvent has a surface tension of 1 compared to water.
This is because the influence of shrinkage deformation is small by being about / 3 smaller, and the drying time is short. However, if a sample containing water is directly placed in a 100% organic solvent, the dehydration will rapidly proceed and the sample will shrink and deform. At first, alcohol (acetone) etc. is diluted with purified water (pure, distilled water). Then, a sample containing water is immersed in a 50% solution to be dehydrated. Similarly, 60%, 70%, 80%, 90%, 95%
A solution is prepared, and the sample is sequentially transferred into a high-concentration solution. The time for immersing the sample in one solution is about 5 to 15 minutes. Finally, the biological sample is immersed in 100% ethyl alcohol and dehydrated. The sample is immersed for about 30 minutes.
Dehydration treatment is performed twice. In addition, the sample which is easily deformed is subjected to a dehydration treatment from a 2% solution.

As described above, the process up to the dehydration treatment of the sample has been completed.
Subsequent procedures differ depending on the critical point drying method and the t-butyl alcohol freeze drying method. The procedure of the critical point drying method will be described in the section, and the procedure of the t-butyl alcohol freeze drying method will be described in the section.

The biological sample subjected to the dehydration treatment is placed in isoamyl acetate (amyl acetate) and left for about 15 minutes.

The sample subjected to the substitution process is placed in a pressure-resistant container, filled with liquid carbon dioxide at room temperature, and the sealed container is heated to a critical temperature or higher, and the pressure of the container increases. When the pressure in the container reaches a certain pressure, carbon dioxide is instantaneously changed from a liquid to a gas. When the gas is gradually discharged out of the container, the sample dries.

The sample subjected to the dehydration treatment is t-
Put in butyl alcohol. The sample replaced with t-butyl alcohol is placed on a sample stage of a freeze-drying apparatus, and the stage is cooled down to 5 ° C (or may be left in a refrigerator for several tens minutes). When the t-butyl alcohol is frozen, the inside of the freeze-drying device is slowly evacuated, and the frozen t-butyl alcohol sublimates. When sublimation is over and the pressure starts to drop sharply, the stage temperature is returned to room temperature, and when it is brought to atmospheric pressure, a dried sample is completed.

[0013]

As described above, the conventional procedure for preparing a biological sample has been described. FIG. 2 is a drawing showing a secondary electron image of ananabena treated by t-butyl alcohol lyophilization method using a scanning electron microscope. It is a substitute photograph. FIG.
(A) is a secondary electron image of anabena in which the fixative is 2% glutaraldehyde and dehydration is performed from 20% alcohol, and the anabaena is contracted. FIG. 2B is a secondary electron image of the anabaena processed under the same conditions, and the anabaena is deformed. As described above, when the anabena is treated by the t-butyl alcohol freeze-drying method, the anabena shrinks or deforms and cannot maintain its original shape (living body). Also, there is no reproducibility in this method, such as shrinkage or deformation. FIG. 2 (c) is a secondary electron image of anabena in which the fixative is 2% glutaraldehyde and dehydration is performed from 10% alcohol, but the anabaena is contracted. In addition,
Although the secondary electron image of the sample processed by the critical point drying method is not given, the same problem as the t-butyl alcohol freeze drying method occurs in the critical point drying method.

Conventionally, the critical point drying method and t
-When the sample is deformed in the process of preparing the sample by the butyl alcohol freeze-drying method, the deformation of the sample cannot be confirmed until a scanning electron microscope image is observed. For this reason, even if a sample is prepared with great effort, the operation may be wasted.

In the preparation of samples by the critical point drying method or the t-butyl alcohol freeze-drying method, pretreatment work including chemical fixation, specialized knowledge and proficiency are required. Is required.

An object of the present invention is to provide a method for preparing a biological sample for a scanning electron microscope and a method for observing a biological sample, which solve such problems.

