JPH09196936A - Optical probe microscope device - Google Patents

Abstract

(57) [Summary] [Objective] To provide an optical probe microscope apparatus capable of microscopic observation and electrophysiological property measurement with high position resolution. [Structure] An optical microscope section 100 for observing a wide area image of a sample 900 and an optical probe 211 installed on top of the sample 900, and a light irradiation section 21 for irradiating the sample 900 with light.
From the scanning near-field optical microscope unit 200 for observing a local image of the sample 900 including 0, the patch clamp measuring unit 300 for deriving and recording the current generated in the sample 900, and the scanning near-field optical microscope unit 200. The acquired local image of the sample and the electrophysiological information obtained from the patch clamp measurement unit 300 for each light irradiation position of the scanning near-field optical microscope unit 200 are collected, analyzed, and recorded, and the scanning type The near-field optical microscope unit 200 is provided with an analysis unit 400 that notifies the irradiation light wavelength instruction and scanning instruction. Then, the water-immersed sample 900 stored in the container 500 is observed.

Description

Detailed Description of the Invention

[0001]

BACKGROUND OF THE INVENTION 1. Field of the Invention The present invention relates to an optical probe microscope apparatus for simultaneously performing electrophysiological measurement and recording of cells and the like and near-field microscopic observation.

[0002]

In observing the characteristics of living cells, attention has been paid to the observation of electrophysiological characteristics in addition to the conventional morphological observation using an optical microscope.

As a device for complex microscopic observation in which the electrophysiological characteristic measurement recording method typified by the patch clamp method and the optical microscopic observation method are combined, a video of a differential interference microscope and an image enhancing apparatus accompanying it are used. A multi-measurement microscope incorporating a laser tweezers and a patch clamp current measuring device in a video-enhanced differential interference microscope for observation and recording has been proposed (Tatsumi et al., Proceedings of the 2nd Research Symposium of the Near Field Optics Research Group, pp75- 80, 1994
November).

In this multi-measurement microscope apparatus, a water immersion type objective lens having a large numerical aperture is used as the first objective lens for collecting the incident illumination light and irradiating it onto the sample. And enables Ar
-The light of wavelength = 643 nm output from the Kr laser light source is introduced into the optical path of epi-fluorescence, and is collected by the second objective lens having a large numerical aperture, which is installed to face the first objective lens with the sample in between. The sample is illuminated with light to perform the laser tweezers function.

In this way, in this apparatus, the optical image of the cell membrane is captured by the video enhanced differential interference microscope, and the change in the biomembrane current when the laser tweezers function is turned on / off is observed.

[0006]

In complex observation, which is a fusion of electrophysiological characteristic measurement represented by the patch clamp method and optical microscopic observation, information on electrophysiological characteristic from a very small area that is precisely defined is selected. It is a condition that the property is acquired and acquired.

In contrast to this condition, in the conventional device using the laser tweezers as the optical probe, since the laser tweezers use a normal imaging optical system used for the optical path of epi-fluorescence, the irradiation diameter (that is, position resolution) is It is known that it is limited by the diffraction effect of light as a wave, and its value is theoretically 0.61 times the wavelength of laser light. Therefore, the resolution is several hundreds.
Laser tweezers that introduce laser light into the optical path of epi-fluorescence that is on the order of nm, condense it with an objective lens with a large numerical aperture, and irradiate the sample, so the area where light acts is limited to the optical axis direction of the irradiation light. There was a problem that it could not be done.

Further, since the conventional apparatus uses a differential interference microscope having a resolution of about the wavelength, it is impossible to observe the morphological or optical physical properties of the sample with the resolution of the wavelength or less.

The present invention has been made in view of the above, and an object thereof is to provide an optical probe microscope apparatus capable of microscopic observation and electrophysiological characteristic measurement with high position resolution.

[0010]

An optical probe microscope apparatus according to claim 1 has (a) an optical microscope section for observing a wide area image of the sample, and (b) an optical probe installed on the upper part of the sample, A scanning near-field optical microscope unit for observing a local image of the sample is provided with a light irradiation unit for irradiating the sample with light, and (c) a single living cell in the sample or a biological membrane from a minute area of the biological membrane. An electrical information measuring unit for deriving and recording a transverse current or a membrane potential, (d) a local image of the sample obtained from the scanning near-field optical microscope unit, and each irradiation position of light of the scanning near-field optical microscope unit In addition to collecting, recording, and analyzing the electrophysiological information obtained from the electrical information measuring unit, an analysis control unit for controlling the entire apparatus is provided.

Here, the electrical information measuring section inputs a patch electrode for accessing the surface of the sample, a micromanipulator for moving the patch electrode, and a current signal across the biomembrane which is a bioelectric signal detected by the patch electrode. A preamplifier that converts the voltage signal, amplifies and outputs the voltage signal, and an electric signal generator that outputs a command signal for measuring the seal resistance, fixing the membrane potential, or fixing the membrane current according to an instruction from the analysis control unit. , The voltage signal output from the preamplifier is input, amplified, processed, and output to the analysis control unit, and the command signal output from the electric signal generator is input and arithmetically processed to It may be characterized in that it comprises a main amplifier for outputting to an on-amplifier.

