JPH0129217Y2 - - Google Patents

Description

[Detailed Description of the Invention] The present invention relates to a microelectrode fabrication device for patch clamps for fabricating hyaline capillaries for measuring ionic currents present in the cells of experimental animals and living organisms.

Generally, it is known that various ions move in and out of the cell membrane when a cell of a laboratory animal or the like is excited, and channels are assumed to selectively pass each ion, and each channel is equipped with a gate. It is thought that by opening, ions can pass through. As shown in FIG. 1, in recent years it has been proposed to extract various types of information inside a cell by extracting an ionic current using a microelectrode 2 made of a hyaline capillary without damaging the cell 1. That is, the method involves fixing the potential of a microcell membrane region under a glass microelectrode, that is, a patch, and measuring the current caused by ions passing through a channel contained within the patch. The tip of the glass microelectrode is 1 μm or less, and the tip is in close contact with the cell membrane so that it can be encapsulated inside the cell membrane.It is also hollow from one tip to the other, making it convenient for extracting and measuring ionic current. It is necessary to have an appropriate resistance value when the electrolytic solution 2a is contained in the hollow space, and to be able to satisfactorily measure even the difficult-to-analyze channel directly under the wall of the glass microelectrode. In order to manufacture glass microelectrodes that meet these conditions, the conventional method has been to heat them with a gas burner and manually process them by pulling both ends by hand, which takes a day to produce 100 to 200 glass microelectrodes. Only two to three glass microelectrodes could be used during production, requiring particularly high skill levels, and did not meet the demands of the times.

Therefore, the present invention was devised in view of the above circumstances, and it eliminates the drawbacks of the old-fashioned glass microelectrode manufacturing method.It allows for the production of high quality glass microelectrodes in large quantities in a short time without requiring any skill. The purpose of the present invention is to provide a microelectrode manufacturing device for patch clamps that can be manufactured using the following methods.

In order to achieve the above object, the present invention has a fixed clamp that holds the upper end of the glass tube material, and a movable clamp that also serves as a weight that holds the lower end of the glass tube material, and has a fixed clamp and a movable clamp that hold the lower end of the glass tube material. A movable heater for heating the glass tube material is disposed in between, and a stopper is disposed below the movable clamp for regulating the lowering limit of the movable clamp during the first stage of heating by the heater; After moving and fixing the heater to the center part of the ultra-thin tube shape of the glass tube material which has been stretched and formed by heating in the first stage, heating is performed in a second stage by the heater, and the glass tube material is cut from the center part of the ultra-thin tube shape. The present invention is characterized by a microelectrode product for a patch clamp in which the movable clamp can be further lowered and a stopper is provided so as to be freely engageable and detachable from the movable clamp.

DESCRIPTION OF THE PREFERRED EMBODIMENTS An embodiment of a patch clamp microelectrode manufacturing device according to the present invention will be described below with reference to the drawings. Second
In the figures, 3 is a pedestal equipped with a leg seat 4, and 5 is a case for accommodating an electric circuit installed on the pedestal 3. The front left side of the case 5 has a first heater temperature regulator 6 and a start button switch 7, as shown in FIGS. 2 and 3. Further, a second heater temperature regulator 8 and a main switch 9 are disposed on the right side of the front surface of the case 5, as shown in FIG. A column 10 is erected on the pedestal 3 in front of the case 5. Guide grooves 13 for guiding the vertical movement of the heater support 11 and the movable clamp 12 are formed on the front end sides of both sides of the support column 10, and a fixed clamp 14 is fixed at the upper end. The fixing clamp 14 has a glass tube material 1 at the front end.
The glass tube material 15 has a holding groove 16 into which the upper end of the glass tube material 15 is fitted, and the glass tube material 15 can be fixed in the holding groove 16 with a tightening screw 16a. A first micro switch SW ₁ is fixed to the upper end of the support column 10 and is turned on by contacting the heater support 11 . The heater support 11 has a pinion that meshes with the rack 17 of the support column 10, and by rotating the pinion with the operating knob 18, the heater support 11 moves up and down along the support column. The vertical movement is guided by the guide groove 13 and the guide protrusion of the heater support 11 that fits into the guide groove 13. A stopper 19 is attached to the column 10 for convenient positioning of the heater support 11, and the stopper 19 is attached in the longitudinal direction of the column 10 so that the setting position of the heater support 11 can be freely varied as described later. It is now possible to move. On the front side of the heater support 11, the central part is U.
A heater 20 is attached that includes an insertion portion 20a of a glass tube material 15 that is bent into a character shape. A movable clamp 12 is attached to the pillar 10 so as to be freely movable up and down, located below the heater support 11. The movable clamp 12 also serves as a weight, and a weight can be added depending on conditions such as the diameter of the glass tube material 15. Movable clamp 12
The vertical movement of is guided by a guide groove 13 and a guide protrusion fitted into the guide groove 13. A support body 21 is fixed to the front surface of the movable clamp 12, and the front surface of the support body 21 has a holding groove 22 into which the lower end of the glass tube material 15 is inserted, and the glass tube material 15 is held down by a tightening screw 23. It can be fixed within the groove 22. A stopper 24 is swingably attached to the stopper 24, which is located slightly above the lower end of the support column 10 and can come into contact with the lower end of the movable clamp 12. The stopper 24 has an adjustment screw 25
By loosening it, it can be moved up and down along the support column 10, and the position of contact with the movable clamp 12 can be freely adjusted.After positioning, it can be fixed by tightening the adjustment screw 25. Also, on one side of the stopper 24, there is a second micro switch.
SW ₂ is fixedly installed, and when the movable clamp 12 and the stopper 24 come into contact, the second micro switch SW ₂
has started to turn off. A cushion member 3a is fixed on the base 3 located in front of the support column 10. Further, a third micro switch SW ₃ is fixedly installed at the lowest end of the support column 10, as shown in FIGS. Switch SW ₃ is set to turn OFF.

