JPH0416200Y2 - - Google Patents

Description

[Detailed description of the device] a. Purpose of the device (industrial field of application) The quick freezing device for biological specimens according to this device is used in the fields of medicine, biology, etc. to process biological specimens excised from living bodies. It is used when making specimens for microscopic observation.

(Prior Art) In the fields of medicine and biology, a biological sample excised from a living body is processed into a microscopic specimen and observed using an optical or electron microscope. In order to perform specimen processing work on such biological samples,
Conventionally, biological tissues have been fixed with various water-soluble fixatives such as formalin, glutaraldehyde, and osminic acid, and degreased with alcohol. However, in the case of biological samples that move quickly, such as muscles, the tissue itself deforms during the fixation process, making it impossible to observe the original morphology of the tissue. Also, during fixation using a water-soluble fixative or degreasing using alcohol, amino acids, sugars,
The elution of water-soluble substances such as various ions is unavoidable, and the biological tissue changes considerably during the treatment process, making accurate microscopic observation impossible.

In order to eliminate these inconveniences, biological tissues are instantly frozen with extremely low temperatures of helium or nitrogen, and then
A quick freezing device as shown in FIG. 10 for fixing tissue is commercially available. This quick freezing device
The interior of a container 2 provided at the center of the base 1 and a helium tank 3 can be freely communicated with each other, and a copper block 4 provided at the upper opening of this container 2 can be freely cooled to an extremely low temperature.
A sample holder 7 is provided at the lower end of an elevating rod 6 which is provided above the container 2 so as to be movable up and down by a pair of upper and lower bearings 5,5.

When rapidly freezing a biological sample collected from a living body, raise the lifting rod 6 as shown in FIG.
A biological sample is attached to the lower surface of the sample holder 7 at the lower end of the lifting rod 6 while the locking piece 8 at the upper end of the lifting rod 6 and the in/out piece 9a of the solenoid 9 are engaged. When the attachment of the biological sample to the sample holder 7 is completed, the shutter 10 that has been covering the top of the copper block 4 is opened and the solenoid 9 is energized to lower the elevator rod 6. As the lifting rod 6 falls, the biological sample adhering to the lower surface of the sample holder 7 comes into contact with the upper surface of the cryogenic copper block 4 and is instantly frozen. Iron ring 1 provided around sample holder 7
1 is attracted to the electromagnet 12 around the container 2 when the lifting rod 6 falls.

However, the conventional rapid freezing apparatus for biological samples constructed and used as described above suffers from the following disadvantages. That is, when a sample attached to the lower surface of the sample holder 7 is brought into contact with the upper surface of the copper block 4, the lifting rod 6 with the sample holder 7 at the lower end is allowed to fall freely, so that the sample and the upper surface of the copper block collide with force. However, the tissue in the sample is easily destroyed and accurate microscopic examination cannot be performed.

Utility Model Application No. 59-77077 discloses a rapid freezing device for biological samples that solves these inconveniences. The device for rapidly freezing biological samples that has been previously devised is equipped with a removable sample holder on a slider that can be moved up and down along a guide vertically disposed in front of the substrate, and the slider is equipped with a removable sample holder that can be moved up and down using an external force. A mechanism is attached to the top surface of the table that protrudes forward from the bottom end of the board.
A block made of a metal with good heat conductivity such as copper or silver and having a mirror surface on the upper surface is fixed so that the mirror surface and the lower surface of the sample holder face each other.Furthermore, a block is provided below the table that can be raised and lowered by external force. A cryogen tank is provided, and an extremely low-temperature liquid cryogen such as liquid helium or liquid nitrogen is placed in this cryogen tank with an open top.When the cryogen tank rises, the metal block on the top of the table is immersed in the liquid cryogen. It is designed to be immersed in.

4 and 5 show a first embodiment of the quick-freezing device for biological samples according to the recent invention. First, the quick-freezing device according to the recent invention will be explained. A slider 17 is fitted onto a rod-shaped guide 16 which is vertically supported on the front surface of a vertical substrate 13 via brackets 14 and 15 so as to be able to move up and down. A bearing is provided at the fitting portion between the guide 16 and the slider 17, so that the slider can be moved up and down smoothly without rattling. A sample holder 1 is attached to an arm 18 projecting to the side of this slider 17.
9 is detachably provided. This sample holder 19
The structure of the attachment/detachment part between the arm 18 and the arm 18 can be of various conventionally known structures, but the attachment/detachment part must be constructed so as not to wobble.

