JP3357551B2 - Scanning probe microscope and method for measuring freezing cleavage / cleavage of frozen sample using the same - Google Patents

Description

DETAILED DESCRIPTION OF THE INVENTION

[0001]

BACKGROUND OF THE INVENTION 1. Field of the Invention The present invention relates to a scanning probe microscope (SPM) for measuring the surface of a sample by receiving a force from the sample such as an atomic force microscope (AFM), and a method for freezing and breaking a biological sample using the same. Belongs to the technical field of cleavage measurement method,
In particular, the present invention belongs to the technical field of a scanning probe microscope capable of vacuum evacuation and capable of cooling and freezing a sample, and a method for measuring the freezing and cleavage of a biological sample using the same.

[0002]

2. Description of the Related Art An AFM for measuring a sample surface by measuring a physical force generated between a probe and a sample has been conventionally developed. FIG. 5 is a diagram schematically and partially showing an example of such a conventional AFM. In the figure, 1 is AFM,
2 is a laser light source, 3 is a probe, 4 is a cantilever having a probe 3 attached to the tip, 5 is a sample, 6 is a sample stage on which the sample is held, 7 is a sample stage 6 provided at the end, and X
Scanner for moving and adjusting in the directions of the axis, the Y axis (the axis orthogonal to the plane of FIG. 5), and the Z axis; 7X, an X-axis piezoelectric scanning element;
Y is a Y-axis piezoelectric scanning element, 7Z is a Z-axis piezoelectric scanning element, 8 is a mirror, and 9 is a 2-division or 4-division photodiode (shown in FIG. 5 as a 4-division).

In the AFM 1 having such a configuration, first, a sample 5 is set on a sample table 6 on an upper surface of a scanner 7, and then a laser beam is emitted from a laser light source 2 to an upper surface of a cantilever 4, and the reflected light is photo The light enters the diode 9 via the mirror 8. Then, the probe 3 and the sample 5
Are brought closer to each other to a distance of 1 nm or less, an interatomic force (attractive force) between the tip atom of the probe 3 and the surface atom of the sample 5 is obtained.
A repulsive force acts to move the probe 3 up and down, and as a result, the cantilever 4 bends vertically. Due to the bending of the cantilever 4, the position where the reflected light of the laser beam enters the photodiode 9 changes. Due to this change, the output of the photodiode 9 changes, and based on this change in the output, the distance between the probe 3 and the sample 5 is kept constant with respect to the Z-axis piezoelectric scanning element 7Z of the scanner 7 (ie, The feedback control is performed so that the atomic force is kept constant. Then, by performing two-dimensional scanning of the probe 3 or the sample 5 while controlling the distance between the probe 3 and the sample 5, an unevenness image (constant force image) of the surface of the sample 5 is not shown. For example, it is obtained in an image display device.

On the other hand, in recent years, biological specimens have been actively observed using a scanning probe microscope. When observing a biological sample using a scanning probe microscope, especially an AFM, the biological sample is fixed on the biological sample and then labeled on a hydrophilic substrate such as mica to observe the biological sample. The greatest feature of the observation using this AFM is that the observation can be performed without coating or staining the sample, and that most observations are performed in the atmosphere, but observation is possible even in liquid. That is.

[0005] Among such techniques for observing a biological sample, there is a technique called a freezing technique. This freezing technique includes a Cryo SEM method in which a biological sample is frozen and observed using a scanning electron microscope (SEM) as it is,
There is a freeze replica method in which a frozen biological sample is cut, a metal is coated on the cut surface, a replica of the surface is formed, and the replica is observed with a transmission electron microscope (TEM). In particular, the freeze replica method is an epoch-making method in which the state inside the cell membrane can be observed because the cleavage is performed between the double cell membranes when the living body is split.

[0006]

However, in the freeze replica method described above, a transmission electron microscope (TEM) is used.
Therefore, it is not possible to directly observe the biological sample that has been frozen and cleaved, and to observe a replica of the biological sample for observation. For this reason, a biological sample cannot always be observed accurately. In addition, since a replica is required, it takes time and cost to observe a biological sample.

Therefore, it is conceivable to directly observe a biological sample frozen and cut using an AFM. Like this, A
The method of observing the surface of a frozen biological sample frozen by FM is a very effective method because a replica of the biological sample is not required and the frozen surface of the biological sample can be directly observed.