[0017]

Means for Solving the Problems A scanning electron microscope biological sample preparation method of the present invention to achieve this object, the surface water
The method is characterized in that the sample covered with is frozen, and the frozen sample is heated to a temperature higher than zero degree in a state where the frozen sample is placed in the evacuated sample chamber. Further, the biological sample observation method of the present invention freezes a sample whose surface is covered with water.
In a state where the frozen sample is placed in a evacuated sample chamber, the temperature of the frozen sample is raised to a temperature higher than zero degree, and the state of the sample during the temperature rising process is measured by a scanning electron microscope. The observation is performed by

[0018]

Embodiments of the present invention will be described below with reference to the drawings. FIG. 3 is a schematic view of a scanning electron microscope shown as an embodiment of the present invention. In FIG. 3, reference numeral 1 denotes a lens barrel of a scanning electron microscope, 2 denotes a sample chamber, and 3 denotes an exhaust device for exhausting the sample chamber 2. 4 is a sample stage, and sample stage 4 is X,
It is movable in the Y and Z directions. 5 is a heat absorber. 6 is a thermo module, the thermo module 6 is
Π in which a P-type semiconductor 7 and an N-type semiconductor 8 are joined by a metal electrode 9
A single or a plurality of Peltier elements composed of a mold series circuit are sandwiched between ceramic plates 10 in parallel. When a current flows in the direction of arrow A, the thermo module 6 generates heat absorption at the joint thereof due to the Peltier effect, and generates heat on the opposite side. The fever is reversed. Therefore, heating and cooling can be performed electronically by changing the direction of the current.
11 is a sample holder and 12 is a biological sample. FIG. 4 shows a biological sample 12 placed on a sample holder 11. Biological sample 12 is covered with water 18. Note that an alcohol solution may be used instead of water. 13 is a temperature detector for detecting the temperature of the sample holder 11, and 14 is a control device. Reference numeral 15 denotes a memory, and the memory 15 stores a target temperature of the sample holder after a lapse of a predetermined time from the start of heating of the sample holder, as shown by T in FIG. 16 is a power supply, and 17 is a vacuum gauge.

The operation of this device will be described below.

The controller 14 sends a cooling signal to the power supply 16, that is, a signal for flowing a current to the thermo module 6 in the direction of the arrow A. As a result, the thermo module 6
The sample holder 11 located above is cooled, and the sample 12 is also cooled. The temperature of the sample holder 11 is detected by a temperature detector 13, and the detection signal is sent to a control device 14.
When the temperature of the sample holder 11 becomes −45 ° C., the control device 14 stops supplying the cooling signal to the power supply 16. The control for stopping the supply of the cooling signal at the time of −45 ° C. by the control device 14 is based on the data stored in the memory 15. By this cooling, the sample 12 freezes while being covered with the water 18. When the cooling of the sample is completed, the controller 1
4 sends an exhaust signal to the exhaust device 3, which exhausts the sample chamber 2. The degree of vacuum in the sample chamber 2 is measured by a vacuum gauge 17, and a signal thereof is sent to the control device 14.
The controller 14 sets the degree of vacuum in the sample chamber 2 to 0.01 Torr or more.
Set to a low vacuum state between 2 Torr.

Next, the control device 14 sends a heating signal to the power supply 16, that is, a signal for flowing a current in the direction of the arrow B. As a result, the sample holder 11 located on the thermo module 6 is heated, and the sample 12 is also heated. The temperature of the sample holder 11 is detected by a temperature detector 13, and the detection signal is sent to a control device 14. Control device 14
Compares the temperature stored in the memory 15 with the temperature sent from the temperature detector 13, and based on the data stored in the memory 15, the temperature of the sample holder that can be considered to be substantially the same as the temperature of the sample is compared. The power supply 16 is controlled so as to gradually increase at a preset speed. In this way, the temperature change of the sample ends when it reaches room temperature, for example, 15 ° C.

Here, the change in the state of the sample 12 due to the heating and the reason why the biological sample does not deform or the like will be described.

