JP5806450B2 - Cell observation method - Google Patents

Description

  The present invention relates to a cell observation method.

  Conventionally, methods for observing membrane potential activity of living cells are known (see, for example, Patent Document 1 and Patent Document 2). In the method for measuring the membrane potential described in Patent Document 1, live cells stained with a membrane potential sensitive dye are irradiated with excitation light, and the absorption reaction or fluorescence of the membrane potential sensitive dye is detected by a two-dimensional photosensor such as a photodiode array or CCD. Take a picture of the reaction. Since the absorbance or fluorescence intensity of the membrane potential sensitive dye changes depending on the membrane potential of the cell to which the membrane potential sensitive dye is attached, it is indirectly determined by measuring the absorbance or fluorescence intensity of the membrane potential sensitive dye. In addition, cell membrane potential activity can be observed. In addition, the multipoint simultaneous measurement method described in Patent Literature 2 combines a Niipou disk type confocal microscope and a CCD to capture a fast membrane potential response as a confocal image.

  Here, in the observation of the membrane potential activity of living cells, a method of causing a membrane potential change by electrically stimulating cells with an insertion electrode such as a patch clamp is generally used. However, electrical stimulation of cells with insertion electrodes is not practical because a large number of electrodes must be inserted into the sample when a plurality of cells are to be stimulated. Moreover, in order to change a stimulation point, it is necessary to re-stab an electrode in another position, and there exists a demerit that it will be a damage to a living cell in addition to an effort and time.

  On the other hand, methods other than electrical stimulation with electrodes are also known as methods for stimulating cells (see, for example, Patent Document 3). The scanning confocal microscope apparatus described in Patent Literature 3 uses a caged compound reagent to stimulate living cells. Specifically, the scanning confocal microscope apparatus described in Patent Document 3 emits a neurotransmitter from a caged compound by irradiating the caged compound with ultraviolet laser light by a galvanomirror scanning method, thereby causing an action potential of the cell. Is triggered.

JP 2004-115633 A JP 2004-212132 A JP-A-7-333515

  However, the scanning confocal microscope apparatus described in Patent Document 3 has a disadvantage in that the membrane potential of a cell cannot be measured with high temporal resolution due to insufficient temporal resolution for imaging of membrane potential. Thus, in the past, only one of cell stimulation and membrane potential measurement could be performed with a high degree of spatial freedom and temporal resolution, but both are compatible with a high degree of spatial freedom and temporal resolution. Could not be done.

  The present invention has been made in view of the above-described circumstances, and provides a cell observation method and a scanning microscope apparatus capable of observing a cell membrane potential response to a stimulus with high spatial freedom and temporal resolution. Objective.

In order to solve the above problems, the present invention employs the following means.
The present invention relates to a reference image acquisition step of acquiring a reference image of a neuron cell into which a channel rhodopsin 2 gene and a membrane potential sensitive dye that changes the light intensity generated according to the magnitude of the cell membrane potential are introduced, and the reference image With respect to the reference image acquired by the acquisition step, a stimulation region for stimulating the nerve cells by laser irradiation and an observation region for exciting the membrane potential sensitive dye by laser irradiation are smaller than the visual field range of the reference image. A region designation step to designate, and the stimulation region designated in the region designation step is irradiated with a laser beam having a wavelength of 400 nm to 500 nm, and the nerve is produced by channel rhodopsin 2 expressed in the nerve cell from the channel rhodopsin 2 gene. A stimulation process for stimulating cells to induce membrane potential activity, and irradiating the observation area with laser light. A cell comprising: an observation image acquisition step of scanning a condensing position to excite the membrane potential sensitive dye, detecting a light intensity of signal light emitted from the membrane potential sensitive dye, and acquiring an observation image of the observation region Provide an observation method.

According to the present invention, by designating a stimulation region and an observation region with respect to a reference image of a biological sample (nerve cell) acquired by a reference image acquisition step, desired cells can be simplified by the stimulation step and the observation image acquisition step. Can be stimulated, and the observation image can be acquired. In addition, by designating the observation region within a range smaller than the visual field range of the reference image, it is possible to shorten the time required for scanning in the observation image acquisition step and observe the observation region quickly.

  In this case, when the membrane potential activity of the cell is induced by the physiologically active substance in the stimulation region in the stimulation process, the membrane potential activity is propagated from the stimulation region to the observation region. In addition, since the light intensity of the membrane potential sensitive dye changes depending on the membrane potential of the cell to which the membrane potential sensitive dye is attached, when the membrane potential activity of the cell is propagated to the observation region, the light of the membrane potential sensitive dye The intensity changes.

  Therefore, by observing the observation image of the observation region obtained by detecting the light intensity of the signal light emitted from the membrane potential sensitive dye, the observation site accompanying the propagation of the membrane potential response generated in the stimulated region (stimulation region) ( It is possible to indirectly observe the temporal change of the membrane potential in the observation region). Thereby, the membrane potential response of the cell to the stimulus can be observed with high spatial freedom and temporal resolution.

  In the observation image acquisition step, scanning may be performed in a direction intersecting the depth direction while changing the condensing position of the laser light applied to the observation region in the depth direction. By doing in this way, an observation area can be observed three-dimensionally and the improvement of space freedom can be aimed at. Note that either of the stimulation process and the observation image acquisition process may be started first, or both processes may be started simultaneously.

In the above invention, the region designating step may designate the line-shaped observation region.
With this configuration, the scanning range can be limited to one direction compared to scanning a three-dimensional or planar observation region, and the membrane potential response of cells to stimuli can be observed with higher temporal resolution. can do.

Moreover, in the said invention, the said area | region designation | designated process is good also as designating the said observation area | region of a point shape.
With this configuration, it is possible to limit the scanning range to a narrower range than when scanning a line-shaped observation region, and it is possible to observe the membrane potential response of cells to a stimulus with higher time resolution.

Moreover, in the said invention, it is good also as performing the laser irradiation to the said stimulation area | region by the said stimulation process, and the laser irradiation to the said observation area | region by the said observation image acquisition process by a different optical system.
With this configuration, it is possible to control the laser irradiation to the stimulation region and the laser irradiation to the observation region independently in terms of time and space. Therefore, temporal and spatial freedom of cell stimulation and cell observation can be increased.

Moreover, in the said invention, the said observation image acquisition process is good also as acquiring the observation image of the same said observation area | region several times in time intervals.
With this configuration, it is possible to observe the movement of the cell membrane potential increase site with time.