[0008] In order to observe a frozen biological sample as it is by AFM, it is necessary to perform measurement while maintaining the temperature of the sample at a frozen temperature (for example, a temperature of about liquid nitrogen). It is necessary to measure in a vacuum to prevent adsorption from taking place on the surface.

However, conventionally, an AFM that operates in a vacuum at a temperature in which a sample is kept frozen has not been developed. Therefore, when directly observing a biological sample that has been frozen and cut using the conventional AFM as it is, first, the biological sample is frozen and cut using a general-purpose cutting device, and then the frozen and cut biological sample is sealed in a vacuum. It is conceivable that the specimen is transported to an AFM that can evacuate and cool the sample in an evacuated state, and observation is performed. It is conceivable that good observation data may not be obtained due to the fact that frost adheres to the sample surface due to the temperature rise of the sample.

The present invention has been made in view of the above circumstances, and has as its object not to need a replica of a sample to be frozen such as a biological sample, to reduce the degree of vacuum and to prevent frost on the sample surface. By providing a scanning probe microscope that can both prevent adhesion and directly observe the fractured surface of a frozen fractured sample, and a method for measuring the freeze-fracture / cleavage of a frozen sample such as a biological sample using the same. is there.

[0011]

According to a first aspect of the present invention, there is provided a scanning probe microscope in which a sample to be frozen such as a living body is placed on a scanner which is moved by an observation sample stage. A cantilever having a probe arranged opposite to the sample is irradiated with a light beam emitted from a light source, and the scanner is scanned while a reflected light of the light beam reflected by the cantilever is received by a photodetector. Thereby, in the scanning probe microscope adapted to perform observation of the sample, the inside of the housing of the apparatus main body can be evacuated, and the sample is placed on the scanner while maintaining the vacuum in the housing. A sample introduction mechanism to be exchangeable, and installed near the observation sample stage in the housing, A cleaving means for splitting the serial sample, provided in the apparatus main body, and a cooling means for cooling freezing the sample while holding the vacuum within the housing, disposed within said housing, by said sample said splitting means A cleaving stage for moving the sample for cleaving.

The invention according to claim 2 is characterized in that the observation sample stage and the cutting stage are shared by one sample stage. Further, the invention of claim 3 is characterized in that the cutting means is attached to a cantilever holder holding the cantilever.

Further, the invention according to claim 4 is characterized in that a heat conductor for transmitting the cold heat of the cooling means to the cleaving means and the sample while maintaining the vacuum in the housing is provided.

According to a fifth aspect of the present invention, a sample holder holding a sample is placed on a scanner which is moved by a sample stage, and a cantilever having a probe arranged opposite to the sample is emitted from a light source. A scanning probe microscope adapted to observe the sample by irradiating a light beam to be irradiated and scanning the scanner while receiving a reflected light of the light beam reflected by the cantilever by a photodetector. While the inside of the housing of the main body can be evacuated,
A sample introduction mechanism for exchanging the sample on the scanner while maintaining the vacuum in the housing; and cooling means provided on the apparatus main body and cooling the sample while maintaining the vacuum in the housing. And a cleaving means for cleaving the sample in the housing, the sample holder comprises a lower holder holding the sample, and an upper holder for sandwiching the sample between the lower holder, The cleavage means sandwiches the sample between the upper and lower holders, freezes the upper, lower holder and the sample, and further cleaves the upper holder with the lower holder attached to the scanner. This is a cleavage means for cleaving the sample.

According to a sixth aspect of the present invention, there is provided a method for measuring the freeze-fracture / cleavage of a frozen sample, comprising the steps of measuring a frozen sample such as a biological sample using a scanning probe microscope according to any one of the first to fifth aspects. A freeze-cleavage / cleavage measurement method, wherein the sample is cooled and frozen in a vacuum inside the housing, then the frozen sample is cleaved or cleaved, and then the cleaved or cleaved sample is measured by the scanning probe microscope. It is characterized by.