On the other hand, a rack 21 is fixed to a slide plate 20 which is supported on the front surface of the base plate 13 so as to be movable up and down by a guide member (not shown). In this rack 21,
A gear 2 pivotally mounted on the front surface of the board 13 by a shaft 22
3 are engaged. Furthermore, a worm wheel 24 is coaxially connected to this gear 23. The worm 25 meshed with the worm wheel 24 is connected to the output shaft of a motor 26, and by rotating the motor 26 forward or reverse, the slide plate 20 can be moved up and down.

A bracket 27 projects from the side of the slide plate 20 closer to the slider 17, and the upper end of a screw rod 28 is fixed to the end of this bracket 27. The lower end of this screw rod 28 is attached to the slider 1 described above.
A double nut 30 is loosely engaged with a bracket 29 projecting laterally from the bracket 7, and a double nut 30 is screwed into a portion projecting downward from the bracket 29. A compression spring 32 is provided between the lower surface of the double nut 31 screwed together between the upper and lower brackets 27 and 29 and the upper surface of the lower bracket 29, and a constant elasticity determined by the screwing position of the upper double nut 31 is provided. so that the slider 17 can be pressed downward.

Below the sample holder 19 attached to the slider 17, there is a metal block 3 whose mirror-finished upper surface faces the biological sample 34 on the lower surface of the holder.
3 is fixed to the upper surface of an L-shaped bracket 35 fixed to the lower end of the substrate 13.

Furthermore, a cryogen tank 36 that can be raised and lowered is provided so as to surround the lower part of a bracket 35 having such a metal block 33 fixed to its upper surface. A cryogen tank 36 storing an extremely low temperature liquid cryogen 37 such as liquid nitrogen.
is placed on a saucer 41, and this saucer 41 is connected to a lifting rod 43 that is movable up and down along guides 42, 42 by engagement of a pinion 39 fixed to the output shaft of a motor 38 and a rack 40. . Therefore, the cryogen tank 36 is raised and lowered by rotating the motor 38 in the forward or reverse direction, and the metal block 33 on the upper surface of the bracket 35 is immersed in the liquid cryogen 37 or exposed outside the liquid cryogen 37. .

Reference numeral 44 denotes a liquid level sensor for detecting whether or not the metal block 33 is immersed in the liquid cryogen 37, and is supported by the substrate 13 via a bracket 45. Also, inside the cryogen tank 36, both ends are connected to the cryogen tank 36.
A flexible pipe 46 is provided which is led out and has one end opened toward the upper surface of the metal block 33 and the other end connected to a pressure gas supply device such as a nitrogen gas cylinder.
An electromagnetic on-off valve 47 is provided in a portion of the flexible pipe 46 that is exposed from the liquid cryogen 37.

The work of freezing and fixing a biological sample using the previously invented biological sample quick-freezing apparatus constructed as described above is carried out as follows.

(1) The motor 38 drives the pinion 39 and the rack 4.
0, the lifting rod 43 is raised to raise the cryogen tank 36 placed on the saucer 41, and the metal block 33 fixed to the upper surface of the bracket 35 is immersed in the liquid cryogen 37. By this operation, if the liquid cryogen 37 is liquid nitrogen, the metal block 33
is cooled to an extremely low temperature of around -196℃.

(2) After attaching a 1 mm square biological sample 34 taken from the tissue to be examined to the lower surface of the removable sample holder 19 using glycerin or other suitable means, the sample holder 19 is attached to the slider 17. Attach it to the arm 18 of.

