JP2007322165A - Electrophysiological measuring instrument, and electrophysiologial measurement method - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To facilitate operation even with respect to small cells, and to measure potential in the cell in a low invasive manner. <P>SOLUTION: This electrophysiological measuring instrument 1 is provided with the first electrode 11, arranged in an outside of the sample cell A under a contact condition, a second electrode 12 inserted into the inside of the sample cell A, and a measuring means 15 for measuring a potential difference between the first electrode 11 and the second electrode 12 inserted into the sample cell A. <P>COPYRIGHT: (C)2008,JPO&INPIT

Description

  The present invention relates to an electrophysiological measurement device and an electrophysiological measurement method.

2. Description of the Related Art Conventionally, a method for detecting changes in membrane potential or membrane current (patch clamp method) is known as a method for analyzing signal transmission and function expression in cells (see, for example, Patent Document 1).
In this method, the tip of the glass tube is brought into contact with the cell and sucked, and the electrolytic solution in the glass tube is used as an electrode that is kept in close contact with the cell by storing the electrolytic solution inside the glass tube. Is.
JP 2004-294221 A

  However, since the patch clamp method sucks cells by bringing the glass tube into contact with the cells, it is difficult to suck the cells with respect to smaller cells, and the thickness of the glass tube is limited. There is a disadvantage that there is. In addition, when the glass tube is thinned, it becomes easier to penetrate the cell membrane and the control of the contact pressure becomes difficult, so that there is a problem that the invasiveness is large and the operation is difficult for a small cell.

  The present invention has been made in view of the above-described circumstances, and can be easily operated even on a smaller cell, and an electrophysiological measurement device capable of measuring an intracellular potential in a minimally invasive manner. The object is to provide an electrophysiological measurement method.

In order to achieve the above object, the present invention provides the following means.
The present invention includes a first electrode disposed in contact with the outside of a sample cell, a second electrode inserted into the sample cell, and the second electrode inserted into the first electrode and the sample cell. And an electrophysiological measuring device including a measuring means for measuring a potential difference between the two.

In the above-mentioned invention, it is good also as providing the sample cell holding means which holds the sample cell inside, and the 1st electrode being provided in the bottom of the sample cell holding means.
In the above invention, the first electrode may be a planar electrode wider than a horizontal cross section of the sample cell.
Moreover, in the said invention, the said 1st electrode is good also as arrange | positioning avoiding the optical path of the light for observing the said sample cell.

Moreover, in the said invention, the said 1st electrode is good also as consisting of a transparent electrode provided on the optical path of the light for observing the said sample cell.
In the above invention, the sample holding means may insulate the first electrode inside the sample holding means from the outside of the sample holding means.

In the above invention, the second electrode may be a needle-like electrode having a tip that is thinner than the sample cell. In the above invention, the second electrode includes a semiconductor material. It is good.

Moreover, in the said invention, the said 2nd electrode is good also as the front-end | tip part inserted in the said sample cell being exposed, and other parts being coat | covered with the insulating film.
Moreover, in the said invention, the said measurement means is good also as measuring the change of the said electrical property measured before and after insertion to the said sample cell of the said 2nd electrode.

Moreover, in the said invention, it is good also as providing the stimulation signal generation means which applies the stimulation signal for giving a stimulus with respect to the said sample cell to a said 2nd electrode.
Moreover, in the said invention, it is good also as providing the optical observation means which optically observes the said sample cell in which a said 2nd electrode is inserted.

  The present invention also includes a step of placing the first electrode in contact with the outside of the sample cell, a step of inserting the second electrode into the sample cell, the first electrode, and the inside of the sample cell. An electrophysiological measurement method comprising: detecting a potential difference between the second electrode inserted into the first electrode.

  According to the present invention, it is possible to easily operate even a smaller cell, and there is an effect that the intracellular potential can be measured with minimal invasiveness.

Hereinafter, an electrophysiological measurement device 1 and an electrophysiological measurement method according to a first embodiment of the present invention will be described with reference to FIGS.
As shown in FIG. 1, an electrophysiological measurement apparatus 1 according to this embodiment is provided in a microscope apparatus 2 for observing cells.

  The microscope apparatus 2 observes a stage 4 on which a container 3 that accommodates cells is placed, an illumination apparatus 5 that illuminates the cells on the stage 4, light reflected or transmitted through the cells, or fluorescence generated from the cells. An observation device 6, a stage controller 7 that drives and controls the stage 4, a microscope controller 8 that controls the illumination device 5 and the observation device 6, an image processing device 9 that processes an image obtained by the observation device 6, and the image And a display device 10 for displaying an image processed by the processing device 9.