In the aspect described above, the observation pattern in which the observation image acquisition process is repeated to obtain a plurality of times the observed image with a predetermined acquisition timing after at regular time intervals after stimulation of the stimulation step and the stimulation step, cells Is performed a plurality of times with a time interval that can be activated again, and the light intensity of the observation image acquired by the observation image acquisition step is determined by the acquisition timing of the plurality of observation images in each observation pattern. An average calculation step of calculating an average by adding the light intensities of the same observation image may be included.
With this configuration, it is possible to reduce the noise component of the light intensity in the observation image over time by the average calculation step, and to observe the time-series change of the membrane potential response of the cell to the stimulus at a high S / N. .

In the aspect described above, the observation pattern in which the observation image acquisition process is repeated to obtain a plurality of times the observed image with a predetermined acquisition timing after at a predetermined time interval following stimulation of the stimulation step and the stimulation step, the The predetermined time interval from the stimulation of the cell to the acquisition of the first observation image is shifted for each observation pattern by a time interval shorter than the time required for one scan, so that the cell can be activated again. at a time interval, have multiple rows may be varied every plurality of images obtained as a result time-series change in fluorescence intensity.
By configuring in this way, it is possible to detect and detect a change in membrane potential faster than the time required for one scan, and to observe the membrane potential response of a cell to a stimulus with higher temporal resolution. .
In the above invention, the membrane potential sensitive dye may be a fluorescent dye FM4-64.
In the above invention, the signal light may be second harmonic (SHG) signal light.

Further, the above invention may include a time change calculating step of calculating a time change of light intensity at each point of the observation region based on the plurality of observation images acquired by the observation image acquiring step. .
With this configuration, it is possible to indirectly and indirectly observe the time-series change of the membrane potential response of the cell at each point in the observation region based on the time change of the light intensity at each point in the observation region.

The reference example of the present invention scans in the direction intersecting the depth direction while changing the condensing position of the light irradiated to the biological sample in the depth direction, and the three-dimensional reference image and observation image of the biological sample are scanned. An observation optical system that can be acquired, a stimulation optical system that stimulates cells by irradiating light to the biological sample, and the cells that are stimulated by the stimulation optical system for the reference image acquired by the observation optical system A region designating unit for designating a stimulation region to be performed and an observation region for obtaining the observation image within a range smaller than the visual field range of the reference image; and after stimulation by the stimulation optical system for the stimulation region of the cell designated by the region designating unit From the plurality of observation images acquired at different times by scanning the observation region of the cell designated by the region designation unit a plurality of times with the observation optical system, the position and light intensity on the scanning path A position characteristic calculation unit that calculates a position characteristic of a plurality of light intensities indicating a relationship, and a time change of the light intensity at a desired position on the scanning path from the position characteristics of the plurality of light intensities calculated by the position characteristic calculation unit. Provided is a scanning microscope apparatus comprising a time characteristic calculation unit for calculating.

According to this reference example , stimulation is applied to the cells in the stimulation region designated by the region designation unit on the three-dimensional reference image of the biological sample acquired by the observation optical system, and similarly. A three-dimensional observation image of the observation region designated by the region designation unit is acquired by the observation optical system.

  Here, for a biological sample into which a bioactive substance that activates cells upon receiving light and induces membrane potential activity and a membrane potential sensitive dye that changes the light intensity generated according to the magnitude of the membrane potential of the cells are introduced. When the stimulation area and observation area are designated by the area designating unit, light is applied to the stimulation area by the stimulation optical system, and the membrane potential activity of the cell is induced by the physiologically active substance, the membrane potential activity is transferred from the stimulation area to the observation area. Propagated. In addition, since the light intensity of the membrane potential sensitive dye changes depending on the membrane potential of the cell to which the membrane potential sensitive dye is attached, when the membrane potential activity of the cell is propagated to the observation region, the light of the membrane potential sensitive dye The intensity changes. Therefore, if an observation image of the observation area is acquired by the observation optical system after stimulating the cells in the stimulation area by the stimulation optical system, the change in the cell membrane potential with respect to the stimulation in the stimulation area is reflected in the change in the light intensity in the observation area. Can be observed indirectly.

  In addition, after stimulating the cells in the stimulation area with the stimulation optical system, the position characteristic calculator calculates a desired position on the scanning path from a plurality of observation images acquired at different times by scanning the observation area multiple times with the observation optical system. By calculating the position characteristics of the light intensity at the time and calculating the time change of the light intensity at the position by the time characteristic calculation section, the time change of the membrane potential response of the cell to the stimulus can be easily performed with high spatial freedom and time resolution Can be observed.

  According to the present invention, it is possible to observe the membrane potential response of a cell to a stimulus with high spatial freedom and temporal resolution.

It is a flowchart of the cell observation method which concerns on the 1st Embodiment of this invention. 1 is a schematic configuration diagram of a scanning laser microscope according to a first embodiment of the present invention. It is a figure which shows the reference image acquired by a reference image acquisition process. It is a figure which shows a mode that the irritation | stimulation area | region and the observation area | region were designated to the reference image by the area | region designation | designated process. It is a figure which shows the operation | movement which acquires an observation image in multiple times by an observation image acquisition process. It is a figure which shows the relationship between the line direction for every acquisition timing in each observation image, and fluorescence intensity. It is the figure which showed each point for observing a time-dependent change of a membrane potential in an observation area | region. It is a figure which shows the relationship between the line direction for every acquisition timing in each observation image, and fluorescence intensity. It is the figure which showed the relationship between time after stimulating a cell and the fluorescence intensity of an observation image. (A) is a figure which shows each observation pattern by the cell observation method which concerns on the 1st modification of this embodiment, (b) is the figure which replaced (a) with the average of each observation pattern by the average calculation process. is there. It is a figure which shows each observation pattern by the cell observation method which concerns on the 2nd modification of this embodiment. It is a figure which shows a mode that the stimulation area | region and the observation area | region were designated to the reference image by the cell observation method which concerns on the 3rd modification of this embodiment. It is an enlarged view of the observation area | region of FIG. (A) shows an observation image 12 ms after stimulation, (b) shows an observation image 14 ms after stimulation, (c) shows an observation image 16 ms after stimulation, (d) shows stimulation The observation image after 18 ms elapses from (i) shows the observation image after 20 ms elapses from the stimulus, and (f) shows the observation image after 22 ms elapses from the stimulation. (A) shows a state in which observation positions (points A to D) are designated in the observation region, and (b) is a diagram showing a change in membrane potential with time at each observation position in (a). It is a schematic block diagram of the scanning laser microscope which concerns on the 2nd Embodiment of this invention. It is a schematic block diagram of the scanning laser microscope which concerns on the 3rd Embodiment of this invention.

