JP2009085981A - Sample stage device having temperature gradient - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To provide a sample stage device which has a sample stage surface where a temperature gradient is formed and the temperature gradient can be intentionally controlled unlike conventional heaters for microscopes, since recently, it has been found out that nematomorph M and tick exhibit thermotaxic behavior. <P>SOLUTION: A sample stage device 1 comprises the sample stage surface 31 where a sample is placed and a heating means for heating the sample stage surface 31. The heating means forms a desired temperature gradient in the sample stage surface 31. The temperature gradient is formed by using a transparent resistance heating film region 35 made of a single material and demarcating the transparent resistance heating film into small regions 39 having different temperatures by varying the separation distance between a pair of feeding electrodes. <P>COPYRIGHT: (C)2009,JPO&INPIT

Description

  The present invention relates to a sample mounting device having a temperature gradient, and more particularly to a sample mounting device having a temperature gradient suitable for microscope observation.

For artificial insemination of animals, samples such as sperm and eggs should be kept at a predetermined temperature and observed while observing them with a microscope. As a device for satisfying this requirement, there is a microscope observation heating device. There are Patent Documents 1, 2 and the like in applications for this type of device.
In this type of device, a transparent resistance heating film is basically formed on a transparent plate by vapor deposition or the like, and the sample placed on the transparent plate is heated by heating the film. Is heated to a predetermined temperature.

Japanese Patent No. 2835422 Utility Model Registration No. 3016894

The above-described microscope heating apparatus is required to heat the sample mounting surface to a certain temperature regardless of the type, and this determines the performance of this type of apparatus. It has become.
By the way, recently, it has been found that behaviors such as nematodes and ticks are thermotaxis.

  Therefore, unlike conventional devices, a sample mounting device is required in which a temperature gradient is formed on the sample mounting surface and the temperature gradient can be intentionally controlled so as to be able to cope with the evaluation of biological samples as described above. ing.

  The present invention has been made in order to solve the above problems, and the invention of claim 1 includes a sample mounting surface on which a sample is mounted and a heating means for heating the sample mounting surface. In the sample mounting device, the heating means forms a desired temperature gradient on the sample mounting surface, and the sample mounting device has a temperature gradient.

  According to a second aspect of the present invention, in the sample mounting apparatus according to the first aspect, the heating means is provided in contact with the resistance heating film region provided on the back side of the sample mounting surface and the resistance heating film region. The sample mounting device is characterized in that the distribution of energization amounts is different within the resistance heating film region.

  According to a third aspect of the present invention, in the sample mounting device according to the second aspect, the resistance heating film region is made of a single material, and the current distribution is changed by changing the distance between the pair of power supply electrodes. The sample mounting device is characterized by being different.

  A fourth aspect of the present invention is the sample mounting apparatus according to the second aspect, wherein the resistance heating film region is partitioned into a plurality of regions while being insulated from each other.

  According to a fifth aspect of the present invention, there is provided the sample mounting apparatus according to any one of the first to fourth aspects, further comprising temperature lowering means for uniformly reducing the temperature of the sample mounting surface. It is a mounting device.

  According to a sixth aspect of the present invention, in the sample mounting device according to the fifth aspect, the temperature lowering means is disposed so as to face the resistance heating film region formed on the back side of the sample mounting surface, and is spaced apart from each other. It is a sample mounting device comprising two partition plates and a refrigerant filled between the partition plates.

  A seventh aspect of the present invention is the sample mounting apparatus according to the sixth aspect, further comprising a refrigerant circulation means for circulating a refrigerant between the partition plates.

  The invention according to claim 8 is the sample placement apparatus according to claim 6 or 7, wherein an air layer is provided between the resistance heating film region and the temperature drop means. It is.

  A ninth aspect of the present invention is the sample mounting apparatus according to any one of the first to eighth aspects, wherein the sample mounting portion is transparent.

  A tenth aspect of the present invention is the sample placement apparatus according to the ninth aspect, wherein the sample placement apparatus is placed on a microscope stage and the sample is observed with a microscope.

  According to the sample mounting device of the present invention, a temperature gradient is formed on the sample mounting surface, and the temperature gradient can be set to a desired value, so that it is suitable for evaluating the thermal mobility of nematodes and the like. Yes.