The illumination device 5 includes a transmission illumination light source 5A that irradiates the cells with illumination light from the side opposite to the observation device 6, and a condenser lens that collects the illumination light emitted from the transmission illumination light source 5A on the cells. 5C and an epi-illumination light source 5B that irradiates the cells with illumination light from the same direction as the observation device 6 are provided.
The observation device 6 includes an observation optical system including an objective lens (not shown), a CCD element 6A that captures an image of light from the cell via the observation optical system, and directly observes the light from the cell. And an eyepiece 6B.

  As shown in FIG. 2, the electrophysiological measurement apparatus 1 according to the present embodiment is placed on the stage 4 of the microscope apparatus 2 and provided on the bottom surface of the container 3 that stores a buffer solution or a culture solution L. A flat plate-like first electrode 11, a second electrode 12 brought close to the cell A cultured on the first electrode 11 from above, a drive unit 13 for moving the second electrode 12, A drive unit control circuit 14 for controlling the drive unit 13 and a measurement circuit 15 for measuring a potential difference between the first electrode 11 and the second electrode 12 are provided.

The first electrode 11 is composed of a transparent electrode, and the cells A are cultured on the surface thereof.
As shown in FIGS. 2 and 3, the second electrode 12 is formed in a cantilever shape having a sharp needle portion 12A at the tip. The second electrode 12 is formed by forming a conductive film 12C on the surface of the silicon substrate 12B. The conductive film 12C formed on the silicon substrate 12B is exposed by a size that can be completely accommodated in the nucleus C of the cell A in the needle portion 12A at the tip, and the other portions are covered with the insulating film 12D. ing. The second electrode 12 is attached to the drive unit 13 by an attachment member 16.

  The drive unit 13 includes, for example, a triaxial linear movement mechanism that moves the needle portion 12A in the X, Y, and Z directions. The container 3 is made of an insulating material, and electrically insulates the first electrode 11 inside from the outside. The first electrode 11 is grounded.

  As shown in FIG. 2, the measurement circuit 15 is connected between the first electrode 11 and the second electrode 12. The measurement circuit 15 generates a stimulation signal (action potential) 15A between the first electrode 11 on the bottom surface of the container 3 and the conductive film 12C exposed on the needle portion 12A of the second electrode 12. And an electric signal measuring device 15B for measuring a response to the stimulus signal.

An electrophysiological measurement method using the electrophysiological measurement device 1 according to this embodiment configured as described above will be described below.
In order to measure the membrane potential of the cell A using the electrophysiological measurement apparatus 1 according to this embodiment, the stage controller 7 and the microscope controller 8 are operated, and the cell A to be measured by the operation of the stage 4 is observed under a microscope. Deploy. Then, the drive unit 13 is operated by the operation of the drive unit control circuit 14 so that the needle portion 12A of the second electrode 12 is brought close to the cell A from above the cell A.

At this time, the measurement circuit 15 connected between the first electrode 11 and the second electrode 12 is operated, and the impedance of the electrochemical system between the conductive film 12C of the needle portion 12A and the first electrode 11 is set. taking measurement.
When the first electrode 11 provided on the bottom surface of the container 3 contacts the cell A, the potential of the cell membrane B is fixed to the potential of the first electrode 11 (ground potential). Then, until the conductive film 12C provided on the needle portion 12A of the second electrode 12 comes into contact with the cell membrane B, the distance between the second electrode 12 and the cell membrane B is as indicated by the symbol P in FIG. Even if it changes, the impedance between both does not change.

  Further, when the second electrode 12 is lowered and the conductive film 12C provided on the needle portion 12A comes into contact with the cell membrane B, the impedance between the second electrode 12 and the cell membrane B is indicated by the symbol Q in FIG. Changes rapidly. Then, when the second electrode 12 is further lowered and the conductive film 12C provided on the needle portion 12A enters the nucleus C of the cell A, the conductive film 12C becomes an intracellular potential, so that the impedance becomes a predetermined value. (Code R). Accordingly, the impedance between the first electrode 11 and the second electrode 12 is measured during the insertion operation of the needle portion 12A into the cell A, and a change in the electrolyte resistance is detected, thereby providing the needle portion 12A. The contact and intrusion of the conductive film 12C into the cell A can be accurately detected.