[First Embodiment]
Hereinafter, a cell observation method and a scanning microscope apparatus according to a first embodiment of the present invention will be described with reference to the drawings.
First, as shown in the flowchart of FIG. 1, the cell observation method according to the present embodiment includes a reference image acquisition step SA1 for acquiring a reference image of a specimen (biological sample), and a stimulation region and a reference region on the acquired reference image. An area designating process SA2 for designating an observation area, a stimulation process SA3 for stimulating cells by irradiating the designated stimulation area with laser light, and a fluorescence (signal) emitted from the cells by irradiating the observation area with laser light after the stimulation process SA3 An observation image acquisition step SA4 for detecting the light) and acquiring an observation image, and a time change calculation step SA5 for calculating the time change of the light intensity at each position in the observation region based on the plurality of acquired observation images. It is out.

  When stimulating cells and observing cells using this cell observation method, a physiologically active substance that activates cells to induce membrane potential activity when exposed to light and light generated according to the magnitude of the membrane potential of the cells A membrane potential sensitive dye whose intensity changes is introduced into the specimen in advance.

In the reference image acquisition step SA1, a three-dimensional fluorescence image of the specimen is acquired as a reference image (pre-scan).
In the area designating step SA2, an arbitrary area in the three-dimensional space is designated as a stimulation area and an observation area within a range smaller than the visual field range of the reference image. Examples of the stimulation region and the observation region include a three-dimensional region, a planar region, a point-shaped region (point region), or a line-shaped region (line region).

  The stimulation process SA3 and the observation image acquisition process SA4 are configured to scan in the direction intersecting the depth direction while changing the condensing position of the laser light applied to the stimulation area and the observation area in the depth direction. In the stimulation step SA3, for example, the stimulation UV laser light having a predetermined wavelength is irradiated to the stimulation region at a predetermined timing. In the observation image acquisition step SA4, observation images in the same observation region are acquired a plurality of times at a predetermined acquisition timing with a predetermined time interval.

Next, a scanning laser microscope (scanning microscope apparatus) 1 according to the present embodiment will be described with reference to FIG.
The scanning laser microscope 1 according to the present embodiment includes, for example, as shown in FIG. 2, a light source unit 10 that emits laser light, and a scanner unit 20 that scans a sample A with laser light emitted from the light source unit 10. The sample unit A is irradiated with laser light scanned by the scanner unit 20, while the irradiation unit 30 for collecting the fluorescence returning from the sample A, the detection unit 40 for detecting the fluorescence from the sample A, and each of these units And a computer 50 having a control unit 51 for control.

  The light source unit 10 includes a stimulation light source unit 12 that emits stimulation light (laser light) that irradiates the specimen A and stimulates cells with a physiologically active substance, and excitation light that irradiates the specimen A and excites a membrane potential-sensitive dye. An observation light source unit 16 that emits a laser beam), and an optical path synthesis unit 19 that synthesizes an optical path of stimulation light from the stimulation light source unit 12 and an optical path of excitation light from the observation light source unit 16.

  The stimulation light source unit 12 includes a UV laser 11 having a wavelength of 351 nm and an acoustooptic device 13 that can modulate the light intensity of stimulation light emitted from the UV laser 11 from 0% to 100%. The acoustooptic device 13 is configured to block the stimulation light or transmit the stimulation light having a predetermined light intensity by setting the transmittance of the stimulation light to 0% under the control of the control unit 51 of the computer 50.

  The observation light source unit 16 includes an argon laser 15 having a wavelength of 515 nm, and an acoustooptic device 17 capable of modulating the light intensity of excitation light emitted from the argon laser 15 from 0% to 100%. The acoustooptic device 17 is configured to block the excitation light or transmit the excitation light having a predetermined light intensity by setting the transmittance of the excitation light to 0% under the control of the control unit 51.

The optical path synthesis unit 19 has a characteristic of reflecting stimulation light having a wavelength of 351 nm and transmitting excitation light having a wavelength of 515 nm.
The scanner unit 20 scans in the direction in which the spot position of the laser light (stimulation light and excitation light) applied to the specimen A intersects the optical axis, that is, the two-dimensional direction of X and Y, under the control of the control unit 51. Can be done.

  The irradiation unit 30 is reflected by the pupil projection lens 31 that forms the laser beam emitted from the scanner unit 20 as an intermediate image, the mirror 33 that reflects the intermediate image formed by the pupil projection lens 31, and the mirror 33. The imaging lens 35 that collects the light flux from the intermediate image to make parallel light, and the laser light that has been made parallel light by the imaging lens 35 irradiate the specimen A, while the fluorescence generated at the irradiation position of the specimen A The objective lens 37 which condenses, and the semi-focusing part 39 which can move the sample A to the optical axis direction (Z direction) of the laser beam inject | emitted from the objective lens 37 are provided.

  The semi-focusing part 39 includes a stage 38 on which the specimen A is placed. The semi-focusing unit 39 can move the stage 38 so that the condensing surface F of the laser light focused on the specimen A is at an arbitrary position with respect to the optical axis direction under the control of the control unit 51. It has become.

  The detection unit 40 transmits the laser light synthesized by the optical path synthesis unit 19, and reflects the fluorescence generated in the specimen A and returning through the irradiation unit 30 and the scanner unit 20, and the dichroic mirror 41. A wavelength separation filter 43 that removes unnecessary wavelength light from the reflected fluorescence, a condensing lens 45 that condenses the fluorescence emitted from the wavelength separation filter 43, and fluorescence condensed by the condensing lens 45 A confocal pinhole 47 that partially passes through and a photodetector 49 that detects fluorescence that has passed through the confocal pinhole are provided.

The dichroic mirror 41 has a characteristic of transmitting stimulation light having a wavelength of 351 nm and excitation light having a wavelength of 515 nm, and reflecting light having a wavelength longer than the wavelength of the excitation light.
The photodetector 49 converts the detected fluorescence into an electrical signal and outputs it to the computer 50.