A sample placement device 1 according to an embodiment of the present invention will be described with reference to the drawings.
As shown in FIG. 1, this sample mounting apparatus 1 uses a nematode M as a sample, and evaluates the thermotaxis of the nematode M by microscopic observation.
In this embodiment, the sample mounting device 1 is installed by being fitted into a hole of the stereomicroscope stage S, and observation is performed with the objective lens R facing from above.

The structure of the sample mounting device 1 will be described.
Reference numeral 3 denotes a frame. The frame 3 is formed in a rectangular frame shape and has a rectangular opening 9. As shown in detail in FIGS. 2 and 3, rectangular water cases 11 and 13 are formed on the opposite longitudinal sides of the frame 3. Although the water cases 11 and 13 have substantially the same structure, the width dimension of the water case 11 is set smaller than that of the water case 13, and the capacity of the water case 11 is smaller than that of the water case 13. It has become.

An upper surface opening 15 is formed on the upper surface of the water cases 11 and 13, and a lower surface opening 17 is formed on the lower surface.
Upper openings 15 and 15 of the water cases 11 and 13 are closed by lids 19 and 19, respectively. Two connection pipes 21 are attached to each lid 19, and one end of a hose 23 is connected to each connection pipe 21. The hose 23 is connected to a tank containing water as a refrigerant (not shown) via a pump.
The upper surfaces of the water cases 11 and 13 are higher than the upper surface 5 of the frame 3.

As shown in FIG. 5, the lower surface of the water cases 11 and 13 on the lower surface opening 17 side is one step higher than the lower surface 7 of the frame 3.
Between the lower surface of the water case 11 where the lower surface opening 17 is formed and the lower surface 7 of the frame 3, the inner surface of the frame 3 bulges inward to form a step surface 25. Also, the inner surface of the inner side wall 4 of the double wall of the frame 3 bulges inwardly between the lower surface of the water case 13 where the opening 17 is formed and the lower surface 7 of the frame 3, and the step surface is similarly 25 is formed.
Further, stepped surfaces 27 are formed at both ends of the short side of the lower surface 7 of the frame 3.

As shown in FIGS. 3 and 4, the sample mounting device 1 is assembled by storing the sample mounting portion 29 in the opening 9 of the frame 3.
Each layer constituting the rectangular sample mounting portion 29 will be described with reference to FIGS.
Reference numeral 33 denotes a first transparent glass plate, and a transparent resistance heating film region 35 made of ITO or the like is formed on the surface of the transparent first glass plate 33 which is visible by vapor deposition. The structure of the transparent resistance heating film region 35 will be described in detail later.
Reference numerals 41 and 41 denote a pair of power supply electrodes, and these power supply electrodes 41 and 41 are disposed in contact with the transparent resistance heating film region 35. Each power supply electrode 41 has a strip shape, and both end portions slightly protrude from the edge of the first transparent glass plate 33 for extraction. Although not shown, one end of the power transmission line is connected to each of the power supply electrodes 41 and 41, and the other end of the power transmission line is connected to the temperature controller.
Reference numeral 43 denotes a black paper for shielding, and the black paper 43 is formed with a rectangular opening 45 having four rounded corners.

Reference numeral 47 denotes a second transparent glass plate.
The lengths of the long side and the short side of the first transparent glass plate 33, the black paper 43, and the second transparent glass plate 47 are all set to be substantially the same.
Reference numeral 49 denotes a first spacer formed in a substantially rectangular frame shape. The length of the long side and the short side of the outer edge of the first spacer 49 is set slightly smaller than that of the second transparent glass plate 47. ing.

Reference numeral 51 denotes a third transparent glass plate, which is larger than the second transparent glass plate 47 by the length of the short side of the third transparent glass plate 51. As shown in FIG. When laminated, it becomes a stepped surface 53 with respect to the second transparent glass plate 47.
Reference numerals 55 and 55 denote a pair of second spacers, and each second spacer 55 has an elongated thin plate shape. The second spacers 55 and 55 have a shape in which the long side of the rectangular first spacer 49 is missing, and the portions corresponding to the remaining short sides are opposed to each other with a predetermined distance therebetween. The length dimension of each second spacer 55 is set slightly smaller than the length dimension of the short side of the third transparent glass plate 51.