[0016]

The scanning probe microscope of the present invention having the above-described structure, and the use of the scanning probe microscope to freeze and cut a sample to be frozen.
In the cleavage measurement method, a sample to be frozen, such as a biological sample, is cooled and frozen in a vacuum by the cold heat of a cooling means provided in the apparatus main body, and further, a temperature rise of the sample is prevented and the frozen state of the sample is maintained. Then, the sample is cleaved or cleaved in the housing of the apparatus body by cleaving means or cleaving means. Therefore, the AFM observation of the fracture surface of the frozen-fractured sample or the cleavage surface of the frozen-cleaved sample is directly performed without producing a replica.

Further, since the evacuation of the inside of the housing of the apparatus main body can be performed, deterioration of the degree of vacuum due to the transport of the sample in the housing is prevented, and the temperature of the sample is prevented from rising. No frost adheres to the surface of the sample, and the sample can be observed more accurately. As described above, in the same apparatus, the sample is cooled and frozen in a vacuum, and the cooled or frozen sample is cut or cleaved and measured, so that the cut or cleaved surface of the frozen sample forms a replica. Without using it, it will be observed directly and more accurately. For example, when the sample is a biological sample, the biological sample is cleaved or cleaved between its cell membranes, so that the state of the cell membrane that could not be observed conventionally can be observed directly and more accurately without attaching frost. become.

Further, the sample is cut on the sample stage, so that the sample can be observed immediately. The efficiency of observation is improved. In particular, according to the first to third aspects of the present invention, when the sample is not properly cleaved and the cleaved surface of the sample cannot be accurately observed, the sample can be immediately cleaved again. It is not necessary to take out from the device, and the efficiency of observation is further improved.

[0019]

Embodiments of the present invention will be described below with reference to the drawings. FIG. 1 is a diagram schematically showing an example of an embodiment in which a scanning probe microscope according to the present invention is applied to an AFM. The conventional AFM shown in FIG.
The same components as those described above are denoted by the same reference numerals, and a detailed description thereof will be omitted.

As shown in FIG. 1, the AFM 1 of this embodiment has a scanner 7 mounted on a sample stage 10 capable of performing manual coarse movement in the XYZ directions. Scanner 7 in this example
Is formed in a cylindrical shape.

A sample table 6 is attached to the tip 7a of the scanner 7.
Can be attached detachably. As shown in FIG. 2, the sample stage 6 includes a main body 6a and the main body 6a.
and a sample holder holder 6b attached to the sample holder a. The main body 6a has left and right side surfaces 6c and 6d formed as inclined surfaces extending downward, and has a trapezoidal left and right transverse section, and a transfer rod 11 (shown in FIG. 1) is screwed to a rear surface thereof. A female screw hole 6e is formed. On the other hand, the tip 7a of the scanner 7 has a right and left cross section of the same shape as the trapezoid of the right and left cross section of the main body 6a of the sample table 6, and has a groove 7b in which the left and right side surfaces 6c and 6d of the main body 6a fit. It is formed as a planer table equipped.

As shown in FIG. 1, a cylindrical first load lock mechanism 13 extending laterally is hermetically attached to the housing 12 of the main body of the AFM device at a position substantially equal to the height of the planner base 7a. In addition, a cylindrical second load lock mechanism 27 is hermetically attached to the first load lock mechanism 13 via a cylindrical member 26. In this case, the transfer rod 11 is held at the open end of the second load lock mechanism 27 in an airtight and slidable manner, and thus the transfer rod 11 is held at the open end of the second load lock mechanism 27. In this state, the second load lock mechanism 27 is attached to and detached from the tubular member 26. Therefore, the second load lock mechanism 2
When the member 7 is attached to the cylindrical member 26, the transfer rod 11 is arranged in the first and second load lock mechanisms 13, 27. First
The load lock mechanism 13 is provided with a gate valve (not shown) for opening and closing the communication between the first load lock mechanism 13 and the cylindrical member 26, and the second load lock mechanism 27 similarly has a gate valve. Although not shown, a gate valve that opens and closes communication between the inside of the second load lock mechanism 27 and the inside of the tubular member 26 is provided. The cylindrical member 26
Is connected to an exhaust pump (not shown) via an exhaust pipe 28.