Further, the electrical information measuring section has a microelectrode for accessing the inside of the sample, a micromanipulator for moving the microelectrode, and a bioelectric signal detected by the microelectrode for inputting, amplifying and outputting the bioelectric signal. The voltage signal output from the amplifier and the preamplifier is input, amplified, processed and output to the analysis control unit, and the command signal output from the electric signal generator is input and processed. It may be characterized in that it comprises a main amplifier for outputting to a preamplifier.

Here, either one of a glass microelectrode and a metal microelectrode can be preferably adopted as the microelectrode.

Further, in the optical microscope section, a Hoffman modulation system, a phase contrast microscope or a differential interference microscope capable of enhancing contrast can be preferably used.

The light irradiator may be provided with a wavelength tunable laser, and a white light source, a spectroscope for spectrally outputting the white light output from the white light source, and a light output from the spectroscope. And a light selector that selects and outputs light having a specific wavelength component from all wavelength components.

According to the optical probe microscope apparatus of claim 1,
First, a wide area image of the sample is obtained with the optical microscope section. Continued
Based on the wide area image of the sample thus obtained, a multi-dimensional micromanipulator or the like is used to bring the electrode tip close to or in contact with a specific position of a biological sample such as a target cell,
Alternatively, it penetrates into the sample.

When the patch clamp method is used, the tip of the patch electrode is brought into close contact with a specific position of a biological sample such as a target cell to form a gigaohm (GΩ) seal. At this time, in order to electrically monitor the process up to the formation of a gigaohm (GΩ) seal, a command pulse signal for measuring the seal resistance, fixing the membrane potential or fixing the membrane current is output to the preamplifier via the main amplifier. In order to
An electric signal generator (pulse generator) controlled by the analysis control unit may be used.

When the microelectrode method is used, the tip of a microelectrode composed of a glass microelectrode or a metal microelectrode is made to penetrate from a specific position of a biological sample such as a target cell. Then, the electric signal detected by the microelectrode during the process from approach to intrusion is electrically monitored through the preamplifier and the main amplifier. An electric signal generator (pulse generator) controlled by the analysis control unit may be used to output the command pulse signal to the preamplifier via the main amplifier in order to confirm that the cell is a living cell. .

Next, the optical probe of the scanning near-field optical microscope section is moved to a position on a biological sample such as a target cell while observing a wide area image of the sample obtained by the optical microscope section. Then, the tip of the optical probe is brought close to the initial position, which is the near-field region of the sample surface. In moving the optical probe to the near-field region, a system such as a non-contact scanning microscope mode or a shear force non-contact scanning microscope mode can be preferably used.

Thus, the geometrical arrangement for observing the sample is completed, and the preparation for optical microscopic observation and electrophysiological property measurement is completed.

First, the irradiation light generated in the light irradiation unit is irradiated through the optical probe set at the initial position. Here, the irradiation light that is irradiated to the sample through the optical probe is evanescent light that is non-emission light, and since a lens for image formation is not used, there is no restriction of diffraction by the aperture of the lens and The resolution is determined only by the aperture diameter at the tip of the optical probe. As a result, if the diameter of the tip of the optical probe is set to several tens of nm and the distance between the optical probe and the sample is set to several tens of nm which is a near-field region, the number of wavelengths of the irradiation light is several tens.
With a resolution of 1/100 to 1/100, it is possible to observe the fine shape and structure of the sample, or the optical characteristics in a minute region. The local image of the sample thus observed is notified to the analysis control unit.

Further, a change in current or membrane potential across the biological membrane due to electrophysiological changes in the sample caused by light irradiation is derived from the electrodes and measured by the electric information measuring section, and the measurement results are analyzed and controlled. The department will be notified. The current or membrane potential derived from the electrodes is converted into a voltage signal by the current-voltage converter in the preamplifier,
Since it is a minute signal, it is preferable to notify the analysis control unit via the main amplifier that performs processing such as amplification, calculation, and reduction of current noise.

The analysis control unit collects the local image information of the sample notified from the scanning near-field optical microscope unit and the electrophysiological characteristic information notified from the electric information measuring unit, and stores it together with the initial position information. The analysis control unit can also control the operation (irradiation / scanning condition) of the main amplifier, the electric signal generator (pulse generator), and the light irradiation unit.

Next, the analysis control unit instructs the scanning near-field optical microscope unit to perform scanning. Then, the local image information and the electrophysiological information of the sample are collected for each scanning position and stored together with the scanning position information.

In order to secure the scanning in the near-field region in scanning, a method such as constant height mode control, non-contact scanning microscope mode control, or shear force non-contact scanning microscope mode control is suitable. Can be used.

After completion of scanning, information collection, and information storage, the analysis control unit analyzes the optical image information of the entire scanning region of the sample and the electrophysiological characteristic information according to the light irradiation position based on the stored information, The microscopic observation image and the bioelectric reaction are made to correspond to each other and reconstructed to display or record. As a result, multidimensional observation of the sample is possible.