The ice frozen in an arched or flat shape begins to sublime due to the relationship between the vacuum in the sample chamber and the temperature value of the sample holder. The speed depends on the temperature rise curve of the holder. It is necessary to slightly change the sublimation speed depending on the structure of the cell membrane of the biological sample. The reason is to match the sublimation speed of the ice in the cells. When the balance is lost, contraction deformation is induced in the biological sample. That is, it is important that all the ice inside the sample is sublimated before the ice covering the sample is sublimated.

By the way, as the ice inside the sample sublimates, the incident electron beam diffuses to the deep part of the sample, and the reflected electrons generated inside can be detected, so that the internal structure can be observed like an optical microscope. is there. Heretofore, since the chemical fixation was performed, the mass of the cells was increased by the chemical reaction, and the diffusion of the incident electrons became difficult, so that the internal observation could not be performed as expected. The sample image during the heating of the sample is displayed on a display device (not shown), and by observing the image, the operator can confirm whether or not the sample is deformed during the temperature rise process.

An example of a reflected electron image in a low vacuum state and a secondary electron image in a high vacuum state of a sample subjected to the above-mentioned temperature treatment under a scanning electron microscope will be described. When observing the sample subjected to the temperature treatment in a high vacuum state,
The sample is coated to prevent image degradation due to charge-up.

FIG. 5 is a photograph substituted for a drawing showing a backscattered electron image of the Spirogyra in a low vacuum state. The Spirogyra does not shrink, and the Spirogyra remains in its original form (living body).
Further, in the preparation of the sample of the present invention, since the sample is not chemically fixed as in the prior art, the inside of the spider can be clearly observed. FIG. 6A is a drawing substitute photograph showing a backscattered electron image of algae generated in water in a low vacuum state, and FIG. 6B is a drawing substitute photograph showing a secondary electron image of the algae in a high vacuum state. It is a photograph. As is clear from this image, there is no shrinkage of the algae, and the algae almost maintains its original form (living body). FIGS. 7A and 7B are drawing-substituting photographs showing the backscattered electron image of the algae in a low vacuum state.
3 is a drawing substitute photograph showing a secondary electron image of algae in a high vacuum state. FIG. 7 is a photograph at a magnification higher than the magnification of the photograph shown in FIG. 6, and it can be further understood that the algae maintain the original form (living body). FIG. 8A is a drawing substitute photograph showing a backscattered electron image of the algae in a low vacuum state.
(B) is a drawing substitute photograph showing a secondary electron image of algae in a high vacuum state. FIGS. 9 (a) and 9 (b) are drawing-substitute photographs showing a backscattered electron image of a daphnia in a low vacuum state, and FIGS. 10 (a) and (b) show secondary electron images of a daphnia in a high vacuum state. It is a drawing substitute photograph shown. As is clear from this image, there is no deformation of the daphnia, and the daphnia almost maintains its original form (living body).

FIG. 1 (b) summarizes the operation procedure for preparing the biological sample of the present invention described above. The time required for preparing the biological sample of the present invention is about several hours, and the time required for preparing the biological sample is greatly reduced as compared with the related art.

In the above embodiment, the memory 1
5 is provided to control the temperature rise of the sample based on the information stored in the memory. However, the present invention can be implemented without providing the memory and the control means. In such a case, it is necessary to select the flow rate of heat from the surroundings to the sample holder and the heat capacity of the sample holder appropriately so that the temperature of the sample rises at approximately the same rate as when controlling the temperature rise of the sample. It is. Further, the temperature of the sample may be controlled so that the temperature of the sample rises within the range of the hatched portion in FIG. Further, in the above description, the preparation of the sample was performed in the scanning electron microscope. However, in the apparatus having the above-described configuration, that is, in the apparatus including the means for cooling and heating the sample, the means for exhausting the sample chamber, and the like. The sample may be prepared by using

[0029]