Further, the sample stage 6 includes the transfer rod 1
When the female screw 6e of the sample table 6 is screwed into the tip of the sample table 6, the sample screw 6 is detachably attached to the transfer rod 11. Then, the transfer rod 11 to which the sample stage 6 is attached is attached to the planer stage 7 of the scanner 7.
The sample table 6 is mounted on the planer table 7a by advancing toward "a" and fitting the left and right side surfaces 6c and 6d of the sample table 6 into the grooves 7b of the planer table 7a.

Further, a housing 14 for the cooling means is hermetically attached to the housing 12 of the AFM apparatus main body, and a liquid nitrogen tank 15 is accommodated in the housing 14 for the cooling means. This liquid nitrogen tank 1
5 is connected to a heat conducting rod 16.
6 is a housing 12 of the main body of the AFM device which is kept airtight.
Is extended inside. Then, the heat conductive rod 16 and the planer table 7a are formed by stacking a copper thin film on the first
The heat conductor 17 is connected by heat conduction.

Further, an AFM head 19 to which a cantilever holder 18 is mounted is mounted on the main body of the AFM apparatus so as to be located above the scanner 7. As shown in FIG. 3, the cantilever holder 18 is formed in an L-shape. A probe is attached to one end of the L-shape of the cantilever holder 18 at its tip, and a cutter is attached to the other side of the L-shape. The blade 21 is attached via a heat insulating material (not shown). In that case, the cantilever holder 18 is disposed such that one side of the L-shape is in the X-axis direction and the other side is in the Y-axis direction, and therefore, the cutter blade 21 is disposed so as to be orthogonal to the X axis. . This cutter blade 21 is also thermally conductively connected to the heat conducting rod 16 via a second heat conductor 22 formed by laminating copper thin films in the same manner as the above-described planer table 7a.

In the AFM 1 of the present embodiment thus configured, when the biological sample 5 is observed by freezing and breaking, first, the cold heat of the liquid nitrogen in the liquid nitrogen tank 15 is applied to the planer table 7 a and the cutter blade 21. The heat conducting rod 16 and the first and second heat conductors 17, 22 respectively
And the planer table 7a and the cutter blade 21
Are cooled together. In this case, the cold heat of the liquid nitrogen is hardly transmitted from the cutter blade 21 to the cantilever holder 18 by the heat insulating material, so that the cantilever 4 is hardly affected by the cold heat of the liquid nitrogen.

On the other hand, as shown in FIG. 3, the biological sample 5 placed on the sample holder 23 is rapidly frozen in liquid nitrogen or liquid nitrogen (slush) which has been subjected to a freezing treatment. Thereafter, the sample holder 23 is attached to the sample table 6 in liquid nitrogen by pressing the flange 23a of the sample holder 23 with the sample holder presser 6b of the sample table 6, and the sample table 6 is attached to the tip of the transfer rod 11. Attached to. In this state, the first load lock mechanism 13 and the cylindrical member 26 are attached to the housing 12, the gate valve of the first load lock mechanism 13 is closed, and the inside of the housing 12 is isolated from the atmosphere. Evacuated and evacuated to a vacuum. Next, the transfer rod 11 to which the sample stage 6 is attached is inserted into the load lock mechanism 13 and the second load lock mechanism 27 is
6 to the first load lock mechanism 13.

In this state, the gate valve of the first load lock mechanism 13 is kept closed, the gate valve of the second load lock mechanism 27 is opened, and the cylindrical member 26 and the second load lock mechanism 27 are operated by the exhaust pump. Exhaust the inside. Thereafter, the gate valve of the first load lock mechanism 13 is opened, and the transfer rod 11 is advanced toward the planer table 7a while the vacuum in the housing 12 is maintained, and the sample table 6 is mounted on the planer table 7a. Is done. As described above, the transfer rod 11 and the first and second load lock mechanisms 13 and 27 constitute a sample introduction mechanism of the present invention. Since the planer table 7a is cooled by the cold heat of liquid nitrogen, the sample table 6 is
When attached to the biological sample 5, the biological sample 5 on the sample stage 6 is kept in a frozen state without increasing its temperature. Further, the transfer rod 11 is turned in a direction in which the screwing by the screw is released, and is removed from the sample table 6.