Further, the wavelength of the irradiation light is changed, and the scanning, the information collection and the information storage are performed for each wavelength in the same manner as described above, and the optical image information of the entire scanning area of the sample and the light irradiation position are determined. The electrophysiological characteristic information is analyzed, and the microscopic observation image and the bioelectric reaction are associated with each other and reconstructed to display or record. As a result, it is possible to observe a multidimensional sample according to the wavelength of the irradiation light.

Further, the optical probe is arranged at a fixed position without performing the scanning, the pinpoint illumination is performed while the range of the action of the light is limited, and the information is collected with time. It is also possible to observe changes in optical properties and changes in electrophysiological characteristics of the sample that occur in response to the above.

Further, by arranging the optical probe at a fixed position without scanning, changing the wavelength of the irradiation light while limiting the range of the action of light, and collecting information over time, It is also possible to observe the wavelength dependence of changes in the optical physical properties of the sample and changes in the electrophysiological properties that occur in response to pinpoint illumination.

The optical probe microscope apparatus according to claim 5 is
(A) an optical microscope section for observing a wide area image of a sample; and (b)
A scanning type near-field optical microscope section for observing a local image of the sample, which has an optical probe installed on the sample, and a variable wavelength light irradiation section for irradiating the sample with light; Obtained from the scanning near-field optical microscope unit for each scanning position of the electrical stimulation unit that applies an electrical stimulation to one living cell or a minute area of the biological membrane, and (d) the sample stimulated by the electrical stimulation unit. A local image of the obtained sample is collected, recorded and analyzed, and an analysis control unit for controlling the entire apparatus is provided.

Here, the electrical stimulator inputs a patch electrode for accessing the surface of the sample, a micromanipulator for moving the patch electrode, and a current signal across the biomembrane which is a bioelectric signal detected by the patch electrode. , A preamplifier that converts to a voltage signal, amplifies and outputs, and outputs an electrical signal that imparts an electrical stimulus to the sample through the patch electrode, and an instruction from the analysis control unit An electric signal generator that outputs a first command signal related to measurement, membrane potential fixation, or membrane current fixation and a second command signal related to applying electrical stimulation to a sample, and a voltage signal output from an amplifier are input, Amplify, process and output to the analysis control unit, and also input and output the first command signal and the second command signal output from the electric signal generator. It may be characterized in that it comprises a main amplifier for outputting toward the preamplifier.

Further, the electrical stimulator receives microelectrodes for accessing the inside of the sample, a micromanipulator for moving the microelectrodes, and bioelectric signals detected by the microelectrodes, amplifies and outputs the bioelectric signals, and A preamplifier that outputs an electrical signal that gives an electrical stimulus to the sample via an electrode, and an electric signal generator that outputs a command signal relating to the electrical stimulus given to the sample by an instruction from the analysis controller. The voltage signal output from the preamplifier is input, amplified, processed, and output to the analysis control unit, and the command signal output from the electric signal generator is input and arithmetically processed to perform the preamplifier. It may be characterized in that it is provided with a main amplifying section for outputting toward.

Here, as the microelectrode, one of a glass microelectrode and a metal microelectrode can be preferably adopted.

In the optical microscope section, a Hoffman modulation system, a phase contrast microscope or a differential interference microscope capable of enhancing contrast can be preferably used.

The light irradiator may be provided with a wavelength tunable laser, and a white light source, a spectroscope for spectrally outputting the white light output from the white light source, and a light output from the spectroscope. And a light selector that selects and outputs light having a specific wavelength component from all wavelength components.

According to the optical probe microscope apparatus of claim 5,
First, a wide area image of the sample is obtained with the optical microscope section. Continued
Based on the wide area image of the sample thus obtained, a multi-dimensional micromanipulator or the like is used to bring the electrode tip close to or in contact with a specific position of a biological sample such as a target cell,
Alternatively, it penetrates into the sample.

When the patch clamp method is used, the tip of the patch electrode is brought close to and in contact with a specific position of a target biological sample such as a cell to form a gigaohm (GΩ) seal. At this time, in order to electrically monitor the process up to the formation of a gigaohm (GΩ) seal, a command pulse signal for measuring the seal resistance, fixing the membrane potential and fixing the membrane current is output to the preamplifier via the main amplifier. In order to
An electric signal generator (pulse generator) controlled by the analysis control unit may be used.

When the microelectrode method is used, the tip of the microelectrode composed of a glass microelectrode or a metal microelectrode is made to penetrate from a specific position of a biological sample such as a target cell. Then, the electric signal detected by the microelectrode during the process from approach to intrusion is electrically monitored through the preamplifier and the main amplifier. An electric signal generator (pulse generator) controlled by the analysis control unit may be used to output the command pulse signal to the preamplifier via the main amplifier in order to confirm that the cell is a living cell. .