  In addition to the control unit 51, the computer 50 includes an image constructing unit 53 that constructs a reference image and an observation image based on an output signal output from the photodetector 49, and a stimulation region on the reference image acquired by the observation optical system 5. And an area designating unit 55 for designating an observation area, and a position characteristic calculation for calculating a position characteristic of light intensity indicating a relationship between a predetermined observation position on the scanning path and light intensity from an observation image acquired by the observation optical system 5 And a time characteristic calculation unit 59 that calculates a temporal change of the light intensity at the observation position from the position characteristic of the light intensity calculated by the position characteristic calculation unit 57. The image construction unit 53, the region designating unit 55, the position characteristic calculation unit 57, and the time characteristic calculation unit 59 perform various processes via the control unit 51.

  The stimulation optical unit 12, the scanner unit 20, and the irradiation unit 30 configured in this way constitute the stimulation optical system 3 and can stimulate the cells of the specimen A. Further, the observation optical unit 16, the scanner unit 20, the irradiation unit 30, the detection unit 40, and the computer 50 constitute the observation optical system 5 and can acquire a reference image and an observation image of the specimen A. ing.

Next, reagents used in the cell observation method according to this embodiment will be described.
In this embodiment, as a physiologically active substance that activates cells upon receiving light and induces membrane potential activity, for example, glutamic acid, which is a physiologically active substance, side chains that inactivate the physiological activity of glutamic acid (caged compound) Use caged glutamic acid with. This caged glutamic acid is usually inactive, but when irradiated with ultraviolet light, its compound is cleaved and glutamic acid is dissociated to induce physiological activity. Moreover, as a membrane potential sensitive dye, for example, a membrane potential sensitive dye RH-795 is used.

  When the living tissue injected with caged glutamic acid is irradiated with UV laser light, the caged compound of caged glutamic acid at the irradiated site is cleaved, and activated glutamic acid is released. The released glutamic acid acts as an excitatory transmitter for the synapses of nerve cells in the vicinity, and causes a post-synaptic potential. When the post-synaptic potential accumulates and exceeds the synaptic firing threshold, the synapse generates an action potential. Therefore, when stimulating light (UV laser light) is irradiated to the stimulation region of the specimen A into which caged glutamic acid has been introduced, excitatory stimulation can be given to the cells in the stimulation region to induce cell action potentials.

  On the other hand, membrane potential sensitive dye RH-795 (hereinafter simply referred to as “membrane potential sensitive dye”) is a fluorescent dye having a maximum excitation wavelength of 530 nm and a fluorescence wavelength of 712 nm. This membrane potential sensitive dye has a characteristic that the fluorescence intensity changes by about 1% at the maximum depending on the magnitude of the membrane potential of the cell. In addition, the membrane potential-sensitive dye increases the fluorescence light intensity as the cell undergoes depolarization and undergoes depolarization to increase the membrane potential of the cell.

Hereinafter, the operation of the thus configured cell observation method and the scanning laser microscope 1 will be described.
First, the cell observation method according to this embodiment and the case where the cells of the specimen A are stimulated by the scanning laser microscope 1 will be described.
When stimulating cells, the stimulation optical system 3 stimulates the cells. Specifically, first, stimulation light having a wavelength of 351 nm is emitted from the UV laser 11 and passes through the acoustooptic device 13 to modulate the emission power. The stimulation light modulated by the acoustooptic device 13 is reflected by the optical path synthesis unit 19, then passes through the dichroic mirror 41, and is deflected in the XY two-dimensional direction by the scanner unit 20.

  The stimulation light deflected by the scanner unit 20 is collected by the pupil projection lens 31 so as to form an intermediate image and reflected by the mirror 33. The stimulation light reflected by the mirror 33 is condensed again by the imaging lens 35 to become parallel light, and is condensed on the condensing surface F on the specimen A by the objective lens 37.

  In this case, the control unit 51 controls the semi-focusing unit 39 to adjust the distance between the objective lens 37 and the sample A so that the condensing surface F on the sample A is at an arbitrary position with respect to the optical axis direction. As a result, it is possible to irradiate the stimulation light to an arbitrary region in the XYZ three-dimensional space on the specimen A at a predetermined timing.

  When stimulating light is irradiated to an arbitrary region of the specimen A, the caged compound of caged glutamic acid at the irradiated site is cleaved to release glutamic acid, and excitatory action potentials of nearby neurons are induced. Thereby, a desired cell can be stimulated.

Next, the cell observation method according to this embodiment and the case where the cells of the specimen A are observed with the scanning laser microscope 1 will be described.
When observing cells, an observation image of the specimen A is acquired by the observation optical system 5. Specifically, first, excitation light having a wavelength of 515 nm is emitted from the argon laser 15 and passes through the acoustooptic device 17 to modulate the emission power. The excitation light modulated by the acoustooptic device 17 passes through the optical path synthesis unit 19 and the dichroic mirror 41, and is then deflected in the XY two-dimensional direction by the scanner unit 20.

  The excitation light deflected by the scanner unit 20 is collected by the pupil projection lens 31 so as to form an intermediate image and reflected by the mirror 33. The excitation light reflected by the mirror 33 is condensed again by the imaging lens 35 to become parallel light, and is condensed on the condensing surface F on the specimen A by the objective lens 37. Then, similarly to the stimulation light, the control unit 51 controls the semi-focusing part 39 so that the excitation light can be irradiated to an arbitrary region in the XYZ three-dimensional space on the specimen A at a predetermined timing.

  In this case, when the excitation light is scanned on the light collection surface F of the specimen A by the scanner unit 20, the membrane potential sensitive dye is excited to generate fluorescence. The generated fluorescence is condensed by the objective lens 37, travels in the opposite optical path as the excitation light, and is reflected by the dichroic mirror 41 through the imaging lens 35, the mirror 33, the pupil projection lens 31, and the scanner unit 20. .

  The fluorescence reflected by the dichroic mirror 41 enters the wavelength separation filter 43, and light having a specific wavelength is selectively transmitted. The light transmitted through the wavelength separation filter 43 is condensed by the condenser lens 45 and enters the confocal pinhole 47, and only the light from the light condensing surface F of the specimen A is selectively passed through to the photodetector. 49.

  When light is detected by the photodetector 49, the output signal is converted into a digital signal synchronized with the scanning control of the scanner unit 20 by an A / D interface (not shown), and taken into the computer 50. The digital signal captured by the computer 50 is displayed on a display (not shown) corresponding to the scanning position of the scanner unit 20 by the image construction unit 53. Thereby, a fluorescence image (two-dimensional distribution of fluorescence luminance) on the light condensing surface F of the specimen A is acquired.