Next, the sample stage 10 is manually moved to X
The surface of the biological sample 5 is moved near the cutter blade 21 by the coarse movement mechanism in the -YZ direction. Further, the biological sample 5 is X
By moving in the direction, the surface of the biological sample 5 is cut by the cutter blade 21. In this case, the cutting of the biological sample 5 by the cutter blade 21 is repeatedly performed little by little while raising the biological sample 5 in the Z direction until an observable plane appears on the sample surface. When some observable fractured surface appears, the biological sample 5 is cleaved, and the sample stage 10 moves the biological sample 5 to the position where the biological sample 5 faces the probe of the cantilever 4 in XYZ.
Moved in the direction. At this time, the transfer of the biological sample 5 is performed in the housing 12 in a state where the degree of vacuum is not deteriorated, and the temperature of the biological sample 5 rises because both the planer table 7a and the cutter blade 21 are cooled by the cold heat of liquid nitrogen. As a result, frost does not adhere to the cut surface of the biological sample 5. After that, AF similar to the conventional AFM observation
M observation is performed.

When the AFM observation of the sample is completed, the sample table 6 is detached from the planer table 7a to exchange the sample, and the second load lock mechanism 27 is detached from the cylindrical male member 26. These removals
The above procedure is performed in the reverse order of mounting the sample table 6. That is, after the transfer rod 11 is screwed into the sample table 6, the transfer rod 11 is pulled to remove the sample table 6 from the planer table 7 a, and the sample table 6 is further moved into the cylindrical member 26. Next, while closing the gate valve of the first load lock mechanism 13, the inside of the tubular member 26 and the inside of the second load lock mechanism 27 are leaked to introduce air. Thereafter, the second load lock mechanism 27 is removed from the cylindrical member 26 together with the transfer rod 11, and the sample table 6 is removed.
Is exposed and removed from the transfer rod 11, and the sample stage 6 for a new sample is attached to the transfer rod 11. Thus, the exchange of the sample is performed.

According to the AFM 1 of this example, while maintaining the frozen state of the biological sample 5 by the cooling and heating of the liquid nitrogen in the liquid nitrogen tank 15 provided in the AFM apparatus main body, the frozen surface of the biological sample 5 AFM observation can be performed directly without making a replica.
In this case, the inside of the housing 12 of the AFM device main body can be evacuated, and the deterioration of the degree of vacuum accompanying the transfer of the biological sample 5 to the cutting position and the observation position in the AFM device can be eliminated. And the temperature rise of the biological sample 5 can be suppressed by the cold heat of the liquid nitrogen.
Frost does not adhere to the surface of the biological sample 5, and the sample 5 can be observed more accurately.

Further, the cutting of the biological sample 5 is performed on the sample stage 1.
I go on 0 so I can observe it immediately,
If the cleavage of the biological sample 5 is not successful and the cleavage surface of the sample 5 cannot be accurately observed, the cleavage of the biological sample 5 can be immediately performed again. This eliminates the need to remove the biological sample 5 from the AFM device, thereby greatly improving observation efficiency. In addition, since it is not necessary to make a replica of the biological sample 5, the labor for observation can be further reduced, and the cost for observation can be reduced.

The place where the cutter blade 21 is attached may not necessarily be the cantilever holder 18 as in the present embodiment, but the inside of the housing 12 of the AFM device in which the cleavage position and the observation position of the biological sample 5 are the same. It can be anywhere. In that case, in addition to the sample stage 10 for moving the scanner 7 in the XYZ directions, a cutting XZ
Another stage that coarsely moves in the direction is provided in the same chamber as the sample stage 10, the sample stage 6 is mounted on a stage for cutting by the transfer rod 11, and the biological sample 5 is cut. The AFM observation can also be performed by moving the table 6 from the cutting stage to the sample stage 10.

FIG. 4 is a view similar to FIG. 3, showing another example of the embodiment of the present invention. The same components as those of the AFM of the examples shown in FIGS. 1 to 3 are denoted by the same reference numerals, and detailed description thereof will be omitted.

As shown in FIG. 4, the AFM 1 of this example is
A pair of upper and lower sample holders 23 each including an upper holder 23b and a lower holder 23c are provided. The upper and lower holders 23b and 23c are formed in a U-shape having the same dimensions. A ring 24 is attached to the upper holder 23b, and a cleavage attachment 25 that can be engaged with the ring 24 is provided. The attachment 25 for cleavage is detachably attached to the tip of the transfer rod 11.