Next, the optical probe of the scanning near-field optical microscope section is moved to a position on a biological sample such as a target cell while observing a wide area image of the sample obtained by the optical microscope section. Then, the tip of the optical probe is brought close to the initial position, which is the near-field region of the sample surface. In moving the optical probe to the near-field region, a system such as a non-contact scanning microscope mode or a shear force non-contact scanning microscope mode can be preferably used.

Thus, the geometrical arrangement for observing the sample is completed, and the preparation for optical microscopic observation and electrophysiological property measurement is completed.

First, an electrical stimulus is applied to the sample by introducing a current signal or a voltage signal from the electrical stimulator to the sample via the electrode.

Next, the irradiation light generated in the light irradiation unit is irradiated through the optical probe set at the initial position. Here, the irradiation light that is irradiated to the sample through the optical probe is evanescent light that is non-emission light, and since a lens for image formation is not used, there is no restriction of diffraction by the aperture of the lens and The resolution is determined only by the aperture diameter at the tip of the optical probe. As a result, if the diameter of the tip of the optical probe is set to several tens of nm and the distance between the optical probe and the sample is set to several tens of nm which is a near-field region, the number of wavelengths of the irradiation light is several tens.
With a resolution of 1/100 to 1/100, it is possible to observe the fine shape and structure of the sample, or the optical characteristics in a minute region. The local image of the sample thus observed is notified to the analysis control unit.

The analysis control unit collects the local image information of the sample notified from the scanning near-field optical microscope unit and stores it together with the initial position information. The analysis control unit consists of a main amplifier,
It is also possible to control the electric signal generator (pulse generator).

Next, the analysis unit instructs the scanning near-field optical microscope unit to perform scanning. Then, local image information of the sample is collected for each scanning position and stored together with the scanning position information.

In order to secure the scanning in the near-field region in scanning, a method such as constant height mode control, non-contact scanning microscope mode control, or shear force non-contact scanning microscope mode control is suitable. Can be used.

After the completion of scanning, information collection, and information storage, the analysis control unit analyzes the optical image information of the entire scanning region of the sample based on the stored information, and reconstructs the microscopic observation image. Display and record. As a result, it is possible to observe changes in optical properties with respect to electrical stimulation.

Further, the wavelength of the irradiation light is changed, and the scanning, information gathering, and information storing are performed for each wavelength in the same manner as described above, and the optical image information of the entire scanning region of the sample is analyzed to obtain a microscopic observation image. Is reconfigured and displayed and recorded. As a result, it is possible to observe changes in optical properties with respect to electrical stimulation depending on the wavelength of irradiation light.

By disposing an optical probe at a fixed position without scanning and collecting information over time, changes in the optical physical properties of the sample that occur in response to electrical stimulation can be over time. You can also observe.

Further, without scanning, the optical probe is arranged at a fixed position, the wavelength of the irradiation light is changed while the range of the action of light is limited, and information is collected with time. It is also possible to observe with time the change in optical properties of the sample that occurs in response to the physical stimulus.

Further, by changing the electrical stimulation condition in various ways, such as changing the amount of electric current to be introduced into the sample or the waveform, it is possible to observe the change in the optical physical properties of the sample according to the different electrical stimulation conditions. .

[0051]

BEST MODE FOR CARRYING OUT THE INVENTION Embodiments of the optical probe microscope apparatus of the present invention will be described below with reference to the accompanying drawings. In the description of the drawings, the same elements will be denoted by the same reference symbols, without redundant description.

(First Embodiment) FIG. 1 is a block diagram of a first embodiment of an optical probe microscope apparatus of the present invention. This device is a device that obtains an optical image of a sample with a high position resolution by a near-field microscope function and measures changes in electrophysiological properties due to optical stimulation by a patch clamp measurement function.

As shown in FIG. 1, this apparatus comprises (a) an optical microscope section 100 capable of observing a wide area image of the sample 900, and (b) an optical probe 211 installed on the upper part of the sample 900. A scanning near-field optical microscope unit that has a variable wavelength light irradiation unit 210 that irradiates the sample 900 with light and an imaging unit 220 that images and images the light from the sample 900, and observes a local image of the sample 900. 200 and (c) sample 90
It is obtained from a patch clamp measurement unit 300 that derives a current across the biological membrane from a single living cell in 0 or a minute region of the biological membrane, calculates and amplifies it, and (d) a scanning near-field optical microscope unit 200. A local image of the sample and electrophysiological information obtained from the patch clamp measurement unit 300 for each light irradiation position of the scanning near-field optical microscope unit 200 are collected, integrated, recorded, analyzed, and scanned. The analysis control unit 400 that notifies the type near-field optical microscope unit 200 of the irradiation light wavelength instruction and scanning instruction and controls the main amplifier and the electric signal generator (pulse generator) of the patch clamp measurement unit 300. Then, the water-immersed sample 900 stored in the container 500 having a heat retaining function is observed.

In the optical microscope section 100, a Hoffman modulation system, a phase contrast microscope, or a differential interference microscope can be preferably used.