As shown in FIG. 5, the electric circuit includes a relay circuit 28 connected to an AC power supply 26 via a fuse 27 and a main switch 9. Main switch 9
A light emitting diode _D2 for indicating power-on is connected to the subsequent stage side via a rectifier _D1 and a current limiting resistor _R1 . The relay circuit 28 includes a start button switch 7 and first to third micro switches.
SW _{1 to 3} and first to second heater temperature regulators 6, 8
is mediated. The heater 20 is connected to the relay circuit 28 via a transformer T.

Next, we will explain the process of producing glass microelectrodes. First, turn the operation knob 18 to raise the heater support 11 to the uppermost position where it comes into contact with the first micro switch SW ₁ , and then tighten the upper end of the glass tube material 15 with the fixing clamp 14 using the screw 1.
3 and insert the glass tube material 15 through the insertion section 20a of the heater 20 until the center of the glass tube material 15 is positioned inside the insertion section 20a and the movable clamp 12 comes into contact with the heater support 11. After lifting it by hand, the lower end of the glass tube material 15 is fixed to the movable clamp 12 with a tightening screw 23. Of course, the heater temperature regulators 6 and 8 are set in advance to appropriate heating temperatures for the first and second stages, respectively. In addition, the stopper 24 stops when the movable clamp 12 falls downward as described below.
The falling distance of the movable clamp 12 is set in advance as described above so that the portion of the glass tube material 15 that is to be stretched by heating and has an extremely small diameter has a preset pipe diameter, and the movable clamp 12 is It is swung to a predetermined position in advance so that it can be engaged when it falls downward. Therefore, the glass tube material 15 is
It is set as shown in FIG. On the other hand, the first
The micro switch SW ₁ is turned on by contact with the heater support 11. Further, the second and third micro switches SW ₂ to _{SW 3} are in the ON state because they are not in any contact with the movable clamp 12, and as a result, the first relay solenoid 29 of the relay circuit 28 switches on the diode D ₃ . The first relay contact 30 is switched as shown in FIG. 5. Next, when the start button switch 7 is pressed for the first time, the second relay solenoid 31 is energized via the diode _D4 and the resistor _R2 , and as a result, the second relay contact 3
2 is switched as _shown in FIG.
heater temperature regulator 6 and further the first relay contact 3
It is energized until it reaches 0. The heater 20 is therefore energized via the secondary winding of the transformer T. By energizing the heater 20, the approximate center of the glass tube material 15 is heated and softened, and the movable clamp 12 is heated as shown in FIG.
As the glass tube material 15 falls until it is stopped by the stopper 24 due to its own weight, the heated portion of the glass tube material 15 is stretched and becomes extremely thin to a predetermined diameter. When the movable clamp 12 is locked to the stopper 24, the second
The micro switch SW ₂ comes into contact with the movable clamp 12 and turns OFF, which de-energizes the first relay solenoid 29 and closes the first relay contact 30.
is switched in the opposite direction to that shown in FIG. 5, and power supply to the heater 20 is cut off. Next, as shown in FIG.
The heater support 11 is lowered so that the point 0 is located at the center of the ultrathin tube portion 15a of the glass tube material 15. With this lowering, the first micro switch _SW1 is turned off and switched to the second heater temperature regulator 8 side. Then, the stopper 24 that is locked to the movable clamp 12 is swung to release the lock, and when the stopper 24 is released from the lock, the second micro switch _SW2 is turned on. Then, if the start button switch 7 is pressed again, the second relay solenoid 31
is energized, and the second relay contact 32 is connected to the fifth
It switches as shown in the figure. Therefore, electricity is supplied from the AC power supply 26 to the second heater temperature regulator 8 and the first relay contact 30 via the second relay contact, the primary winding of the transformer T, and the first microswitch _SW1 . . As a result, the second stage heating of the heater 20 is performed from the secondary winding of the transformer T, and as shown in FIG.
The center of the tube softens and falls to the cutout material 25 due to the weight of the movable clamp 12, so that the ultra-thin tube portion 1
It is cut from the center of 5a. The cutting portion of the glass tube material 15 has a shape with an open tip as shown in FIG. 10, and the opening diameter _S1 of the tip is 5 μm, which is preset
Preferably it is 3 μm or less. The tip of the glass microelectrode formed in this way is heated with another microfuge (not shown) to make the tip smooth and suitable for patch clamping, as shown in FIG. The aperture S ₂ is also formed to be 1 μm or less. To position the heater 20 at the center of the ultrathin tube portion 15a of the glass tube material 15, if the heater 20 is positioned in advance using the stopper 19, the heater support 11 can be positioned by simply lowering it until it comes into contact with the stopper 19. Extremely convenient. or,
The heating temperature of the first stage and the heating temperature of the second stage are
Although the heating temperature is set in advance to a predetermined value, it goes without saying that the heating temperature in the first stage is set to a higher value than the heating temperature in the second stage. The heating temperature in the second stage is set to a value sufficient to cut the ultra-thin tube portion 15a from the center.