The AFM 1 of this embodiment is the same as the AFM 1 of the above-described embodiment.
Cutter blade 21 such as M1 and this cutter blade 21
Is not provided. Another configuration of the AFM1 of the present example is the same as that of the AFM1 of the aforementioned example.
Is the same as

In the AFM 1 of the present embodiment thus configured, the biological sample 5 is placed on the lower holder 23c, and the biological sample 5 is sandwiched by the upper holder 23b. In this state, after the biological sample 5 is rapidly frozen in liquid nitrogen, the sample holder 23 is attached to the sample table 6 by pressing the flange of the lower holder 23c by the sample holder holding metal 6b of the sample table 6. .
Next, the sample stage 6 is attached to the tip of the transfer rod 11 in the same manner as in the above-described example, and the transfer rod 11 is inserted into the load lock mechanism 13, and then a vacuum is drawn in the housing 12 of the AFM device main body.

Next, the sample table 6 is mounted on the planer table 7a by the transfer rod 11, and the transfer rod 11 is removed from the sample table 6. At this time, since the planer table 7a is cooled by the cold heat of the liquid nitrogen, the temperature rise of the biological sample 5 is prevented, and the biological sample 5 is kept in a frozen state. Next, after the attachment 25 for cleavage is attached to the tip of the transfer rod 11,
The transfer rod 11 is operated and the attachment 25 is hooked on the ring 24. When the transfer rod 11 is pulled, the biological sample 5 is cleaved. Thereafter, the sample stage 10 is moved by the coarse movement mechanism in the XYZ directions, and the biological sample 5 is moved to a predetermined position facing the cantilever 4. Conveyed. Thereafter, AFM observation is performed as in the conventional case.

In the AFM 1 of this embodiment, the cutter blade 21 is not provided, and the biological sample 5 is simply cleaved. Therefore, the sample surface is repeatedly cleaved by the cutter blade 21 as in the above-described example. Can not.

Other functions and effects of the AFM 1 of this embodiment are as follows.
Each is the same as the previous example.

[0041]

As is clear from the above description, according to the scanning probe microscope of the present invention and the method for measuring the freezing / cleavage of a sample to be frozen using the same, the cooling means provided in the main body of the apparatus is used. A sample such as a biological sample that is frozen by cold heat is cooled and frozen in a vacuum, and further, while preventing the temperature of the sample from rising and maintaining the frozen state of the sample, the sample is cut or cleaved in the housing of the apparatus body. Since the sample is cleaved or cleaved, the A of the cleaved surface of the frozen cleaved sample or the cleaved surface of the frozen cleaved sample is
FM observation can be performed directly without making a replica, and since a replica of the sample is not required, the time and effort for observation can be further reduced and the cost for observation can be reduced.

Further, since the inside of the housing of the apparatus main body can be evacuated, the degree of vacuum can be prevented from deteriorating due to the transfer of the sample inside the housing, and the temperature of the sample can be prevented from rising. Can be prevented from adhering to the frost. Thereby, the observation of the sample can be performed more accurately. As described above, in the same apparatus, the sample is cooled and frozen in a vacuum, and the cooled and frozen sample is cut or cleaved. Since the measurement is performed by using a replica, the cut or cleaved surface of the sample to be frozen is used as a replica. And can be observed directly and more accurately.
For example, when the sample is a biological sample, the biological sample is cleaved or cleaved between its cell membranes, and the state of the cell membrane, which could not be observed conventionally, can be observed directly and more accurately without attaching frost. become able to.

Further, since the sample is cut on the sample stage, the observation of the sample can be immediately observed, and the observation efficiency can be improved. In particular, according to the first to third aspects of the present invention, the sample is not cleaved properly,
Even if the cleaved surface of the sample cannot be accurately observed, the sample can be immediately cleaved again.
It is not necessary to take out from the observation device, and the observation efficiency can be further improved.

[Brief description of the drawings]

FIG. 1 is a diagram schematically showing a first example of an embodiment of a scanning probe microscope according to the present invention.

FIG. 2 is a perspective view showing a mounting structure of a sample stage in the scanning probe microscope of the example shown in FIG.

FIG. 3 is a diagram for explaining mounting and cutting of a sample in the scanning probe microscope of the example shown in FIG. 1;

FIG. 4 is a diagram illustrating mounting and cutting of a sample in a scanning probe microscope according to another example of the present invention.