FIG. 2 is a block diagram of the variable wavelength light irradiation section 210. FIG. 2A shows a variable wavelength light source with an analysis control unit 4
00 shows a case in which the wavelength tunable laser 212 that outputs the light of the wavelength instructed from 00 is used, and FIG. 2B shows the wavelength tunable light source as the white light source 215 and the white light source 215.
A spectroscope 216 for spectrally outputting the white light output from
Movable slit 2 for selecting and outputting light having a specific wavelength component from all wavelength components of the light output from the spectroscope 216
17 shows the case of being composed of 17 and.

Further, the optical probe 211 is provided with a glass tube having an opening of several tens nm at its tip.

The image pickup section 220 includes an objective lens 221 that collects light from the sample, an image forming optical system 222 that forms an image of the light passing through the objective lens 221, and an image pickup device 223 having a light receiving surface on the image forming surface. With.

The patch clamp measuring section 300 includes a patch electrode 310 for accessing a sample and a patch electrode 310.
A multidimensional micromanipulator 320 for moving the
A preamplifier that converts and amplifies a current, which is a bioelectric signal detected by the patch electrode 310, into a voltage signal and outputs the voltage signal;
A preamplifier 330 that inputs a current signal that is a bioelectric signal that is detected by the patch electrode 310 across the biological membrane, converts the current signal into a voltage signal, amplifies and outputs the voltage signal, and an analysis control unit 40.
According to instructions from 0, seal resistance measurement, membrane potential fixation,
And an electric signal generator 380 that outputs a command signal for fixing the membrane current, and the voltage signal output from the preamplifier 300 is input, amplified, processed, and analyzed and controlled.
And a main amplifier 340 which outputs a command signal output from the electric signal generator 380, performs arithmetic processing, and outputs the processed signal to the preamplifier 330.

In the optical probe microscope apparatus of this embodiment, first, the optical microscope section 100 obtains a wide area image of the sample.
Then, based on the obtained wide area image of the sample, the multi-dimensional micromanipulator 320 is used and the patch electrode 310 is used.
The tip of the is brought into contact with a specific position of the biological sample 900 such as a target cell. Then, the patch electrode 310
A negative pressure is applied to the inside of the sample to suck the sample 900, and the cell membrane is sucked and penetrates to form a gigaohm (GΩ) seal (see FIG. 3). At this time, in order to electrically monitor the process until the formation of the GΩ seal, command pulses for measuring the seal resistance value, fixing the membrane potential, and fixing the membrane current are supplied to the main amplifier 3.
Pulse generator 380 under the control of the analysis control section 400 for outputting to the preamplifier 330 via 40.
Is used.

After confirming the formation of the GΩ seal, the electrode 31
The cell membrane sucked and invaded into the inside of 0 is destroyed to make a whole cell recording state (see FIG. 4).

Next, the optical probe 211 of the scanning near-field optical microscope unit 200 is moved to a position on the biological sample 900 such as a target cell while observing a wide area image of the sample obtained by the optical microscope unit 100. To do. Then, the optical probe 211 is scanned by scanning the three-dimensional micromanipulator.
Of the sample 900 whose tip is the near-field region of the surface of the sample 900
Then, the initial position of a distance of several tens nm is approached. For moving the optical probe 211 to the near-field region, a method such as a non-contact scanning microscope mode or a shear force non-contact scanning microscope mode can be preferably used.

Thus, the geometrical arrangement for observing the sample 900 is completed, the optical microscopic observation and the electrophysiological measurement are ready, and the electrophysiological characteristic measurement is started.

First, the analysis control section 400 notifies the scanning near-field optical microscope section 200 of the wavelength of the irradiation light. In response to this instruction, the irradiation light generated by the light irradiation unit 210 is irradiated onto the sample 900 via the optical probe 211 set at the initial position. Here, the sample 9 is passed through the optical probe 211.
The irradiation light applied to 00 is non-radiation evanescent light, and since a lens for image formation is not used, there is no restriction of diffraction due to the aperture of the lens, and the positional resolution is that of the tip of the optical probe 211. It is determined only by the aperture diameter. As a result, if the diameter of the tip of the optical probe 211 is set to several tens nm and the distance between the tip of the optical probe 211 and the sample 900 is set to several tens nm, which is a near-field region, then several tenths to several hundreds of wavelengths of irradiation light. With a resolution of one-half, it is possible to observe the fine shape and structure of the sample, or the optical characteristics in a minute region. The local image of the sample 900 thus observed is notified to the analysis unit 400.

Further, changes in current and membrane potential across the biological membrane due to electrophysiological changes in the sample caused by light irradiation are derived from the patch electrode 310 and measured by the patch clamp measuring section 300. Is notified to the analysis control unit 400. The current / potential derived by the patch electrode 310 is converted into a voltage signal by the current / voltage converter in the preamplifier 330, but since it is a minute signal, it is mainly processed by amplification, calculation, reduction of current noise, etc. Amplifier 3
The analysis control unit 400 is notified via 40.

The analysis control unit 400 collects the local image information of the sample notified from the scanning near-field optical microscope unit 200 and the electrophysiological characteristic information notified from the patch clamp measurement unit 300, and together with the initial position information. Store.