  For example, it is possible to observe the temporal change in the fluorescence intensity of the observation region of the specimen A by programming the control unit 51 to repeatedly acquire the fluorescence image of the same observation region. In this case, when there is no change in the membrane potential of the cells present in the observation region, the fluorescence intensity does not change with time. On the other hand, when synaptic excitatory activity caused by irradiation of stimulation light is propagated to cells in the observation area and the membrane potential of the cells rises, the increase in membrane potential is also reflected in the change in fluorescence intensity over time. Observed as a rising change.

Next, a method for observing changes in the membrane potential of the specimen A due to stimulation using the cell observation method and the scanning laser microscope 1 according to the present embodiment will be described.
First, in the reference image acquisition step SA1, a reference image of the specimen A as shown in FIG. 3 is acquired by the observation optical system 5 (pre-scan). Here, the reference image will be described as an XY plane image. The symbol C indicates a cell.

  Subsequently, in the area designating step SA2, the area designating unit 55 designates two ROIs of the stimulation area and the observation area for the reference image. These two ROIs can be designated as arbitrary three-dimensional figures in the XYZ three-dimensional space by the scanning of the scanner unit 20 and the operation of the semi-focus unit 39. The higher the ROI dimension and the larger its volume (volume, area, length), the longer it takes to scan the ROI uniformly with laser light. Conversely, the lower the ROI dimension is, and the smaller the volume is, the shorter the time required to uniformly scan the ROI with laser light.

  Therefore, the ROI has the lowest possible dimension within a necessary and sufficient range, and the volume of the ROI can be reduced to maximize the time resolution of stimulation and observation. In the present embodiment, for example, as shown in FIG. 4, a point-like stimulation region P and a line-like observation region L are designated on the reference image.

  The point-like stimulation region P can be applied, for example, when stimulating a single spine of a dendrite of a nerve cell. In addition, by specifying the line-like observation region L along, for example, the axon of a nerve cell, it is possible to observe how the action potential of the cell propagates through the axon.

  Next, in the stimulation process SA3, the stimulation optical system 3 irradiates the stimulation region P with stimulation light at a predetermined timing (point scan). In the observation image acquisition step SA4, the observation optical system 5 repeatedly executes scanning (line scan) in the observation region L after irradiation of the stimulation light to the stimulation region P, and the observation image in the observation region L is set at a predetermined time interval. Open multiple times at a predetermined acquisition timing.

  For example, if the time required for the line scan of one line in the observation region L is 1 ms, as shown in FIG. 5, the first line scan (L-1) is performed 1 ms after the stimulation light irradiation, and the stimulation light irradiation is performed. 2 ms later, the second line scan (L-2) is performed. Thereafter, the same operation is repeated at an acquisition timing of 1 ms interval.

  Note that the line scan of the observation region L may be performed before the stimulation light is irradiated, but the irradiation of the excitation light by the line scan causes the fluorescent discoloration of the membrane potential sensitive dye. It is desirable not to scan, that is, not to irradiate excitation light.

  The light intensity of the fluorescence detected at each acquisition timing is taken into the computer 50 from the photodetector 49 in synchronization with the scanning control of the line scan, and each of the lines from the start point to the end point of the line in the observation region L by the image construction unit 53. It is displayed on the display as a plot of fluorescence intensity versus scanning position.

  For example, assuming that an action potential, that is, a membrane potential rises at the start point of the observation region L 1 ms after irradiation of the stimulation light, and moves and propagates toward the end point of the observation region L over 6 ms, each acquisition timing A plot of the fluorescence intensity observed in FIG. 6 is as shown by Line 1 to Line 6 in FIG.

  Next, a temporal change in light intensity at each point in the observation region L is calculated in a temporal change calculation step SA5. When an observer calculates | requires the time-dependent change of the membrane potential in each position of the observation area | region L, the plot showing it is a plot of the fluorescence intensity with respect to a time axis. Since FIG. 6 is a plot of the fluorescence intensity with respect to the observation position, a method for converting this into a plot of the fluorescence intensity with respect to the time axis will be described with reference to FIGS.

  For example, as shown in FIG. 7, assuming that the observation positions on the scanning path for observing the change in membrane potential with time are point A, point B, point C, and point D, the position characteristic calculator 57 activates each observation position. Position characteristics of a plurality of light intensities indicating the relationship between the points A, B, C, D, and the light intensity are calculated. At this time, the calculated position characteristic of the light intensity may not be displayed on the display, or the calculated position characteristic of the light intensity may be displayed on the display as shown in FIG. FIG. 8 illustrates the positions of points A to D on the horizontal axis of the plot.

  Next, the temporal change of the light intensity at each observation position A point, B point, C point, and D point is calculated from the position characteristic of the light intensity calculated by the position characteristic calculation unit 57 by the operation of the time characteristic calculation unit 59. The For example, when the time-dependent change in the light intensity at point A is calculated, the fluorescence intensities of Line 1 to Line 6 at point A in FIG. 8 are plotted with time (ms) on the horizontal axis and their fluorescence intensity on the vertical axis. Can be redone.

  Strictly speaking, since time has elapsed even while the excitation light is scanned from the start point to the end point of the observation region L, the time characteristic calculator 59 determines the scan point of the excitation light from the start of the first line scan. The time to reach point A is calculated, and the time at which fluorescence is detected at point A is faithfully taken on the time axis.

  Similarly, for the second line scan to the sixth line scan, the time when the fluorescence detection at the point A is faithfully calculated is calculated, and the fluorescence intensity at each time is plotted. As a result, a plot showing the change with time of the membrane potential as shown in FIG. 9 is obtained. By displaying this plot on the display, the observer can know the change over time of the membrane potential at point A.

  In the present embodiment, the process of plotting the temporal change of the membrane potential at only four points A to D has been described. However, if this process is performed at all positions in the observation region L, all of the observation regions L will be displayed. A time-dependent change plot of the membrane potential at the pixel point can be obtained.

  As described above, according to the cell observation method and the scanning laser microscope 1 according to the present embodiment, the observation image acquisition step SA4 is performed by designating the stimulation region P and the observation region L that are smaller than the visual field range of the reference image. It is possible to shorten the time required for scanning and to observe the observation region L in a three-dimensional manner. In addition, by observing the observation image of the observation region L using caged glutamic acid and a membrane potential sensitive dye, it is possible to indirectly observe the temporal change in the membrane potential of the cells C in the stimulated stimulation region P. Thereby, the time change of the membrane potential response to the stimulus can be easily observed with high spatial freedom and time resolution.

  In the present embodiment, the observation image acquisition process is started after the stimulation process. Instead, for example, the stimulation process may be performed after the observation image acquisition process is started first, or the stimulation process. It is good also as starting an observation image acquisition process simultaneously.

  In the present embodiment, a three-dimensional fluorescence image is acquired in the reference image acquisition step SA1, an arbitrary region in the three-dimensional space is specified as a stimulation region and an observation region in the region specification step SA2, and the stimulation step SA3 and In the observation image acquisition step SA4, the three-dimensional stimulation region and the observation region are irradiated with stimulation light or excitation light. Instead, in the reference image acquisition step SA1, two-dimensional An arbitrary fluorescent image is acquired, an arbitrary region in the two-dimensional space is specified as a stimulus region and an observation region in the region specifying step SA2, and these two-dimensional stimulus region and observation region are specified in the stimulus step SA3 and the observation image acquisition step SA4. It is good also as irradiating stimulus light or irradiating excitation light.

  In the present embodiment, the stimulus region P and the observation region L are each designated one place on the reference image. Instead, for example, the stimulus region P and the observation region L are designated on the reference image. A plurality of locations may be designated, and each stimulation region P and observation region L may be scanned in time series. For example, one stimulation region P may be designated on the reference image, and a plurality of observation regions P may be designated. Alternatively, a plurality of stimulation regions P may be designated on the reference image, and a plurality of observation regions P may be designated. It may be specified.

Further, the present embodiment can be modified as follows.
For example, as a first modification, when reproducibility is recognized repeatedly in the change of the membrane potential with respect to the stimulus of the specimen A, the average of the light intensity of the observation image acquired a plurality of times by the observation image acquisition step SA4 is calculated. It is good also as including the average calculation process to calculate.

  In this case, after the stimulation process SA3, the observation image acquisition process SA4 performs the observation pattern for acquiring the observation image a plurality of times at a predetermined acquisition timing a plurality of times at the same timing, and is acquired by the observation image acquisition process SA4 by the average calculation process. What is necessary is just to calculate the average by adding the light intensity of several observation images for every acquisition timing of each observation pattern.

  Specifically, as shown in FIG. 10 (a), after a stimulus to the stimulation region P, after a predetermined time interval, a series of line scans are acquired a plurality of times at a certain timing in the observation region L. The operation is set as one trial, and after the completion of one trial, the next trial is repeatedly performed after a time interval that allows the cells to be activated again.

  Then, in the average calculation step, the fluorescence intensities of the first line scan (L1-1, L2-1, L3-1,...) Of each trial are added and averaged, and as shown in FIG. The fluorescence intensity distribution AveL-1 of the first line scan is calculated. Similarly, the fluorescence intensity of the second line scan (L1-2, L2-2, L3-2,...) Of each trial is also added and averaged, and the fluorescence intensity distribution AveL-2 of the second line scan after stimulation is obtained. calculate. In the same manner, the time course plot of the membrane potential may be replaced with the intensity obtained by averaging each trial.

  By doing so, it is possible to reduce the noise component of the light intensity in the observation image for each passage of time by the average calculation step, and to obtain a highly accurate change plot of the membrane potential over time. Thereby, the time-sequential change of the membrane potential response of the cell with respect to a stimulus can be observed with high S / N.

  Further, as a second modification, for example, an observation pattern in which the observation image acquisition step SA4 acquires the observation image a plurality of times at a predetermined acquisition timing after the stimulation step SA3 is acquired, and the first observation image is acquired after stimulating the cells. The timing up to this may be shifted several times for each observation pattern at a time interval shorter than the time required for one scan.

Specifically, as shown in FIG. 11, the observation images are acquired while shifting the timing from the stimulation to the start of the line scan little by little in steps smaller than the time taken for each line scan. What is necessary is just to replace what put the result in order of the timing from a stimulus to the start of a line scan as a time series change of fluorescence intensity.
By doing so, it is possible to complement and detect a change in membrane potential that is faster than the time required for one scan, and it is possible to observe the membrane potential response of a cell to a stimulus with higher temporal resolution.

  In the present embodiment, the point-like stimulation region P and the line-like observation region L are designated in the region designation step SA2. For example, as a third modification example, shown in FIG. 12 and FIG. As described above, in the area designating step SA2, the area designating unit 55 may designate the stimulation area M shaped like a cell body of a certain cell C and the observation area S having a square shape (clip scan area). FIG. 13 is an enlarged image of the observation region S of FIG.

  FIG. 14A shows a state in which an increase in membrane potential has propagated to the dendrite in the observation region S 12 ms after the stimulation to the stimulation region M in the observation region S shown in FIG. As shown in FIGS. 14 (b) to 14 (f), the rising portion of the membrane potential that has propagated to the observation region S 12 ms after stimulation propagates through the cell membrane of the cells C in the observation region S over 22 ms from the stimulation. Go. In this modification, the time required for one image acquisition of the observation region S is 2 ms.

  FIG. 15A shows a state in which four observation positions A to D are designated in the observation region S, and FIG. 15B is a diagram showing changes in membrane potential with time at each observation position. For example, the time-dependent change in membrane potential at point A shown in FIG. 15 (a) indicates that the excitation light is A in each fluorescent image from 12ms to 22ms after stimulation as shown in FIGS. 14 (a) to (f). The time during which the points are scanned is calculated by the time characteristic calculation unit 59, and the luminance is re-plotted on the time axis with the time when the stimulation light is applied as the time origin, as shown in FIG. Can be reproduced. The same applies to points B to D. Here, the time taken to acquire the fluorescence image of the observation region is 2 ms.

  In addition, in this modification, when the state of propagation of the membrane potential with respect to the stimulus has reproducibility, an average of a plurality of trials may be taken. By doing so, it is possible to improve the accuracy of the obtained membrane potential change with time. In addition, the fluorescence image is acquired while gradually shifting the timing from the start of stimulation to the start of acquisition of the fluorescence image in smaller time steps than the time required for one scan, and the computer 50 rearranges the fluorescence images in time order. Also good. In this way, it is possible to measure the temporal change in membrane potential with a time resolution finer than the time required for one scan.

  As a fourth modification, for example, when the user designates a certain position in the fluorescence image on the display displaying the fluorescence image, a time-dependent plot of the membrane potential at the designated position is displayed on the display. Alternatively, the program may be programmed in the control unit 51 of the computer 50. In this way, the user can more intuitively recognize each position of the sample A and the state of the membrane potential change with time at each position.

[Second Embodiment]
Next, a cell observation method and a scanning microscope apparatus according to the second embodiment of the present invention will be described.
The cell observation method according to the present embodiment is different from the first embodiment in that the laser irradiation to the stimulation area by the stimulation process SA3 and the laser irradiation to the observation area by the observation image acquisition process SA4 are performed by different optical systems.
In the following, portions having the same configuration as those of the cell observation method and the scanning laser microscope 1 according to the first embodiment are denoted by the same reference numerals and description thereof is omitted.

  First, as shown in FIG. 16, a scanning laser microscope (scanning microscope apparatus) 100 according to the present embodiment includes a scanner unit 120 that scans stimulation light separately from the scanner unit 20 that scans excitation light. The pupil projection lens 131 that forms the stimulus light scanned by the scanner unit 120 as an intermediate image, and the dichroic mirror 133 that combines the optical path of the light from the intermediate image formed by the pupil projection lens 131 and the optical path of the excitation light. And.

  In the cell observation method according to the present embodiment, the stimulation light emitted from the UV laser 11 is controlled to be blocked / transmitted by the acoustooptic device 13 under the control of the control unit 51, and then the sample A is scanned by the scanner unit 120. Are scanned in a two-dimensional direction of XY on the light condensing surface F. Then, the stimulation light deflected by the scanner unit 120 is condensed so as to form an intermediate image by the pupil projection lens 131, synthesized with the optical path of the excitation light by the dichroic mirror 133, and then collimated by the imaging lens 35. It is returned and condensed by the objective lens 37 so as to focus on the light condensing surface F of the specimen A. Thereafter, as in the first embodiment, the fluorescence generated in the specimen A is detected by the detector 49, processed by the computer 50, and observed as a change in membrane potential over time.

  As described above, according to the cell observation method and the scanning laser microscope 100 according to the present embodiment, the stimulation light and the excitation light are scanned by the independent scanner units 20 and 120 to the stimulation region. The laser irradiation and the laser irradiation to the observation region can be controlled independently in terms of time and space. Therefore, in the first embodiment, after stimulating the cells, it is not possible to irradiate the observation region with the excitation light until after the operation of deflecting to the observation region of the scanner unit 20 is completed. Then, the scanner units 20 and 120 can irradiate excitation light to the observation area at the same time while irradiating stimulation light to the stimulation area, for example. As a result, temporal and spatial degrees of freedom of cell stimulation and cell observation can be increased.

[Third Embodiment]
Next, a cell observation method and a scanning microscope apparatus according to the third embodiment of the present invention will be described.
As shown in FIG. 17, the cell observation method according to this embodiment is the same as that of the first embodiment in that cell observation is performed using a scanning laser microscope (scanning microscope apparatus) 200 capable of multiphoton excitation observation. Different from the second embodiment.
Hereinafter, the same reference numerals are given to the same parts as those in the cell observation method and the scanning laser microscope 1, 100 according to the first embodiment or the second embodiment, and the description thereof is omitted.

First, reagents used in the cell observation method according to this embodiment will be described.
In the present embodiment, for example, caged glutamic acid is used as a physiologically active substance, and fluorescent dye FM4-64 is used as a membrane potential sensitive dye. When the fluorescent dye FM4-64 (hereinafter simply referred to as “fluorescent dye”) is placed in an environment where the photon density is very high, such as when an infrared ultrashort pulse laser is focused by a lens, the nonlinear optical effect causes the fluorescent dye FM4-64. It is known to emit a second harmonic generation (hereinafter referred to as “SHG”) signal light having a wavelength half that of the incident light.

  The light intensity of the SHG signal light emitted by this fluorescent dye (fluorescent dye FM4-64) varies depending on the membrane potential of the cell membrane. For example, when the membrane potential of the cell membrane changes by 100 mV, the light intensity of the SHG signal light changes by about 10%. The light intensity of the fluorescence emitted by a general membrane potential sensitive dye is a very large change rate of light intensity compared to 0.1% to 1% when the cell membrane potential changes by 100 mV. .

  In the cell observation method according to the present embodiment, the change in the light intensity due to the membrane potential sensitivity of the SHG signal light is used as an optical measurement method for the membrane potential, thereby measuring the signal intensity several times to several tens times the conventional intensity. It becomes possible. Therefore, by generating SHG signal light for specimen B into which caged glutamic acid and a fluorescent dye have been introduced using an ultrashort pulse laser light source, the time-dependent change in cell membrane potential is indirectly reflected as the time change of SHG signal light. Can be observed.

Next, the scanning laser microscope 200 according to the present embodiment will be described.
The scanning laser microscope 200 includes an infrared pulse laser light source 215 instead of the argon laser 15. The infrared pulse laser light source 215 is a femtosecond pulse laser light source having a wavelength of 950 nm. A photodetector 49 is arranged on the transmission side of the specimen B, and between the specimen B and the photodetector 49, condensing lenses 242 and 245 for condensing SHG signal light generated in the specimen B, and an object And a wavelength selection filter 243 that selectively transmits only the wavelength of the SHG signal light.

Hereinafter, the operation of the thus configured cell observation method and the scanning laser microscope 200 will be described.
In the stimulation step SA2, the stimulation light emitted from the UV laser 11 is applied to the stimulation area on the light collection surface F of the sample B at a predetermined timing. On the other hand, in the observation image acquisition step SA4, the excitation infrared pulse laser beam emitted from the infrared pulse laser light source 215 is scanned in the XY two-dimensional direction at the point focused on the light collection surface F of the sample B. Is done.

  Here, since the infrared pulse laser focused on the focusing surface F of the sample B has a very high photon density in terms of time and space, a nonlinear optical effect is exerted on the fluorescent dye introduced into the sample B. Bring. Thereby, SHG signal light S having a wavelength of 475 nm is generated from the fluorescent dye in the same direction as the direction in which the infrared pulse laser travels. The part where the SHG light is generated on the specimen B is on the light condensing surface F of the objective lens 37 where the spatial density of the infrared pulsed laser light is very high.

  The SHG signal light is collected by the condenser lens 242, light having a wavelength other than the SHG light is blocked by the wavelength separation filter 243, and is collected by the condenser lens 245 on the detection portion of the detector 49. Thereby, the detector 49 can detect only the SHG light signal generated on the light condensing surface F of the specimen B. Therefore, according to the cell observation method and the scanning laser microscope 200 according to the present embodiment, pseudo confocal observation is performed even if there is no confocal pinhole in front of the detector 49.

  When the SHG signal light is detected by the photodetector 49, the output signal is converted into a digital signal synchronized with the scanning control of the scanner unit 20 by the A / D interface, and is taken into the computer 50. The digital signal captured by the computer 50 is displayed on the display by the image construction unit 53 corresponding to the scanning position of the scanner unit 20. Thereby, the SHG light image (two-dimensional distribution of SHG light luminance) in the condensing surface F of the sample B is acquired.

  Since the SHG light image acquired in this way changes the SHG light intensity depending on the membrane potential of the cells existing on the light collection surface F of the sample B, the SHG light is stimulated after the sample B is stimulated by the UV laser 11. If the control unit 51 is programmed to repeatedly acquire an optical image, the temporal change in the light intensity at a certain position in the acquired SHG optical image reflects the temporal change in the membrane potential at that position in the specimen B. It can be replaced and observed.

  As described above, according to the cell observation method and the scanning laser microscope 200 according to the present embodiment, by using a SHG optical signal having a large light intensity change with respect to a membrane potential change as a membrane potential-dependent light intensity change medium, The change in membrane potential over time can be observed at high S / N.

  As mentioned above, although embodiment of this invention was explained in full detail with reference to drawings, the specific structure is not restricted to this embodiment, The design change etc. of the range which does not deviate from the summary of this invention are included. For example, the present invention is not limited to those applied to the above-described embodiments and modifications, and may be applied to embodiments in which these embodiments and modifications are appropriately combined, and is particularly limited. is not.

  Further, for example, in each of the above-described embodiments and modifications, caged glutamic acid has been exemplified and described as a physiologically active substance that activates cells and induces membrane potential activity when receiving light, but instead, for example, Alternatively, a sample into which a protein having photosensitivity such as channelrhodopsin 2 is introduced may be used. When channel rhodopsin 2 is expressed in nerve cells by gene transfer and irradiated with light having a wavelength of 400 nm to 500 nm, channel rhodopsin 2 becomes permeable to cations and depolarizes nerve cells (excitatory membrane potential). Rise).

  Accordingly, when a laser light source that emits a stimulation laser beam having a wavelength of 400 nm to 500 nm is used as a light source and a living cell specimen in which channel rhodopsin 2 is uniformly expressed is irradiated with a stimulation laser beam of 400 nm to 500 nm, the stimulation laser beam It is possible to apply a stimulus to the cell that brings about an increase in membrane potential only at the position irradiated with.

Further, in each of the above-described embodiments and modifications, instead of caged glutamic acid in which glutamic acid is linked to caged compound, for example, amino acids that induce physiological activity, cyclic nucleotides (cNMPs), calcium, or the like are linked to caged compound. A caged reagent may be employed.
In addition to RH-795, many other various dyes having different molecular structures, excitation wavelengths, fluorescence wavelengths, or light intensity changes have been developed as membrane potential sensitive dyes. As long as excitation and observation of the present invention are possible, an appropriate dye may be employed depending on the purpose of observation and the specimen.

DESCRIPTION OF SYMBOLS 1,100 Scanning microscope apparatus 3 Stimulation optical system 5 Observation optical system 55 Area | region designation | designated part 57 Position characteristic calculation part 59 Time characteristic calculation part 200 Scanning laser microscope (scanning microscope apparatus)
SA1 Reference image acquisition step SA2 region designation step SA3 stimulation step SA4 observation image acquisition step SA5 time change calculation step C cell L, S observation region P, M stimulation region

Claims (10)

A reference image acquisition step of acquiring a reference image of a neuron cell into which a channel rhodopsin 2 gene and a membrane potential sensitive dye that changes in light intensity generated according to the magnitude of the membrane potential of the cell are introduced;
From the reference image acquired in the reference image acquisition step, a stimulation region that stimulates the nerve cells by laser irradiation and an observation region that excites the membrane potential-sensitive dye by laser irradiation from the visual field range of the reference image An area designating process for designating a small range;
The stimulation region designated in the region designation step is irradiated with a laser beam having a wavelength of 400 nm to 500 nm, and the nerve is produced by channel rhodopsin 2, which is a protein having photosensitivity expressed in the nerve cell from the channel rhodopsin 2 gene. A stimulation process that stimulates cells to induce membrane potential activity;
The observation region is irradiated with a laser beam, the condensing position is scanned to excite the membrane potential sensitive dye, the light intensity of the signal light emitted from the membrane potential sensitive dye is detected, and an observation image of the observation region A cell observation method including an observation image acquisition step of acquiring the image.   The cell observation method according to claim 1, wherein the region designating step designates the observation region in a line shape.   The cell observation method according to claim 1, wherein the region specifying step specifies the point-shaped observation region.   The cell observation method according to any one of claims 1 to 3, wherein the laser irradiation to the stimulation region by the stimulation step and the laser irradiation to the observation region by the observation image acquisition step are performed by different optical systems.   The cell observation method according to claim 1, wherein the observation image acquisition step acquires an observation image of the same observation region a plurality of times. The time during which the cells can re-activate the observation pattern in which the observation image acquisition step repeatedly acquires the observation image a plurality of times at a predetermined acquisition timing after a predetermined time interval after stimulation in the stimulation step and the stimulation step. Repeat several times at intervals,
Average for calculating the average by adding the light intensity of the observation image acquired by the observation image acquisition step to the light intensity of the observation image having the same acquisition timing among the plurality of observation images in each observation pattern The cell observation method according to claim 5, comprising a calculation step.   An observation pattern in which the observation image acquisition step repeatedly acquires the observation image a plurality of times at a predetermined acquisition timing after a predetermined time interval after the stimulation in the stimulation step and after the stimulation in the stimulation step is first performed after stimulating the cells. The predetermined time interval until the observation image is acquired is shifted for each observation pattern by a time interval shorter than the time required for one scan, and a time interval sufficient to activate the cells again is provided, The cell observation method according to claim 5, wherein the cell observation method is performed a plurality of times, and a plurality of images acquired as a result are replaced as a time-series change in fluorescence intensity.   8. The method according to claim 5, further comprising a time change calculation step of calculating a time change of light intensity at each point in the observation region based on the plurality of observation images acquired by the observation image acquisition step. The cell observation method described.   The cell observation method according to any one of claims 1 to 8, wherein the membrane potential sensitive dye is a fluorescent dye FM4-64.   The cell observation method according to claim 9, wherein the signal light is second harmonic (SHG) signal light.

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