(3) The elevator rod 43 that reverses the motor 38 is lowered, and the cryogen tank 36 storing the liquid cryogen 37 is lowered to expose the upper surface of the metal block 33 to the outside of the liquid cryogen. Droplets of liquid cryogen remaining on the upper surface of the metal block 33 are blown away by a small amount of cooling gas blown out from the end of the corrugated pipe 46 by opening the electromagnetic on-off valve 47 for a short time. Since the cooling gas is cooled by the liquid cryogen 37, it does not increase the surface temperature of the metal block 33, and since a small amount of gas is sufficient to blow off the cryogen droplets, only the gas cooled in the corrugated pipe 46 is used. It is enough.

(4) Next, rotate the motor 26 in the normal direction, and turn the worm 2
5, worm wheel 24, gear 23, rack 21,
The screw rod 28 is lowered via the slide plate 20 and the bracket 27 at a constant speed of about 250 mm/sec. As a result, the slider 17 descends with the descent of the screw rod 28 while keeping the bracket 29 sandwiched between the compression spring 32 and the double nut 30, and at the same time, the sample holder 19 with the biological sample 34 attached to its lower surface also moves toward the metal block 33. descend.

(5) When the biological sample 34 comes into contact with the upper surface of the metal block 33, the sample holder 19 and slider 17 stop descending, and the slide plate 20 and bracket 2
7. Only the screw rod 28 and the double nuts 30, 31 are slightly lowered, and a portion of these abuts against a micro switch (not shown), thereby stopping the motor 26.
Therefore, the biological sample 34 is pressed against the upper surface of the metal block 33 with a constant force of, for example, about 1.5 kg due to the elasticity of the compression spring 32.
The above pressing force can be changed by adjusting the position of the upper double nut 31. on the other hand,
By this pressing, the biological sample 34 is rapidly frozen as heat is removed by the metal block 33 having good thermal conductivity, and the movement of the tissue shape along with the water and fat content is stopped.

(6) After that, the motor 38 is energized to raise the cryogen tank 36, the biological sample is immersed in the liquid cryogen for about 10 seconds, and then the slider 17 is slightly raised by a manual knob (not shown), and the sample is removed from the arm 18. Remove holder 19.

(7) After removing the sample holder 19 from the arm 18, the biological sample 34 is thawed over a long period of time and fixed using a processing solution. Good microscopic specimens can be obtained by performing this fixing operation under the following conditions, for example.

That is, first, it was immersed in a mixture of 5% osminic acid solution and acetone at -100 to -80°C for 3 days, then the temperature was gradually raised, and it was heated to -20°C for 2 hours.
After 2 hours at +4°C and 2 hours at room temperature, it is washed with acetone at room temperature. This cleaned biological sample is embedded in resin such as epoxy resin, sliced, and then electrostained using heavy metals.
Use as a specimen for electron microscopy.

Further, FIG. 6 shows a second embodiment of the previous invention. In the embodiment shown in FIG. 6, since the above-mentioned first embodiment used a rack and pinion for the elevating mechanism of the sample holder, the sample holder had to be finished with ultra-precision so that there would be no bumps on the meshing teeth. This is to solve the problem of the inconvenience that the lowering movement of the holder is not smooth, and as shown in Fig. 6, it is fixed vertically to the substrate 13 (see Figs. 4 and 5, omitted in Fig. 6). On the guide rail 48,
As shown in detail in FIG. 7, a slider 50 is provided so as to be movable in the vertical direction via bearings 49, 49. A locking tool 5 provided on the side surface of the slider 50 is located halfway through a cable 52 that is stretched around guide pulleys 51, 51 that are supported on both the upper and lower ends of the board 13.
It is fixed at 3. The cable 52, which is stretched around the guide pulleys 51, 51, is further stretched around a tension pulley 54 and a drive pulley 56, which is rotationally driven by a motor 55. Therefore, by rotating the motor 55 in the forward or reverse direction, the slider 50 can be moved up and down via the cable 52.