Next, the analysis control section 400 instructs the scanning near-field optical microscope section 200 to perform XY scanning. And
Similar to the above, local image information and electrophysiological information of the sample 900 are collected for each scanning position and stored together with the scanning position information.

In order to secure the scanning in the near-field region in scanning, a method such as constant height mode control, non-contact scanning type microscope mode control, or shear force non-contact scanning type microscope mode control is suitable. Can be used. When a sample in a water-immersed state is to be observed, scanning by constant height mode control is particularly suitable.

Next, the analysis control unit 400 notifies the scanning near-field optical microscope unit 200 of the change in the wavelength of the irradiation light.
Then, in the same manner as described above, the sample 9 for the designated wavelength is
00 local image information and electrophysiological information are collected and stored together with scanning position information.

Thereafter, local image information and electrophysiological information of the sample 900 are collected and stored together with the scanning position information for all the irradiation light wavelengths for the purpose of observation in the same manner as described above.

After the completion of scanning, information collection, and information storage, the analysis control unit 400 determines the sample 90 based on the stored information.
The optical image information of the entire 0 scanning area and the electrophysiological characteristic information according to the light irradiation position are analyzed, and the microscopic observation image and the bioelectric reaction are reconstructed in correspondence to display or record.

By using the apparatus of this embodiment, the optical probe 211 is arranged at a fixed position without performing scanning, and pinpoint illumination is performed while limiting the range of the action of light. By collecting information, it is also possible to observe changes in optical physical properties and electrophysiological characteristics of the sample corresponding to this pinpoint illumination over time.

Further, by using the apparatus of this embodiment, the optical probe 211 is arranged at a fixed position without scanning, and the wavelength of the irradiation light is changed while limiting the range of light action. By collecting information over time, it is also possible to observe over time the wavelength dependence of changes in the optical physical properties and changes in electrophysiological properties of the sample that occur in response to this pinpoint illumination.

Further, by using the apparatus of this embodiment, it is possible to observe the irradiation light with one wavelength species.

Although the patch electrode is used in the present embodiment, the glass electrode or the metal microelectrode may be used in place of the patch electrode in the same manner as in the present embodiment. , It is possible to observe the change in the optical aspect of the sample and the electrophysiological aspect depending on the light stimulation position with high position resolution.

When the microelectrode is used, it is not necessary to form the GΩ seal in the case of the patch electrode. Further, when it is not confirmed that the cells are living cells, the pulse generator, which is the electric signal generator, can be excluded from the configuration.

(Second Embodiment) FIG. 5 is a block diagram of a second embodiment of the optical probe microscope apparatus of the present invention. This device is a device for observing an optical image of a sample to which an electrical stimulus has been applied, with a high position resolution by a near-field microscope function.

As shown in FIG. 5, the apparatus of this embodiment is
(A) An optical microscope unit 100 capable of observing a wide area image of the sample 900, and (b) a light irradiation unit 2 having an optical probe 211 installed on the upper part of the sample 900 and irradiating the sample with light.
10, a scanning near-field optical microscope section 200 for observing a local image of the sample 900, and (c) applying electrical stimulation to the sample from a single living cell in the sample 900 or a minute region of a biological membrane. A patch clamp stimulating unit 350 for performing (d)
A local image of the sample obtained from the scanning near-field optical microscope unit 200 is collected, recorded, and analyzed for each scanning position of the sample 900 stimulated by the patch clamp stimulation unit 350, and analysis for controlling the entire apparatus is performed. And a control unit 450. Then, the water-immersed sample stored in the container 500 having a heat retaining function is observed.

In the optical microscope section 100, a Hoffman modulation system, a phase contrast microscope, or a differential interference microscope can be preferably used as in the first embodiment.

The patch clamp stimulator 350 includes a patch electrode 360 for accessing the sample and a patch electrode 360.
A multi-dimensional micromanipulator 370 for moving the
An electric signal that is a bioelectric signal detected by the patch electrode 360, that is, a current signal that traverses a biological membrane is input, converted into a voltage signal, amplified, and output, and at the same time, an electric signal is applied to the sample through the patch electrode. Preamplifier 33 for outputting
5 and instructions from the analysis control unit 450 to measure the seal resistance, fix the membrane potential, and fix the membrane current.
On the application of electrical stimulus to the sample command signal and sample
And the voltage signal output from the preamplifier 335 is input, amplified, processed, and output to the analysis controller 450, and the electric signal generator 385. The main amplification unit 345 that receives the first command signal and the second command signal output from the input unit, performs arithmetic processing, and outputs the processed signal to the pre-amplification unit 335.

In the optical probe microscope apparatus of this embodiment, a wide area image of the sample is obtained by the optical microscope section 100 before the sample is observed. Subsequently, based on the obtained wide area image of the sample, the multi-dimensional micromanipulator 370 is used,
The tip of the patch electrode 360 is brought close to and in contact with a specific position of a biological sample 900 such as a target cell to form a gigaohm (GΩ) seal, and the whole cell state is obtained as in the first embodiment.

