JPH09184807A - Method and apparatus for detecting weak light - Google Patents

Abstract

PROBLEM TO BE SOLVED: To provide a weak light detection method accurately measuring the state in a sample in a non-contact state. SOLUTION: After a photo-decomposable substance is introduced into a sample 900, the decomposition light outputted from a first light source 100 passes through a first pinhole 511 and a beam scanner 400 and subsequently reaches the almost diffraction limit in a visual field by an object lens 20 and the image of the first pinhole is stopped down to one point in the sample to irradiate the sample 900. When the photo-decomposable substance is present at one point in the sample 900, the stopped down decomposition light sufficient to perform photodecomposition irradiates the photo-decomposable substance to develop a decomposition produce. The intensity of the light generated from the sample accompanying the development of the decomposition product is detected to be stored along with the data of a scanning position. By detecting the intensities of fluorescence accompanying the decomposition product developed at the respective positions in the sample 900, the state in the sample is measured.

Description

Detailed Description of the Invention

[0001]

BACKGROUND OF THE INVENTION 1. Field of the Invention The present invention relates to a weak light detection technique used for measuring a substance state in a sample such as a cell.

[0002]

2. Description of the Related Art In measuring the state of a substance in a sample such as cells, a method of introducing a fluorescent substance into the sample, directly irradiating this fluorescent substance with excitation light to excite it, and observing fluorescence emitted from the fluorescent substance (Hereinafter, also referred to as Conventional Example 1) is frequently used.

A laser scanning confocal microscope is preferably used for such fluorescence observation. FIG. 5 is an explanatory diagram of fluorescence measurement using a laser scanning confocal microscope. FIG.
As shown in FIG.
0, the objective lens 920, and the beam splitter 930.
, Beam scanner 940, pinholes 951, 95
2 is an optical microscope having 2 and. Then, the pinhole 951 is irradiated with the excitation light output from the light source 910, the image of the pinhole 951 is narrowed down to near the diffraction limit in the field of view of the microscope by the objective lens 920, and the image of the pinhole 951 is obtained by the beam scanner 940. Freely scan within the field of view. This irradiation directly excites the fluorescent substance previously introduced into the sample 990, and the fluorescence emitted from the fluorescent substance is detected by the photodetector 9 through the pinhole 952.
60, and the processing unit 970 interlocks the detected light amount with the beam scanner 940 to perform mapping in the sample 990.

Also, instead of measuring the generated light such as fluorescence, a method of measuring the electrical properties (resistance, current, voltage) generated on the surface of the sample (cell etc.) (hereinafter also referred to as conventional example 2: W. Denk, Proc. Natl. Acad. Sci. USA Vol.91, p
p6629-6633, July 1994) has been proposed.

FIG. 6 is an explanatory diagram of this method. As shown in FIG. 6, in this method, laser light focused to the diffraction limit is irradiated to the sample 990 in the same manner as in FIG. 5, and this is scanned by the beam scanner 940 and introduced into the sample 990 in advance. A caged substance (usually a substance that does not have a chemical activity, but that chemically changes (decages) by a photochemical reaction by absorbing light of a specific wavelength and has a specific activity) To cancel. As a result of uncaged caged material, sample 9
The action occurs within 90. As a result, the electrical properties (resistance, current, voltage) generated on the surface of the sample are measured by the glass microelectrode 9.
Detect at 70. Then, the inside of the sample is mapped by linking the electrical signal with the beam scanner 950.

[0006]

Since the conventional method for measuring the state in the sample is constructed as described above, it has the following problems.

That is, in the method of Conventional Example 1, when a state change in the sample detected using a certain fluorescent substance is induced by light having a wavelength different from the wavelength of the excitation light of the fluorescent substance, detection is performed. Was difficult.

The method of Conventional Example 2 requires skill because glass microelectrodes are used. Further, since it is a contact measurement, there is a concern that the influence on the sample may be large, and the measurement in a preferable environment cannot always be performed.

The present invention has been made in view of the above, and it is an object of the present invention to provide a weak light detection method for accurately measuring the state in a sample by fluorescence measurement without contact.