FIG. 5 is a diagram partially and schematically showing an example of a conventional AFM.

[Explanation of symbols]

1 ... Atomic force microscope (AFM), 2 ... Laser light source, 3
... probe, 4 ... cantilever, 5 ... biological sample, 6 ... sample stand, 7 ... scanner, 7a ... plane stand, 8 ... mirror, 9 ...
Photodiode, 10: sample stage, 11: transfer rod, 12: housing, 13: rod lock mechanism, 14: housing for cooling means, 15: liquid nitrogen tank, 16: heat conductive rod, 17: first heat conductor, 18 ... Cantilever holder, 19 ... AFM head,
21: cutter blade, 22: second heat conductor, 23
... sample holder, 23b ... upper holder, 23c ... lower holder, 24 ... ring, 25 ... attachment for cleavage

Continuation of front page (56) References JP-A-5-79833 (JP, A) JP-A-7-192677 (JP, A) JP-A-5-133858 (JP, A) JP-A-4-249905 (JP) , A) Keiichi Nakanoto, C.I. B. Mooney, Masashi Iwatsuki, Developm ent of low-tempera ture and high vacu um atomic force mi croscope with free ze-fracture functi on, Review of Scien tific Instruments, the United States, American Instit ute of Physics, 2 May 2001, Vol. 72, No. 2, pp. 1445-1448 (58) Fields investigated (Int. Cl. ⁷ , DB name) G01N 13/10-13/24 G12B 21/00-21/24 G01N 1/28 G01B 21/30

Claims (6)

(57) [Claims] 1. A frozen sample such as a biological sample is placed on a scanner moved by an observation sample stage, and a light beam emitted from a light source is applied to a cantilever having a probe arranged opposite to the sample. And a scanning probe microscope configured to scan the scanner while receiving a reflected light of the light beam reflected by the cantilever by a photodetector, thereby observing the sample. A sample introduction mechanism that allows the inside to be evacuated and allows the sample to be exchanged on the scanner while maintaining the vacuum in the housing, and is installed near the observation sample stage in the housing, Cleaving means for cleaving the sample, provided on the main body of the apparatus, and reducing the vacuum in the housing. A cooling means for cooling frozen before <br/> SL samples lifting state, provided in the housing, and a fracture stage for moving the sample to fracture by the sample said fracture means A scanning probe microscope, characterized in that: 2. The scanning probe microscope according to claim 1, wherein the observation sample stage and the cutting stage are shared by one sample stage. 3. The scanning probe microscope according to claim 1, wherein the cutting means is attached to a cantilever holder that holds the cantilever. 4. The apparatus according to claim 1, further comprising a heat conductor for transmitting cold heat of said cooling means to said cutting means and said sample while maintaining a vacuum in said housing. 2. The scanning probe microscope according to claim 1. 5. A sample holder holding a sample is placed on a scanner moved by a sample stage, and a cantilever having a probe arranged opposite to the sample is irradiated with a light beam emitted from a light source. A scanning probe microscope configured to perform observation of the sample by scanning the scanner while receiving the reflected light of the light beam reflected by the cantilever with a photodetector. A sample introduction mechanism that enables the sample to be exchanged on the scanner while maintaining the vacuum in the housing while allowing exhaust to be performed, and a sample introduction mechanism that is provided in the apparatus main body and maintains the vacuum in the housing. Cooling means for cooling the sample; and cleavage means for cleaving the sample in the housing. A lower holder the sample holder, holding the sample,
Upper holder for holding the sample between the lower holder
Consists of a, the cleavage means, the upper, the sample between the lower holder
Sandwiching and freezing these upper and lower holders and sample
And the lower holder is attached to the scanner.
Cleave the sample by cleaving the upper holder with
A scanning probe microscope characterized in that it is a cleavage means . 6. The run according to any one of claims 1 to 5,
Trial for freezing biological samples using a probe microscopy
A method for measuring the freezing / cleavage of a sample , wherein the sample is cooled and frozen in a vacuum inside the housing,
Cleavage or cleavage of the frozen sample
Is for the cleaved sample measured with the scanning probe microscope
Freezing and cleavage of frozen samples
Measuring method.