Then, similarly to the first embodiment, the tip of the optical probe 211 is brought close to the initial position which is the near-field region of the sample 900.

Thus, the geometrical arrangement for observing the sample 900 is completed.

First, an electrical stimulus is applied to the sample 900 by introducing a current / voltage signal from the patch clamp stimulator 350 to the sample 900 via the patch electrode 360.

Next, the irradiation light generated by the light irradiation unit 210 is irradiated through the optical probe 211 set at the initial position. As in the first embodiment, the irradiation light that is irradiated onto the sample 900 via the optical probe 211 is evanescent light that is non-emissive light and does not use a lens for image formation. The position resolution is determined only by the opening diameter of the tip of the optical probe without being restricted by the above. As a result, several tenths to several hundreds of wavelengths of irradiation light
The fine shape and structure of the sample 900, or the optical characteristics in a minute region can be observed with a resolution of one-half. The observed local image of the sample 900 is notified to the analysis control unit 450.

The analysis control section 450 collects the local image information of the sample 900 notified from the scanning near-field optical microscope section 200 and stores it together with the initial position information.

Next, the analysis control section 450 instructs the scanning near-field optical microscope section 200 to perform scanning. Then, local image information of the sample 900 is collected for each scanning position and stored together with the scanning position information.

Next, the analysis control section 450 notifies the scanning near-field optical microscope section 200 of the change in the wavelength of the irradiation light.
Then, in the same manner as described above, the sample 9 for the designated wavelength is
00 local image information is collected and stored together with the scanning position information.

Thereafter, local image information of the sample 900 is collected and stored together with the scanning position information in the same manner as described above for all the irradiation light wavelengths for observation.

After the completion of scanning, information collection, and information storage, the analysis control unit 450 causes the sample 90 based on the stored information.
The optical image information of the entire 0 scanning area is analyzed, and the microscopic observation image is reconstructed for display or recording. As a result, it is possible to observe changes in optical properties with respect to electrical stimulation depending on the wavelength of irradiation light.

Further, by arranging the optical probe 211 at a fixed position without performing scanning and collecting information over time, changes in optical physical properties of the sample 900 that occur in response to an electrical stimulus are over time. You can also observe it.

Further, by arranging the optical probe 211 at a fixed position without scanning, changing the wavelength of the irradiation light while limiting the range of the action of light, and collecting information over time, Sample 900 generated in response to electrical stimulation
It is also possible to observe the change in the optical physical properties of the above with time depending on the wavelength.

Further, by changing the electrical stimulation condition in various ways such as changing the amount of current or the waveform introduced to the sample 900, it is possible to observe changes in the optical physical properties of the sample according to different electrical stimulation conditions. it can.

Further, although the patch electrode is used in the present embodiment, similar to the first embodiment, a micro electrode such as a glass micro electrode or a metal micro electrode which penetrates into the sample may be used instead of the patch electrode. In the same manner as this embodiment, it is possible to observe changes in the aspect of the sample due to electrical stimulation with high position resolution.

If microelectrodes are used, it is not necessary to form the GΩ seal in the case of patch electrodes.

[0095]

As described in detail above, according to the optical probe microscope apparatus of the first aspect of the present invention, the scanning near-field optical microscope section is used for optical microscope observation, and the scanning near-field optical microscope function is provided. The electrophysiological changes caused by the irradiation of light on a microscopic area by an optical probe with a micro-aperture, which is essential for measurement, are measured by the electrical information measurement unit, and both information are integrated by the analysis unit, so the optical aspect of the sample with high positional resolution. When,
It is possible to observe changes in electrophysiological aspects depending on the light stimulation position.

According to the optical probe microscope apparatus of the fifth aspect of the present invention, since the sample electrically stimulated by the electrical stimulator is observed by the scanning near-field optical microscope with an optical microscope, the electrical stimulation is performed. It is possible to observe the change in the aspect of the sample due to the high positional resolution.

[Brief description of drawings]

FIG. 1 is a configuration diagram of an optical probe microscope according to a first embodiment of the present invention.

FIG. 2 is a configuration diagram of a wavelength variable light irradiation unit.

FIG. 3 is an explanatory diagram of a geometrical arrangement near a sample when forming a GΩ seal.

FIG. 4 is an explanatory diagram of a geometrical arrangement in the vicinity of a sample in a whole cell recording state.

FIG. 5 is a configuration diagram of an optical probe microscope according to a second embodiment of the present invention.

[Explanation of symbols]

100 ... Optical microscope part, 200 ... Scanning near-field optical microscope part, 210 ... Wavelength variable light irradiation part, 211 ... Optical probe, 212 ... Wavelength variable laser, 213 ... White light source, 21
4 ... Spectrometer, 215 ... Slit, 300 ... Patch clamp measurement part, 310 ... Patch electrode, 320 ... Multidimensional micromanipulator, 330 ... Preamplifier, 340 ... Main amplifier, 350 ... Patch clamp stimulator, 360 ... Patch electrode 370 ... Multidimensional micromanipulator, 38
0,390 ... Electric signal generator, 400, 450 ... Analysis control unit.