A support plate 57 is protruded and fixed to the front surface of the slider 50, and a removable sample holder 59 is attached to the lower surface of a mounting portion 58 fixed to the lower surface of the front end of the support plate 57.
is installed. Various conventionally known structures can be adopted as a structure for making the sample holder 59 removably attachable to the lower surface of the mounting part 58, but for example, the structure shown in FIGS. 8 to 9 is used. You can also do that. That is, a locking groove 60 having a narrow lower end opening and a wider depth is formed in the lower surface of the mounting portion 58, and a locking plate portion 61 formed at the upper end of the sample holder 59 can be freely engaged and detached from this locking groove 60. The outer diameter of the holder 59 is smaller than the width of the lower end opening of the locking groove 60. A pin 63 is provided on the lower surface of the locking groove 60 and is given elasticity in the protruding direction by a spring 62.
is provided. Attach the sample holder 59 to the mounting section 58
When locking to the lower surface of the holder, insert the locking plate 61 into the locking groove 60 while aligning the length direction of the locking plate 61 at the upper end of the holder with the direction of the locking groove 60, and then insert the locking plate 61 into the locking groove 60. Rotate the entire 59 90 degrees. When removing, the length direction of the locking plate part 61 and the direction of the locking groove 60 are aligned and the locking plate part 61 is pulled out from the locking groove 60.

In this way, a metal block 3 is placed on the upper surface of the mounting section 58, which is mounted on the sample holder 59 and moves up and down.
A bracket 35 supporting 3 is fixed.
Further, limit switches 64 and 65 are provided on the sides of both upper and lower ends of the guide rail 48, respectively, so that rising and falling of the slider 50 can be detected. The cryogen tank storing liquid cryogen for cooling the metal block 33 and the mechanism for raising and lowering this cryogen tank are the same as in the first embodiment shown in FIGS. It is composed of a mechanism.

The operation of freezing and fixing a biological sample using the biological sample quick-freezing device of the second embodiment configured as described above is the same as that of the first embodiment described above, except that the slider 50 is raised and lowered by the cable 52. be.

As the mechanism for raising and lowering the slider, various other conventionally known means can be used. For example, the slider can be raised and lowered by a linear motor, and the position of the slider can be digitally controlled.

(Problems to be solved by the invention) However, in the biological sample quick-freezing device of the earlier invention, which is configured and used as described above,
However, the following inconvenience still occurs.

That is, if the sample holder 59 to which the frozen-fixed biological sample 34 is attached is left removed from the apparatus section 58, the frozen biological sample 34 will be removed while other biological samples are being frozen-fixed. Because it melts, it must be kept in an extremely cold place. For this reason, in the past, the sample holder 59 to which the frozen and fixed biological samples 34 were attached was placed into the liquid cryogen 37 stored in the cryogen tank 36, and the cryogen was added after all the biological samples 34 had been frozen and fixed. The sample holder 59 that has settled to the bottom of the tank 36 is picked up. However, the upper surface of the liquid cryogen 37 is covered with the evaporated cryogenic gas and cannot be visually observed, making it difficult to pick up the sample holder that has settled to the bottom of the cryogen tank 37.

Furthermore, when the biological sample 34 on the lower surface of the sample holder is pressed against the upper surface of the metal block 33 and frozen, the sample 34 is frozen on the upper surface of the metal block 33.
Part of the tissue No. 4 adheres to the top surface of the metal block 33, which stains the top surface of the metal block 33. Therefore, the top surface of the metal block 33 must be cleaned before freezing the next sample.

For this reason, even when using the previously devised quick freezing device, it was troublesome and time consuming to freeze and fix a plurality of biological samples.

The rapid freezing device for biological samples of the present invention solves the above-mentioned disadvantages.

b. Structure of the invention (means for solving problems) The rapid freezing device for biological samples of the present invention is structured as shown in FIG. That is, the substrate 13 (fourth
A motor 66 is fixed to the bracket 35 fixed to the lower end of the motor 66 (Figs. 5 to 5), and the rotating shaft 67 is intermittently rotatable by the motor 66. A central portion of a circular and horizontal support plate 68 is fixed to the lower end of the rotating shaft 67. This support plate 68 is made of a rigid metal or the like, and has two types of circular holes 69 and 70 each having a plurality (four in the illustrated example) in the outer circumferential portion as shown in FIG. In the example, both circular holes 69 and 7 are provided alternately.
It is good if the 0's are in close pairs. ) is perforated. Further, on the upper surface of this support plate 68, an auxiliary plate 71 as shown in FIG. 3 is placed. This auxiliary plate 71 has a notch 72 that reaches from the outer periphery to the center through which the rotating shaft 67 can be inserted.
and two types of circular holes 73 formed near the outer periphery,
74. Among these, two types of circular holes 7
Reference numerals 3 and 74 denote a circular hole 73 that is approximately the same size as the circular hole 69 bored in the support plate 68 described above, and a circular hole 74 that is the same as or larger than the circular hole 70 of the support plate 68. The same number of circular holes 69 and 70 bored in hole 68 are provided in the same arrangement. Such an auxiliary plate 71
The circular hole 73 and the circular hole 69 are the circular hole 74 and the circular hole 7 are the circular hole 74 and the circular hole 7.
0 and placed on the upper surface of the support plate 68 in a state where they are aligned with each other.