[0010]

The weak light detection method according to claim 1 comprises: (a) a first step of introducing a photodegradable substance into a sample; and (b) a first step of outputting from a first light source. A second step of irradiating the first light limiting member having the first pinhole with the decomposed light of the first wavelength; and (c) an image of the first pinhole with respect to the decomposed light that has passed through the first pinhole. Is narrowed down to the diffraction limit within the field of view, the narrowed down image of the first pinhole is scanned in the sample, and the sample is irradiated with the excitation light of the second wavelength output from the second light source. Step, and (d) the photolytic substance is irradiated with release light,
A fourth step of detecting, for each scanning position, the intensity of the fluorescence of the third wavelength generated by the irradiation of the excitation light according to the change in the state of the sample due to the expression of the degradation product.

In the weak light detection method of the first aspect, the photodegradable substance is introduced into the sample to be measured prior to the detection of the weak light. Then, as the photodegradable substance, a substance whose decomposition product after photolysis interacts with a substance existing in the sample to change the state in the sample and, as a result, changes the fluorescence characteristic of the substance in the sample is selected. To do.

After introducing the photodegradable substance into the sample, the sample is placed at the measurement position. Then, the decomposed light is output from the first light source to irradiate the first light restricting member having the first pinhole. The decomposed light that has passed through the first pinhole has an image of the first pinhole that has reached the diffraction limit within the field of view narrowed down to one point in the sample. The position within the sample to be narrowed down is set by the beam scanner. At the same time that the sample is irradiated with the decomposed light, the sample is irradiated with the light having the second wavelength (excitation light) output from the second light source.
The irradiation position of the excitation light may be the entire sample,
It may be limited to the vicinity of the narrowed down position of the decomposed light.

When a photodegradable substance is present at one point in the sample where the decomposed light is narrowed down, the photodegradable substance is irradiated with sufficient decomposed light, and photodegradation is performed to develop a degradation product. Due to the expression of this degradation product, the state in the sample changes due to the interaction between the degradation product and the substance in the sample. As a result, the emission characteristic of the fluorescence having the third wavelength due to the irradiation of the excitation light in the sample changes, so that the emission situation of the fluorescence changes. The intensity of this fluorescence is detected and stored together with the narrowed down position of the decomposed light in the sample.

Next, the beam scanner is operated to sequentially scan the sampled position of the decomposed light in the sample. Then, the fluorescence intensity is detected at each scanning position and stored together with the information on each scanning position.

In this way, the third generated at each position in the sample
By detecting the intensity of light of the wavelength
Measure the condition in the sample.

It is possible to detect the generated light which has passed through the second light restricting member having the second pinhole located at the confocal position with respect to the first pinhole.

In this case, as in the case of using the confocal microscope, the generated light at the position in the sample where the release light is narrowed down can be detected with high S / N.

The weak light detection device according to claim 3 is (a) the first
A first light source that outputs decomposed light having a wavelength of
A second light source that outputs excitation light having a second wavelength;
(C) Inputting the decomposed light output from the first light source,
A first light restricting member for selectively passing the decomposed light input to the pinhole of (1) and (d) an output direction instruction,
Beam scanner for controlling the output direction of the decomposed light toward the sample through the pinhole of (4), and (e) the decomposed light through the beam scanner and the excitation light output from the second light source are input to the sample. (F) a beam splitter that outputs light in a direction toward and outputs light incident from the sample side in a direction different from the input direction of decomposed light and excitation light.
An objective lens that inputs the decomposed light through the beam splitter and narrows down the image of the first pinhole to a point in the sample near the diffraction limit in the field of view, and (g) irradiation of the decomposed light and irradiation of the excitation light. Which is the fluorescence generated by and which receives the fluorescence through the objective lens and the beam splitter in order and detects the light quantity of the fluorescence, and (h) notifies the beam scanner of the output direction and also detects the light. And a processing unit that collects light detection results output from the container and performs image reconstruction.