Reference numerals 57 and 61 denote fourth and fifth transparent glass plates, respectively, and the lengths of the long side and the short side of the fourth and fifth transparent glass plates 57 and 61 are set to be substantially the same. The length of the short side is slightly larger than the third transparent glass plate 51, and the difference portion of the fourth transparent glass plate 57 is a stepped surface 59 with respect to the third transparent glass plate 51.
Reference numeral 63 denotes a third spacer formed in a substantially rectangular frame shape, and the third spacer 63 is disposed between the fourth and fifth transparent glass plates 57 and 61. The lengths of the long side and the short side of the outer edge of the third spacer 63 are set slightly smaller than the fourth and fifth transparent glass plates 57 and 61.

The above-described members are stacked and stored in the frame 3.
The accommodation state of the short side part of the sample mounting part 29 is demonstrated according to FIG. Since the structure in the vicinity of the water cases 11 and 13 is substantially the same, only the vicinity of the water case 11 is shown in an enlarged view.
Both ends of the pair of short sides of the first glass plate 33, the black paper 43, and the second transparent glass plate 47 are close to the inner surfaces of the water cases 11 and 13 that bulge inward from the frame 3.
The step surface 53 of the third transparent glass plate 51 is in contact with the inner lower surface of the water cases 11 and 13, and further extends toward the frame 3 to close a part of the lower surface opening 17 of the water cases 11 and 13. .
Further, both ends of the short sides of the fourth and fifth transparent glass plates 57 and 61 are close to the inner surface of the frame 3. Then, the step surface 59 of the fourth transparent glass plate 57 contacts the step surface 25 of the frame 3. The side wall of the frame 3 is doubled on the water case 13 side, and the inner surface of the frame 3 is the inner surface of the inner side wall 4.

In addition, the storage state of the longitudinal side portion of the sample mounting portion 29 will be briefly described.
Both ends of the pair of long sides of the first transparent glass plate 33 abut on the stepped surface 27 of the frame 3, and the black paper 43, the second transparent glass plate 47, the third transparent glass plate 51, and the fourth transparent glass are placed thereon. A plate 57 and a fifth transparent glass plate 61 are sequentially laminated. The first transparent glass plate 33, the black paper 43, the second transparent glass plate 47, the third transparent glass plate 51, the fourth transparent glass plate 57, and the fifth transparent glass plate 61 are all close to the inner surface of the frame 3. .

First transparent glass plate 33, black paper 43, second transparent glass plate 47, first spacer 49, third transparent glass plate 51, second spacer 55, fourth transparent glass plate 57, third spacer 63, fifth transparent Insulating silicone-based transparent adhesive is filled between adjacent layers of the glass plate 61 and bonded to each other.
Further, an insulating silicone black adhesive 62 is filled between the frame 3 and the inner surfaces of the water cases 11 and 13 and bonded to each other.

As shown in FIG. 3, the air layer 65 sealed by the first spacer 49 is formed between the second transparent glass plate 47 and the third transparent glass plate 51 by the above-described lamination.
An air layer 67 is also sealed between the fourth transparent glass plate 57 and the fifth transparent glass plate 61 by the third spacer 63.
On the other hand, the step surface 53 of the third transparent glass plate 51 closes only a part of the lower surface opening 17 of the water cases 11 and 13, and between the third transparent glass plate 51 and the fourth transparent glass plate 57. Since the pair of second spacers 55, 55 interposed between the water cases 11, 13 are not arranged on the side of the water cases 11, 13, between the water cases 11, 13, the third transparent glass plate 51, and the fourth transparent glass plate 57. The space communicates. Since water as a coolant flows through the space, in this embodiment, the third transparent glass plate 51 and the fourth transparent glass plate 57 each function as a partition plate.

As shown in FIGS. 1 and 2, the upper surface of the first transparent glass plate 33 that forms the sample mounting surface 31 of the sample mounting portion 29 is one step lower than the upper surface 5 of the surrounding frame 3. A portion of the sample placement surface 31 that faces the opening 45 of the black paper 43 is a microscope observation range, that is, the sample placement portion 32.
On the other hand, as shown in FIG. 4, the lower surface of the fifth transparent glass plate 61 that forms the lower surface of the sample mounting portion 29 is substantially flush with the lower surface 7 of the surrounding frame 3.