As described above, a plurality of (four in the illustrated example) metal blocks 33, 33 are attached to the support plate 68 on which the auxiliary plate 71 is placed. This metal block 33 is connected to the circular hole 69 of the support plate 68 and the auxiliary plate 7.
A flange 76 is formed at the upper end of a cylindrical portion 75 that can be inserted through the circular hole 73 of No. 1, and the upper surface of this flange 76 is a mirror surface. metal block 33
The circular holes 70 and 74 that are not equipped with are for supporting a sample holder 59 as shown in FIG. 9 to which a frozen and fixed biological sample 34 is attached.
0.74, the inner diameter of the circular hole 70 of the support plate 68 is so large that the cylindrical portion of the sample holder 59 can be inserted therethrough, but the locking plate portion 61 provided at the upper end of this sample holder 59 cannot be passed through. , circular hole 74
is the same size as the circular hole 70 or larger.

(Operation) A biological sample is frozen and fixed using the biological sample quick-freezing device of the present invention configured as described above as follows.

Metal blocks 33 are installed in advance in the circular holes 69, 69 of the support plate 68 and the circular holes 73, 73 of the auxiliary plate 71, and when freezing work starts, the upper surface of one of these metal blocks 33 and the vertical Flexible mounting part 5
The biological sample 34 is frozen and fixed in the same manner as in the case of the quick-freezing device according to the earlier invention described above.

When the biological sample has been frozen and fixed, the mounting part 58 is slightly raised to separate the lower surface of the sample holder 59 from the upper surface of the metal block 33, and the sample holder 59 is removed.
is removed from the mounting portion 58. The removed sample holder 59 is inserted into the circular holes 70 and 74 located next to the metal block 33 used for freezing the sample.
It is held on a support plate 68. In this state, the biological sample 34 attached to the lower surface of the sample holder 59 is immersed in the liquid cryogen 37, or even if it is not immersed, it is surrounded by extremely low temperature evaporated gas, so that other biological samples cannot be frozen and fixed. Freeze-fixed biological samples will not thaw during the procedure.

Once the sample holder 59 to which the frozen and fixed biological sample is attached is inserted into the circular holes 70 and 74, the support plate 68 is slightly rotated by the motor 66, and a new biological sample is attached to the next metal block. The sample holder 59 is moved directly below the sample holder 59 attached to the attachment section 58, and the next biological sample is frozen and fixed.

All metal blocks 3 attached to the support plate 68
3, 33, after freezing and fixing a predetermined number of biological samples, the metal blocks 33, 33 and the sample holder 5 to which the frozen and fixed biological samples are attached are removed.
9, 59 and each metal block 33, 3.
At the same time as cleaning the top surface of metal block 33, the biological sample is sent to the next processing step.
33 and the sample holders 59, 59 can be easily removed by lifting the auxiliary plate 71. Furthermore, if a locking portion is provided on the upper surface of the auxiliary plate 71, the operation of lifting the auxiliary plate 71 can be easily performed. However, if a plurality of sample holders 59, 59 are taken out of the cryogen tank 36 at the same time, and there is a risk that part of the biological sample 34 will melt before being sent to the next processing step, the sample holders 59, 59 will be removed from the auxiliary plate 71. Take out one piece at a time. In this case, the sample holder 5
9 and 59 may be taken out one by one before the auxiliary plate 71 is taken out, or one by one after the auxiliary plate 71 is taken out. When the sample holder 59 is taken out from behind the auxiliary plate 71, the circular hole 74 provided in the auxiliary plate 71 is connected to the locking plate portion 61 at the upper end of the sample holder.
be large enough to pass through.

c. Effects of the invention The quick-freezing device for biological samples of the present invention is constructed and operates as described above, but it uses a plurality of metal blocks that are replaced one after another, and a sample holder to which a frozen-fixed sample is attached. Since the specimen can be maintained in a state where it can be easily taken out, freezing and fixation of multiple biological samples can be carried out efficiently.