  In this case, according to the electrophysiological measurement method according to the present embodiment, after it is detected that the conductive film 12C provided on the needle portion 12A has entered the cell A, the movement of the needle portion 12A is stopped, and then again. The action potential is measured by the operation of the measurement circuit 15. That is, a predetermined stimulus signal is generated by the operation of the stimulus signal generation device 15A in the measurement circuit 15, and a response signal to the stimulus signal is measured and recorded by the operation of the electric signal measurement device 15B.

  According to the electrophysiological measurement method using the electrophysiological measurement device 1 according to the present embodiment, since only one thin needle portion 12A having a thin tip based on the silicon substrate 12B enters the cell A, the measurement is performed. The invasiveness to the cell A can be kept low. In addition, the intrusion of the needle portion 12A into the cell A can be easily determined based on the change in impedance, and there is an advantage that a stable operation can be performed even on a small cell A with little variation among operators. is there. Furthermore, the conductive film 12C exposed from the insulating film 12D can be limited to an extremely small size, and the entire exposed portion of the conductive film 12C can enter the cell A, so that stable measurement with low noise can be performed.

  In the present embodiment, the intrusion of the needle portion 12A into the cell A is detected by a change in impedance between the first electrode 11 and the second electrode 12. However, the present invention is not limited to this. The intrusion may be determined while confirming the position of the tip of the needle portion 12A and the position of the cell A by optical means.

  Moreover, the transparent electrode which culture | cultivates the cell A on the surface as the 1st electrode 11 was employ | adopted. Thereby, epi-illumination and transmission illumination via the first electrode 11 and the bottom surface of the container 3 are possible. Instead of this, an opaque electrode may be employed as the first electrode 11 and may be disposed avoiding an optical path through which illumination light passes.

1 is an overall configuration diagram showing an electrophysiological measurement device according to a first embodiment of the present invention. It is an enlarged view which shows the structure of the cantilever vicinity of the electrophysiology measuring apparatus of FIG. It is a figure explaining the insertion operation to the cell of the needle part provided in the cantilever of the electrophysiology measuring device of Drawing 1, (a) before contact, (b) at the time of contact, (c) after the penetration. Each is shown. It is a graph which shows the impedance change between the 1st electrode and 2nd electrode accompanying the insertion operation | movement of FIG.

Explanation of symbols

A cells (sample cells)
1 Electrophysiological measurement device 2 Microscope device (optical observation means)
3 Container (Sample cell holding means)
11 First electrode (transparent electrode)
12 Second electrode 12A Needle part (needle-like electrode)
12D insulating film 15 measurement circuit (measurement means)
15A Stimulation signal generation circuit (stimulation signal generation means)

Claims (13)

A first electrode disposed in contact with the outside of the sample cell;
A second electrode inserted into the sample cell;
An electrophysiological measurement apparatus comprising: a measurement unit that measures a potential difference between the first electrode and the second electrode inserted into the sample cell. A sample cell holding means for holding the sample cell inside;
The electrophysiological measurement apparatus according to claim 1, wherein the first electrode is provided on a bottom portion of the sample cell holding means.   The electrophysiological measurement apparatus according to claim 2, wherein the first electrode is a planar electrode wider than a horizontal section of the data cell.   The electrophysiological measurement apparatus according to claim 2, wherein the first electrode is disposed so as to avoid an optical path of light for observing the sample cell.   The electrophysiological measurement apparatus according to claim 2, wherein the first electrode is a transparent electrode provided on an optical path of light for observing the sample cell.   The electrophysiological measurement apparatus according to claim 2, wherein the sample holding means insulates the first electrode inside the sample holding means from the outside of the sample holding means.   The electrophysiological measurement apparatus according to claim 1, wherein the second electrode is a needle-like electrode having a tip that is thinner than the sample cell.   The electrophysiological measurement apparatus according to claim 1, wherein the second electrode includes a semiconductor material.   2. The electrophysiological measurement device according to claim 1, wherein a tip portion of the second electrode inserted into the sample cell is exposed and the other portion is covered with an insulating film.   The electrophysiological measurement apparatus according to claim 1, wherein the measurement unit measures a change in the electrical characteristics measured before and after insertion of the second electrode into the sample cell.   The electrophysiological measurement apparatus according to claim 1, further comprising a stimulation signal generation unit that applies a stimulation signal for applying stimulation to the sample cell to the second electrode.   The electrophysiological measurement apparatus according to claim 1, further comprising an optical observation unit that optically observes the sample cell into which the second electrode is inserted. Arranging the first electrode in contact with the outside of the sample cell;
Inserting a second electrode into the sample cell;
An electrophysiological measurement method comprising a step of detecting a potential difference between the first electrode and a second electrode inserted into the sample cell.

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