Next, the structure of the transparent resistance heating film region 35 and the pair of power feeding electrodes 41 and 41 formed on the first transparent glass plate 33 will be described in detail with reference to FIG.
A transparent resistance heating film region 35 is formed of a single material with a constant film thickness on the surface opposite to the sample mounting surface 31 of the two surfaces of the first transparent glass plate 33, Resistance temperature coefficient is the same. The transparent resistance heating film region 35 is formed in a line-symmetric trapezoidal shape, and the bottom side and the top side are respectively on the long side of the first transparent glass plate 33.
The trapezoidal transparent resistance heating film region 35 is partitioned into a plurality of small strip-shaped regions 39 by a plurality of ultrathin line-shaped film missing portions 37 extending in parallel at a predetermined distance apart in the longitudinal direction. The film missing portion 37 penetrates the transparent resistance heating film region 35, and the plurality of small regions 39 are electrically insulated from each other. The width dimensions of the plurality of small regions 39 are the same except for those on both sides.

The pair of power supply electrodes 41, 41 are arranged in contact with each other on the trapezoidal transparent resistance heating film region 35 as indicated by a dotted line. Accordingly, each small region 39 becomes a current-carrying region between the electrodes 41 and 41.
Of the small regions 39 (a), (b), (c),..., (I), (a) is the highest temperature, the temperature decreases in order, and (i) is the lowest temperature. become. That is, a temperature gradient is formed.

Such a temperature gradient is formed because there is a difference in the separation distance between the electrodes 41 and 41 for each of the small regions 39 (a), (b), (c),. This is because the quantity distribution is different. In the small area 39 (a), since the separation distance is the shortest, the electric resistance is the smallest and the energization amount is the largest, that is, the current flows most easily and the heat generation amount is the largest. On the other hand, in the small region 39 (i), since the separation distance is the longest, the electric resistance is the largest and the energization amount is the smallest.
As the upper side of the trapezoid of the transparent resistance heating film region 35 is shortened, the inclination angle of the side is increased, the degree of change in the separation distance between the electrodes is increased, and the temperature gradient is also increased.

The usage method of the sample mounting apparatus 1 is demonstrated.
It is mounted on the stage S of the microscope, and the transparent resistance heating film region 35 is energized through a pair of power supply electrodes 41 and 41 from a temperature controller (not shown) to generate heat.
In addition, water is supplied into the water case 11 through the hose 23, and water is discharged from the water case 13 through the hose 23. Water as a refrigerant flows from the water case 11 into the space between the second transparent glass plate 47 and the third transparent glass plate 51 as shown by the arrows in the enlarged view of FIG. 3, and circulates across the space. Then, the water flows out into the water case 13 and is discharged from the water case 13 through the hose. The water filling the space is sequentially replaced, and the water temperature is maintained at a substantially constant temperature. Since the capacity of the water case 11 on the water supply side is smaller than the capacity of the water case 13 on the water discharge side, water does not overflow.

Therefore, the sample mounting surface 31 receives a heating action by the transparent resistance heating film and a temperature lowering action by the refrigerant.
Moreover, an air layer 65 sealed by the first spacer 49 is provided between the transparent resistance heating film region 35 and the third transparent glass plate 51, and the transparent resistance heating film region 35 is excessively cooled. There is no. Moreover, since the air layer 67 is also provided between the 4th glass plate 57 and the 5th glass 61, the water as a refrigerant | coolant is not warmed from the lower side.

The sample placement surface 31 facing the small area 39 (i) has the lowest temperature. When the transparent resistance heating film region 35 is energized and heated, the temperature cannot be lowered to an ambient temperature, for example, 25 ° C. or less. However, by using the refrigerant together as described above, for example, the small region 39 (i) The temperature gradient can be formed so that the small region 39 (a) is 52 ° C. by setting the temperature to 20 ° C. below normal temperature and increasing it by 4 ° C.
A temperature sensor (not shown) is affixed to the first transparent glass plate 33 in an insulating state, and the temperature controller sequentially receives temperature information from the temperature sensor to adjust the current supply amount and maintain the temperature at a predetermined temperature. be able to.

As shown in FIG. 2, as described above, the agar medium K on which the nematode is placed on the microscope observation range 32 (= the sample placement portion) of the sample placement surface 31 on which the desired temperature gradient is formed is applied to a plurality of different temperatures. It arrange | positions so that the small area | region 39 of this may be crossed.
Then, as shown in FIG. 8, the nematode M gathers in a specific temperature region due to the thermotaxis of the nematode M.