As described above, according to the patch clamp microelectrode manufacturing device of the present invention, by simply setting the heater temperature in advance, all of the manufactured glass microelectrodes are of the same quality and of high quality, and no unusable glass microelectrodes can be produced. It has never been said that something can be done with it, and it does not require any special skills, is extremely simple to operate, and anyone can use it.

[Brief explanation of the drawing]

Fig. 1 is an explanatory diagram showing an example of the use of a general glass microelectrode, Fig. 2 is a perspective view showing an embodiment of a patch clamp microelectrode manufacturing device according to the present invention, and Fig. 3 is a patch clamp microelectrode manufacturing device. Figure 4 is a perspective view of the microelectrode fabricator for patch clamps as seen from the other side, Figure 5 is an electric circuit diagram of the microelectrode fabricator for patch clamps, and Figures 6 to 9 are microelectrode fabricators for patch clamps. Fig. 10 is an enlarged cross-sectional view of the main part of the tip of the patch clamp microelectrode after cutting, and Fig. 11 shows the microelectrode for patch clamping shown in Fig. 10 after being heated to fire polish. FIG. 3 is an enlarged cross-sectional view of processed main parts. 3...Pedestal, 4...Legs, 5...Case, 6...
...First heater temperature regulator, 7...Start button switch, 8...Second heater temperature regulator, 9
. . . Main switch, 10 . , 22... Presser groove, 17... Rack, 18... Operation knob, 1
9, 24... Stopper, 20... Heater, 20
a...insertion part, 21...support body, 16a, 23...
... Tightening screw, 25 ... Adjustment screw, 26 ... AC power supply, 28 ... Relay circuit, SW ₁ ... First micro switch, SW ₂ ... Second micro switch, SW ₃ ... Third micro switch Switch, T...Trance.

Claims (1)

[Scope of utility model registration request] a fixing clamp that holds the upper end of the glass tube material;
It has a movable clamp that also serves as a weight to hold the lower end of the glass tube material, and a movable heater that heats the glass tube material is disposed between the fixed clamp and the movable clamp, and a movable heater that heats the glass tube material is provided below the movable clamp. is provided with a stopper that restricts the lowering limit of the lower moving clamp during the first stage of heating by the heater, and the stopper is installed in the center portion of the ultrathin tube shape of the glass tube material that has been stretched and formed by the first stage of heating. After moving and fixing the heater, a second stage of heating is performed by the heater, and the movable clamp is further lowered in order to cut from the central portion of the ultrathin tube shape, and a stopper is provided so as to be freely engaged and detached from the movable clamp. A microelectrode manufacturing device for patch clamps, which is characterized by: