JPH06308118A - Cell sorting and electrode insertion method and device - Google Patents

Abstract

(57) [Abstract] [Object] An object of the present invention is to provide a simple and reliable method and apparatus configuration for cell selection and electrode insertion method and apparatus. [Structure] A reflected light image and a transmitted light image of cells are observed using an epi-fluorescence microscope for a sample consisting of a plurality of cells to which a fluorescent dye is applied and which emits fluorescence and a plurality of cells that do not emit fluorescence, and a television camera respectively. And the reflected light image of the imaged cells is binarized with an image analyzer to distinguish between cells that emit fluorescence and cells that do not, and the binarized fluorescence image of cells that emit fluorescence is framed. A cell positioning method is characterized by comprising a step of recording in a memory and a step of superimposing or binarizing a binarized fluorescence image and a transmitted light image on a monitor.

Description

Detailed Description of the Invention

[0001]

BACKGROUND OF THE INVENTION 1. Field of the Invention The present invention relates to a method for selecting specific cells, an electrode insertion method, and a structure of an insertion device. In the study of neurophysiology, glass microelectrodes filled with a high-concentration electrolyte solution are inserted into specific cells for the purpose of measuring the membrane potential of cells and administering drugs.

[0002]

2. Description of the Related Art Initially, as a method for piercing cells with glass microelectrodes, it was common to puncture glass microelectrodes using a manipulator operated by water pressure or hydraulic pressure while observing the cells with a microscope. The operation required skill and experience. After that, as a method to simplify this operation, you can insert a glass microelectrode into a specific cell by designating multiple cells projected on the TV screen using a mouse (handheld position input device) on the screen. , Devices that can inject drugs have become commercially available.

Recently, however, there is an increasing need for marking specific cells in advance by dyeing with a fluorescent dye or the like and inserting a glass microelectrode into the specific cells. It is necessary to select and memorize the cells emitting fluorescence underneath and insert the glass microelectrode under the transmission microscope.However, because of the large number of cells, the fluorescence image and transmitted light image alternate. It is complicated to observe the above and insert the electrode, and there is a risk of accidentally inserting into different cells.

[0004]

It is possible to stain a specific cell required from a plurality of types of cells with a fluorescent dye, and this specific cell can be displayed on a monitor (monitoring device). If it is possible to superimpose or to be juxtaposed with the transmitted light image, it is possible to puncture the glass microelectrode without mistake.

Further, a computer controls a manipulator for driving glass microelectrodes, cell penetration of the electrodes is detected by an electrical phenomenon of cells detected from the electrodes, and the information is fed back to the computer to automatically detect the glass. It is possible to insert the electrode. Therefore, it is an issue to put the insertion method and the device into practical use.

[0006]

[Means for Solving the Problems] Among the above-mentioned problems, as a method for selecting specific cells, an epifluorescence microscope is used for a sample consisting of a plurality of cells to which a fluorescent dye is added and which emit fluorescence and a plurality of cells which do not emit fluorescence. Observing the fluorescence image and transmitted light image of the cell using the, respectively, the step of photographing with a TV camera, and the fluorescence image of the photographed cell is binarized by the image analysis device, cells that emit fluorescence and cells that do not emit fluorescence It is possible to select specific cells by distinguishing between the two, and recording a binarized image of fluorescent cells in a frame memory and superimposing the binarized image and the transmitted light image on a monitor.

Further, as a device configuration for accurately inserting a glass microelectrode, a means for connecting the glass microelectrode to a probe through an electrode holder and driving the probe with a three-dimensional manipulator, and a three-dimensional manipulator A stepping motor is attached to the control handle, a means for controlling this motor with a computer, a means for applying a pulse from the electrostimulator and a high frequency signal from the buzz controller to the probe while controlling it with a computer, and a potential change detected by the probe It is only necessary to use an electrode puncturing device including means for amplifying and displaying an image, inputting it to a computer, and comparing the electric characteristics set in advance to detect puncturing.

As a method for inserting a glass microelectrode, an electrode is inserted into a cell using an electric stimulator and a high-frequency oscillator and controlled by a computer using a three-dimensional manipulator. In addition, any of the methods of detecting from resting membrane potential, judging from changes in membrane potential, judging from action potential, and judging from changes in electric tension potential due to membrane resistance and membrane capacitance are used. You can

[0009]

[Operation] FIG. 1 shows a process of selecting specific cells. That is, after a fluorescent dye is incorporated only into cells of a specific type in which a glass microelectrode is to be inserted, this cell group is observed with an epifluorescence microscope, and this image is SIT (Silicon
Intensifier Target Tube) When input to the image analysis device through the camera, this image analysis device has functions such as brightness adjustment, contour enhancement, and threshold processing, so only cells that emit fluorescence are selected (binarization). Processing) and the information of this fluorescent image is stored in the frame memory.

On the other hand, a transmission image of the epi-illumination fluorescence microscope is picked up by a CCD camera, and this transmission image is input to an image analysis device,
After adjusting the brightness and emphasizing the contours, etc., if the transmitted light image and the fluorescence image are superimposed or placed side by side on the monitor, only the specific cells that are going to be inserted into the glass microelectrode become clear, and the insertion of the electrode becomes clear. You can definitely do it. FIG. 2 shows a device configuration for accurately inserting a glass microelectrode into a cell. A glass microelectrode 1 for inserting a cell is held by an electrode holder 2 and connected to a probe (Probe) 3. The probe 3 is driven by a three-dimensional manipulator 4. The drive source is a manipulator controller 5, a stepping motor 7 is attached to this handle portion, and it is controlled by a computer 9.

Next, the potential change detected by the probe 3 is amplified by the amplifier 10, monitored by the memory oscilloscope 11, and input to the computer 9 for data feedback. Further, in order to puncture the cell 13 with the glass microelectrode 1, it is necessary to observe potential changes inside and outside the cell 13, and the indifferent electrode (earth electrode) 14 as the counter electrode of the glass microelectrode 3 is connected to one end of the Ringer solution 15. And connect to the probe 3 in advance.

Next, in order to insert the glass microelectrode 1 into the cell 13, it is necessary to give a slight vibration to the electrode holder 2, and a pulse for measurement is not applied to the glass microelectrode 1. It is necessary to provide between the electrodes 14 and 15. Therefore, the former is performed by applying a high-frequency signal from the buzz controller 17 to the electrode holder 2 through the amplifier 10 and controlling it by the computer 9, and the pulse is transmitted from the electric stimulator 18 through the isolator 19 and the amplifier 10 to the probe. 3 is applied. With such a configuration, it becomes possible to accurately insert the glass microelectrode 1 into the cell 13. As an index for inserting a glass microelectrode into a cell, detection of resting membrane potential, detection of action potential, and change of electric tension potential due to membrane resistance and membrane capacitance can be mentioned. In general, the inside of a quiescent cell has a potential of −60 to −80 mV compared to the outside of the cell. Therefore, observing the potential between the glass microelectrode 1 and the indifferent electrode 14 in FIG. There is no potential difference while the cells are in contact with extracellular fluid (Ringer's solution 15), but the potential suddenly changes in the negative direction when they penetrate the cell membrane. Therefore, if the computer 9 is set in advance so that the three-dimensional manipulator 4 stops when the potential changes by -60 to -80 mV, the glass microelectrode 1 can be stopped without penetrating the cell 13. is there.

Next, when a nerve cell is excited, the potential inside the cell membrane changes from the resting membrane potential of -60 to -80 mV in the positive direction. This phenomenon is called depolarization, and there is a membrane potential. When it exceeds the threshold value, it is rapidly depolarized, the membrane potential is temporarily reversed and becomes positive, and after a very short duration of about 2 to 3 mS, it immediately returns to the resting potential. This change in potential is called action potential. According to the present invention, no action potential is detected in the probe 3 before the insertion of the cell 13 of the glass microelectrode 1,
Since it is detected when it is inserted, the potential change due to the action potential is pulse-shaped through the Schmitt trigger circuit, and if this pulse is set in the computer 9 as a signal for stopping the three-dimensional manipulator 4, the glass microelectrode 1
Can be stopped without penetrating the cell 13.

Next, there is resistance in the cell membrane, and cells 13
There is a membrane capacity inside and outside of. Therefore, it is used that a rectangular wave current pulse is passed through the recording electrode, and the electric tension potential recorded at that time is changed by the membrane resistance and the membrane capacitance. FIG. 3 shows this relationship. FIG. 3 (A) shows the relationship between the glass microelectrode 1 and the cell 13 before and after insertion.
Further, FIG. 3B shows a pulse waveform and a voltage change applied between the glass microelectrode 1 and the indifferent electrode. In addition,
The Ringer's solution 15 is present between the glass microelectrode 1, the cell 13 and the indifferent electrode.

First, when the glass microelectrode 1 is not in contact with the cell 13, the resistance is 0 (only the resistance of the electrolytic solution) even if a constant current pulse is applied, so that there is no voltage change. (Above A-
1, B-1), next, when the glass microelectrode 1 comes into contact with the cell 13, the tip of the electrode is sealed to maximize the resistance and the potential change amount increases rapidly, but due to the influence of the relaxation time due to the membrane capacitance. It becomes a deformed potential waveform. (Above A-2, B-2), then, when the glass microelectrode 1 penetrates the cell 13, a potential waveform as shown in the figure is produced by the membrane resistance. (Above A-3, B-3) Therefore, if the manipulator is input so that the potential change due to the energizing pulse is predicted and the constant voltage value is reached, the glass microelectrode 1 will cause the cells 13 It is possible to stop without penetrating.

[0016]

【Example】

Example 1: (corresponding to claim 1 and FIG. 1) Using an earthworm as an experiment, a fluorescent dye (DiI) crystal was embedded in muscle, and after leaving it for 10 hours, the abdominal ganglion was cut out to put a fluorescent substance on nerve cells. I took it in. As a result, nerve cells began to emit red fluorescence in response to the green excitation light of the epifluorescence microscope. This fluorescent image is SIT
It led to the image analysis device through the camera, binarized and selected the cells emitting fluorescence and stored them in the frame memory. Then, this fluorescent image and the transmitted light image were superimposed and displayed on a monitor, and the electrodes were pierced with the naked eye, but it was possible to puncture the marked cells with the glass microelectrodes. Example 2: (corresponding to claim 2 and FIG. 2) The glass microelectrode 1 for inserting cells has a diameter of 1 m.
A Pyrex glass tube with a core of m was drawn by using a microelectrode maker (type name PN-3, made by Narishige).
Then, an aqueous solution of KCl 3 having a concentration of 3 mol was filled therein, and an Ag wire coated with AgCl was inserted thereinto and connected to the probe 3. In addition, for the cell 13, a Ringer's solution was used as the electrolyte solution 15, and as the indifferent electrode 14, an Ag wire coated with AgCl was used.

Next, the model name MO-103M (manufactured by Narishige) was used as the three-dimensional manipulator 4 for moving the probe 3, and the model name PC-9801FA (NEC) was used as the computer 9 for controlling this. Next, a model name MEZ-9301 (Nihon Kohden) was used as an amplifier for amplifying the potential change detected by the probe 3, and a model name VC-11 (Nihon Kohden) was used as the memory oscilloscope 11. Also, an electric stimulator that gives a pulse
The model name SEN-7203 (Nippon Kohden) is used for 12, the model name SS-201J (Nippon Kohden) is used for the isolator 19, and the model name SS-1433 (Nippon Kohden) is used for the buzz controller 17 that performs high-frequency oscillation. did. Example 3: (corresponding to claims 3 and 4) The abdominal ganglion of red earthworm was used as a sample, and 1 ganglion of this ganglion was used.
% Of the enzyme (protease) for 1 minute to dissolve and remove the surface oblique muscle. Then, MGF (Median Giant Fiber) was selected as the nerve cell to be inserted. First, while observing with a stereoscopic microscope, the glass microelectrode was brought close to the surface of the ganglion with a three-dimensional manipulator, and it was confirmed that the tip of the electrode entered the Ringer's solution, and then the induced potential was reset to 0 mV.

Since the resting membrane potential of the MGF membrane is -50 to -60 mV, input to the computer so that the three-dimensional manipulator will stop when the induced potential becomes -50 mV or less.
Moreover, since the thickness of the ganglion is about 100 μm, the manipulator's movable range was input so as not to exceed this range. The penetration speed of the glass microelectrode was set to 1 μm per second, and the stepping motor of the manipulator controller was turned on aiming at the midline from the back of the ganglion. As a result, the membrane potential showed -57 mV near the dorsal surface of the ganglion, and at the same time the stepping motor stopped and the glass microelectrode was successfully inserted. Example 4: (corresponding to claim 5) The abdominal ganglion of red earthworm was used as a sample, and a glass microelectrode was brought close to the surface of the ganglion with a three-dimensional manipulator as in Example 3, and the tip of the electrode was in Ringer's solution. After confirming that it entered the threshold, set the threshold value to about 1/2 of the action potential overshoot.
The potential is set to +10 mV, and a pulse is output when this value is exceeded by the Schmitt trigger circuit, and the three-dimensional manipulator is stopped when continuous pulses are generated. As a result, the glass microelectrode was inserted and a pulse train was generated, and at the same time, the stepping motor was stopped, and the glass microelectrode was successfully inserted. Example 5 (corresponding to claim 6 and FIG. 3) When the waveform of the electric tension potential expected from the membrane resistance and the membrane capacitance is input to the computer in advance, and a voltage waveform equal to or higher than the level synchronized with the stimulation appears. The manipulator is now stopped. Pulses generated by using an electric stimulator width 20 mS, frequency 25H _Z, the amplitude 0.5 nA. As a result, the glass microelectrode was inserted and a pulse train was generated, and at the same time, the stepping motor was stopped, and the glass microelectrode was successfully inserted.

[0019]

EFFECTS OF THE INVENTION The practice of the present invention facilitates the positioning of specific cells when inserting glass microelectrodes into cells, and the use of the insertion device of the present invention enables accurate insertion. It was

[Brief description of drawings]

FIG. 1 is a block diagram showing a method for selecting cells according to the present invention.

FIG. 2 is a configuration diagram of a puncturing device according to the present invention.

FIG. 3 is a schematic diagram showing a potential change due to electrode insertion.

[Explanation of symbols]

 1 glass microelectrode 3 probe 4 three-dimensional manipulator 7 stepping motor 9 computer 11 memory oscilloscope 13 cell 14 indifferent electrode 15 Ringer's solution 17 buzz controller 18 electrostimulator

─────────────────────────────────────────────────── ─── Continuation of the front page (51) Int.Cl. ⁵ Identification code Internal reference number FI Technical display location G01N 21/64 Z 7414-2J 21/78 C 7906-2J 27/28 341 Z 7363-2J G02B 21 / 06 7625-2K 21/32 7625-2K G06F 15/62 395 9287-5L

Claims (6)

[Claims] 1. A fluorescence image and a transmitted light image of the cells are observed using an epi-fluorescence microscope for a sample consisting of a plurality of cells to which a fluorescent dye is applied and which emits fluorescence and a plurality of cells that do not emit fluorescence. The process of photographing with a TV camera and the fluorescence image of the photographed cell are binarized by an image analyzer to distinguish between cells that emit fluorescence and cells that do not emit fluorescence, and the binarized image of cells that emit fluorescence is stored in a frame memory. And a step of superimposing the binarized image and the transmitted light image on a monitor or arranging them side by side on a monitor. 2. A means for connecting a glass microelectrode to a probe through an electrode holder and driving the probe with a three-dimensional manipulator, and a stepping motor attached to a control handle of the three-dimensional manipulator, the motor being operated by a computer. A means for controlling, a means for applying a pulse from the electrostimulator and a high frequency signal from the buzz controller to the probe while controlling the computer, and amplifying the potential change detected by the probe, inputting it to the computer together with displaying an image, An electrode puncturing device comprising: a means for detecting puncture by comparing with a preset electrical characteristic value. 3. An electric stimulator and a high-frequency oscillator are controlled by a computer, and an electrode is inserted into a cell by using a three-dimensional manipulator, and detection is performed based on a change in electrical characteristics of a cell membrane. Electrode insertion method. 4. The electrode insertion method according to claim 3, wherein the presence or absence of insertion into cells is determined by detecting the resting potential. 5. The electrode insertion method according to claim 3, wherein the presence / absence of insertion into a cell is determined from an action potential. 6. The electrode insertion method according to claim 3, wherein the presence or absence of the contact of the electrode with the cell surface and the insertion into the cell is determined from the membrane resistance and the membrane capacitance.