Claims (11)

[Claims] 1. An optical microscope unit for observing a wide area image of a sample, an optical probe installed on the upper part of the sample, and a light irradiation unit for irradiating the sample with light, and observing a local image of the sample. A scanning near-field optical microscope unit, an electric information measuring unit for deriving and recording a current or a membrane potential across a biological membrane from a single living cell in the sample or a minute region of the biological membrane, and the scanning type A local image of the sample obtained from the near-field optical microscope unit and electrophysiological information obtained from the patch clamp measurement unit for each irradiation position of light of the scanning near-field optical microscope unit are collected and recorded. The optical probe microscope apparatus is characterized by comprising: an analysis control section for performing analysis and controlling the entire apparatus. 2. The electrical information measuring unit traverses a patch electrode that accesses the surface of the sample, a micromanipulator that moves the patch electrode, and the biomembrane that is a bioelectric signal detected by the patch electrode. A preamplifier that inputs a current signal, converts it into a voltage signal, amplifies and outputs it, and measures the seal resistance according to an instruction from the analysis control unit.
An electric signal generator that outputs a command signal related to membrane potential fixation or membrane current fixation, and a voltage signal output from the preamplifier is input, amplified, processed, and output toward the analysis control unit. 2. The optical probe microscope apparatus according to claim 1, further comprising: a main amplifier that receives the command signal output from the electric signal generator, performs arithmetic processing, and outputs the processed signal toward the preamplifier. . 3. The electrical information measuring unit inputs a microelectrode for accessing the inside of the sample, a micromanipulator for moving the microelectrode, and a bioelectric signal detected by the microelectrode, and amplifies the bioelectric signal. A preamplifier to output and a voltage signal output from the preamplifier are input, amplified, processed, and output toward the analysis control unit, and the command signal output from the electrical signal generator. 2. The optical probe microscope apparatus according to claim 1, further comprising: a main amplifier that inputs, performs arithmetic processing, and outputs toward the preamplifier. 4. The optical probe microscope apparatus according to claim 3, wherein the microelectrode is one of a glass microelectrode and a metal microelectrode. 5. A local image of the sample, comprising an optical microscope unit for observing a wide area image of the sample, an optical probe installed on the upper part of the sample, and a variable wavelength light irradiation unit for irradiating the sample with light. A scanning near-field optical microscope section for observing, an electrical stimulating section for applying an electrical stimulus to a single living cell in the sample or a minute area of a biological membrane, and the electrical stimulating section A local image of the sample obtained from the scanning near-field optical microscope unit for each scanning position of the sample is collected, recorded and analyzed, and an analysis control unit for controlling the entire apparatus, Optical probe microscope device. 6. The electrical stimulation unit comprises a patch electrode for accessing the surface of the sample, a micromanipulator for moving the patch electrode, and a current across the biomembrane which is a bioelectric signal detected by the patch electrode. A signal is input, converted into a voltage signal, amplified and output, and a preamplifier that outputs an electrical signal that imparts an electrical stimulus to the sample via the patch electrode, and from the analysis controller. According to the instructions, measure the seal resistance,
An electric signal generator that outputs a first command signal for fixing the membrane potential and fixing the membrane current and a second command signal for applying an electrical stimulus to the sample; and a voltage signal output from the preamplifier. While inputting, amplifying, processing, and outputting to the analysis control unit, the first command signal and the second command signal output from the electric signal generator are input and arithmetic processing is performed. The optical probe microscope apparatus according to claim 5, further comprising: a main amplification unit that outputs the pre-amplification unit. 7. The electrical stimulator receives microelectrodes that access the inside of the sample, a micromanipulator that moves the microelectrodes, and a bioelectric signal detected by the microelectrodes that is input, amplified, and output. In addition, a preamplifier that outputs an electrical signal that gives an electrical stimulus to the sample via the microelectrode, and a command signal relating to the electrical stimulus given to the sample by an instruction from the analysis controller. An electrical signal generator for output, and the voltage signal output from the preamplifier is input, amplified, processed and output toward the analysis controller, and the output from the electrical signal generator is output. An optical probe microscope apparatus according to claim 5, further comprising: a main amplification unit that receives a command signal, performs arithmetic processing, and outputs the processed signal toward the pre-amplification unit. 8. The optical probe microscope apparatus according to claim 7, wherein the microelectrode is one of a glass microelectrode and a metal microelectrode. 9. The optical microscope unit comprises any one of a Hoffman modulation system, a phase contrast microscope, and a differential interference microscope.
Alternatively, the optical probe microscope apparatus according to item 5. 10. The optical probe microscope apparatus according to claim 1, wherein the light irradiation unit includes a variable wavelength laser. 11. The light irradiator includes a white light source, a spectroscope for spectrally outputting white light output from the white light source, and a specific wavelength from all wavelength components of the light output from the spectroscope. An optical probe microscope apparatus according to claim 1 or 5, further comprising: a light selector that selects and outputs component light.