[Brief explanation of drawings]

Figures 1 and 2 show the main parts of the quick freezing device of the present invention, with Figure 1 being a longitudinal side view, Figure 2 being a sectional view taken along line A-A in Figure 1, and Figure 3 being a plan view of the auxiliary plate. Figure, 4th
Figures 5 to 5 show the first embodiment of the quick freezing device according to the previous invention, Figure 4 is a front view, Figure 5 is a vertical side view, and Figures 6 to 9 are the second embodiment. Fig. 6 is a perspective view of the main part, Fig. 7 is a cross-sectional view showing the engagement part between the guide rail and the slider, Fig. 8 is a side view of the lower end of the attachment, and Fig. 9 is a perspective view of the sample holder. , FIG. 10 is a partially vertical front view showing a conventional quick freezing device. 1... Base, 2... Container, 3... Helium tank, 4... Copper block, 5... Bearing, 6...
... Lifting rod, 7 ... Sample holder, 8 ... Locking piece, 9
...Solenoid, 9a...Input/output piece, 10...Shutter, 11...Iron ring, 12...Electromagnet, 13...
Substrate, 14, 15...Bracket, 16...Guide, 17...Slider, 18...Arm, 19...Sample holder, 20...Slide plate, 21...Rack, 22...Shaft, 23...Gear , 24... Worm car, 25... Worm, 26... Motor, 27
... Bracket, 28 ... Screw rod, 29 ... Bracket, 30, 31 ... Double nut, 32 ... Compression spring, 33 ... Metal block, 34 ... Biological sample, 35 ... Bracket, 36 ... Cryogen tank, 37
...liquid cryogen, 38 ... motor, 39 ... pinion, 40 ... rack, 41 ... saucer, 42 ... guide, 43 ... lifting rod, 44 ... liquid level sensor, 4
5... Bracket, 46... Serpentine pipe, 47... Solenoid on-off valve, 48... Guide rail, 49... Bearing, 50... Slider, 51... Guide pulley,
52... Cable, 53... Locking tool, 54... Tension pulley, 55... Motor, 56... Drive pulley, 57... Support plate, 58... Mounting part, 59
... Sample holder, 60 ... Locking groove, 61 ... Locking plate portion, 62 ... Spring, 63 ... Pin, 64, 65
...Limit switch, 66...Motor, 67...
... Rotating shaft, 68 ... Support plate, 69, 70 ... Circular hole, 71 ... Auxiliary plate, 72 ... Notch, 73, 7
4... Circular hole, 75... Cylindrical part, 76... Flange.

Claims (1)

[Scope of utility model registration request] A slider that can be vertically raised and lowered along the front surface of the substrate, a mechanism that raises and lowers the slider, a sample holder that can be attached to and detached from the slider and that attaches a biological sample to the bottom surface, and a sample holder that is fixed to the substrate below the sample holder. A bracket equipped with a metal block,
In a quick-freezing device for biological samples, which consists of a cryogen tank that moves up and down below this bracket and immerses the metal block in the liquid cryogen stored inside when it rises, a rotating shaft installed in the center inside the cryogen tank is used. A rotatable and horizontal support plate is provided, an auxiliary plate is placed on the upper surface of the support plate, and a plurality of first circular holes are provided at positions on the outer periphery of the auxiliary plate and the support plate that are aligned with each other. A cylindrical part of a metal block with a flange part provided at the upper end of the cylindrical part is inserted, and a sample having a locking plate part at the upper end is placed near the first circular hole adjacent to the auxiliary plate and the support plate. A rapid freezing device for a biological sample, characterized in that the same number of second circular holes into which holders can be inserted as the first circular holes are provided.