The embodiment of the present invention has been described in detail above. However, the specific configuration is not limited to this embodiment, and the present invention can be changed even if there is a design change without departing from the gist of the present invention. included.
For example, in the above embodiment, since the thermal mobility of the nematode M is evaluated, the transparent resistance heating film region 35 is partitioned by the film missing portion 37 to clarify the boundary of the temperature difference. Depending on the type of observation and the purpose of observation, it is not always necessary to clarify the boundary of the temperature difference.
In the above embodiment, a glass plate is used, but the material is not limited to this and may be a plastic plate. Moreover, it does not need to be transparent unless it is for microscope observation.

  According to the sample mounting apparatus of the present invention, since a desired temperature gradient can be formed on the sample mounting surface, it can be widely used for microscopic observation and classification of biological samples having temperature dependency.

It is a perspective view which shows the use condition of the sample mounting apparatus which concerns on embodiment of this invention. It is explanatory drawing of the usage method of the sample mounting apparatus of FIG. It is sectional drawing of the sample mounting apparatus of FIG. It is the perspective view which looked at the sample mounting apparatus of FIG. 1 from the downward direction. It is the disassembled perspective view which looked at the sample mounting apparatus of FIG. 1 from the downward direction. It is the disassembled perspective view which looked at the sample mounting part of the sample mounting apparatus of FIG. 1 from the downward direction. It is explanatory drawing of the structure of the transparent transparent heating film area | region of FIG. It is an observation figure of the nematode in the sample mounting apparatus of FIG.

Explanation of symbols

DESCRIPTION OF SYMBOLS 1 ... Sample mounting device 3 ... Frame 5 ... (Frame) upper surface 7 ... (Frame) lower surface 9 ... (Frame) opening 11, 13 ... Water case 15 ... (Case upper surface) opening 17 ... (Case bottom) Opening 19 ... Lid 21 ... Connection pipe 23 ... Hose 25 ... (Water case side) Step surface 27 ... (Frame side) Step surface 29 ... Sample mounting part 31 ... Sample mounting Placement surface 32 ... Observation range for microscope
33... First transparent glass plate 35... Transparent resistance heating film region 37... Extra-fine linear film missing portion 39... Small region 41, 41. Paper) Opening 47 Second transparent glass plate 49 First spacer 51 Third transparent glass plate 53 Step surface 55, 55 Pair of second spacers 57 Fourth transparent glass plate 59 Step surface 61 Fifth transparent glass plate 63 Third spacer 65 Air layer 67 Air layer S Microscope stage R Objective lens M Nematode K Agar medium W ‥water

Claims (10)

  In a sample mounting device comprising a sample mounting surface for mounting a sample and a heating means for heating the sample mounting surface, the heating means forms a desired temperature gradient on the sample mounting surface A sample mounting device having a temperature gradient formed therein. In the sample mounting apparatus according to claim 1,
The heating means comprises a resistance heating film region provided on the back side of the sample mounting surface and a pair of power supply electrodes provided in contact with the resistance heating film region. A sample mounting device characterized by having different distributions. In the sample mounting apparatus according to claim 2,
The sample mounting device, wherein the resistance heating film region is made of a single material, and the distribution of energization amount is varied by changing the distance between the pair of power supply electrodes. In the sample mounting apparatus according to claim 2,
The sample mounting device, wherein the resistance heating film region is partitioned into a plurality of regions in a state of being insulated from each other. In the sample mounting apparatus according to any one of claims 1 to 4,
Furthermore, the sample mounting apparatus is provided with temperature lowering means for uniformly lowering the temperature of the sample mounting surface. In the sample mounting apparatus according to claim 5,
The temperature lowering means is arranged to oppose a resistance heating film region formed on the back side of the sample mounting surface, and includes two partition plates spaced apart from each other and a refrigerant filled between the partition plates. A sample placement device. In the sample mounting apparatus according to claim 6,
A sample placement device comprising a coolant circulation means for circulating a coolant between partition plates. In the sample mounting apparatus according to claim 6 or 7,
A sample mounting device, wherein an air layer is provided between the resistance heating film region and the temperature lowering means. In the sample mounting apparatus according to any one of claims 1 to 8,
A sample mounting device, wherein the sample mounting portion is transparent. In the sample mounting apparatus according to claim 9,
A sample mounting apparatus characterized by being placed on a microscope stage and observing a sample with a microscope.

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