According to the present invention, a sample whose surface is covered with water is provided.
In a state where the frozen sample is frozen and the frozen sample is placed in a evacuated sample chamber, the temperature of the frozen sample is raised to a temperature higher than zero degree, so that a scanning electron microscope image of a biological sample that maintains its original shape without deformation Can be obtained, and since the chemical fixation is not performed as in the related art, the internal structure of the sample can be observed.
Further, in the present invention, the conventional fixation, dehydration, replacement, critical point drying and freeze drying do not have to be performed, so that there is no danger due to chemicals. Further, since the preparation of the sample of the present invention is very simple, the quality of the sample is not affected by the technical knowledge and proficiency, and the time for preparing the sample is significantly reduced as compared with the conventional case. Also, the present invention freezes a sample whose surface is covered with water.
And the frozen sample was placed in the evacuated sample chamber .
State, allowed to warm the frozen sample to a temperature above zero degrees,
Since the state of the sample during the temperature rise process is observed with a scanning electron microscope, if the sample is slightly deformed in the process of sample preparation, it is possible to know whether the sample has been deformed, and the biological sample obtained with the scanning electron microscope Image of the original form (living body)
It can be determined whether or not the image of the biological sample is maintained. In the present invention, chemical fixation is not performed,
The completed sample can be stored.

[Brief description of the drawings]

FIG. 1 shows a conventional procedure for preparing a biological sample and a procedure for preparing a biological sample of the present invention.

FIG. 2 is a drawing substitute photograph showing a backscattered electron image of ananabena treated by t-butyl alcohol lyophilization method with a low vacuum scanning electron microscope.

FIG. 3 is a schematic view of a scanning electron microscope shown as an example of the present invention.

FIG. 4 shows a biological sample placed on a sample holder.

FIG. 5 is a drawing substitute photograph showing a backscattered electron image of a Spirogyra subjected to the treatment of the present invention in a low vacuum state.

FIG. 6 is a drawing substitute photograph showing a scanning electron microscope image of algae treated according to the present invention.

FIG. 7 is a drawing substitute photograph showing a scanning electron microscope image of algae treated according to the present invention.

FIG. 8 is a drawing substitute photograph showing a scanning electron microscope image of the algae treated according to the present invention.

FIG. 9 is a drawing substitute photograph showing a backscattered electron image of a daphnia treated with the present invention in a low vacuum state.

FIG. 10 is a drawing substitute photograph showing a secondary electron image of a daphnia subjected to the treatment of the present invention in a high vacuum state.

FIG. 11 is a diagram for explaining a sample holder heating temperature after a lapse of a predetermined time from the start of sample holder heating.

[Explanation of symbols]

 DESCRIPTION OF SYMBOLS 1 Column 2 Sample chamber 3 Exhaust device 4 Sample stage 5 Heat sink 6 Thermo module 7 P-type semiconductor 8 N-type semiconductor 9 Metal electrode 10 Ceramic plate 11 Sample holder 12 Sample 13 Temperature detector 14 Controller 15 Memory 16 Power supply 17 Vacuum Gauge 18 water

──────────────────────────────────────────────────続 き Continuation of front page (56) References JP-A-3-165435 (JP, A) JP-A-63-269039 (JP, A) JP-A-61-751 (JP, U) JP-A-2-75 147 (JP, U) Hikaru Hei 2-2649 (JP, U) (58) Fields investigated (Int. Cl. ⁷ , DB name) G01N 1/28 G01N 33/48 G01N 23/225 H01J 37/20

Claims (2)

(57) [Claims] 1. A method of freezing a sample whose surface is covered with water,
Frozen sample placed in a evacuated sample chamber
In a scanning electron microscope biological sample preparation method characterized by raising the temperature of the frozen sample to a temperature above zero degrees. 2. A method of freezing a sample whose surface is covered with water,
Frozen sample placed in a evacuated sample chamber
A method for observing a biological sample, comprising: raising the temperature of the frozen sample to a temperature higher than zero degree, and observing the state of the sample in the process of increasing the temperature with a scanning electron microscope.