In the weak light detection device of the third aspect, the decomposed light output from the first light source causes the first pinhole to pass through the sample into which the photolytic substance and the fluorescent substance sensitive to the membrane potential are introduced. The image of the first pinhole carried by the first light limiting member, the beam scanner, the beam splitter, and the objective lens is narrowed down to the initial position in the sample to the diffraction limit in the field of view. .

Further, the excitation light output from the second light source is applied to a specific portion of the sample via the beam splitter.

When a photodegradable substance is present at one point in the sample in which the decomposed light is narrowed down, the photodegradable substance is irradiated with sufficient decomposed light and photodegraded to express a photodegraded product, for example, a cell membrane. The change of the electric potential of occurs. As a result, the fluorescence characteristics of the fluorescent substance change due to the irradiation of the excitation light, and the amount of generated fluorescence changes.

Of the fluorescence, the fluorescence that has sequentially passed through the objective lens and the beam splitter is input to the photodetector, and the light intensity is detected and notified to the processing unit. The processing unit collects the light amount detection result output from the photodetector and stores it together with the initial position information in the sample.

Next, the processing unit notifies the beam scanner of the output direction instruction, and sequentially scans the narrowing position of the release light in the sample. Then, at each scanning position, the fluorescence intensity is detected in the same manner as above, and stored together with the information on each scanning position.

Here, the second fluorescence is inputted through the objective lens and the beam splitter in sequence, and the second fluorescence is selectively passed through the second pinhole located at the confocal position with the first pinhole. It is possible to further include a light limiting member.

In this case, similarly to the case of using the confocal microscope, the generated light at the position in the sample where the release light is narrowed down can be detected with high S / N.

The weak light detection device according to claim 5 is (a) the first
A first light source that outputs decomposed light having a wavelength of
A second light source that outputs excitation light having a second wavelength;
(C) Inputting the decomposed light output from the first light source,
The first light restricting member that selectively passes the decomposed light input to the pinhole of (1), and (d) the decomposed light that has passed through the first light restricting member and the excitation light that is output from the second light source. And outputs the light in the direction toward the sample and outputs the incident light from the side of the sample in a direction different from the input direction of the decomposed light and the excitation light, and (e) the light passing through the beam splitter. A beam scanner for controlling the output direction of the decomposed light directed to the sample via the first pinhole according to the input and output direction instruction, and (f) the decomposed light via the beam scanner is input to the first pinhole. Objective lens that narrows down the image of 1 to one point in the sample within the field of view to near the diffraction limit, and (g) fluorescence generated by irradiation of the decomposition light and irradiation of the excitation light, the objective lens and the beam splitter being sequentially arranged. Through firefly (H) Notifying the output direction instruction to the photodetector for detecting the amount of fluorescence and the output direction instruction to the beam scanner, and collecting the photodetection results output from the photodetector to perform image reconstruction. And a section.

In the weak light detection device of the fifth aspect, the decomposed light output from the first light source causes the first pinhole to enter the sample into which the photolytic substance and the fluorescent substance sensitive to the membrane potential are introduced. The image of the first pinhole carried by the first light limiting member, the beam splitter, the beam scanner, the beam splitter, and the objective lens, which is carried to the diffraction limit in the field of view, is initially located in the sample. Is narrowed down to.

Further, the excitation light output from the second light source is applied to a specific site in the sample via the beam splitter and the beam scanner.

When a photodegradable substance is present at one point in the sample where the decomposed light is narrowed down, the photodegradable substance is irradiated with sufficient decomposed light, and photodegradation is performed to produce a photodegradation product, for example, a cell membrane. The change of the electric potential of occurs. As a result, the fluorescence characteristics of the fluorescent substance change due to the irradiation of the excitation light, and the amount of generated fluorescence changes.

Among the fluorescence, the objective lens, the beam scanner,
The fluorescence that has passed through the beam splitter and the beam splitter is input to the photodetector, and the light intensity is detected and notified to the processing unit. The processing unit collects the light amount detection result output from the photodetector,
Store with the initial position information in the sample.

Next, the processing section notifies the beam scanner of the output direction, and sequentially scans the narrowing position of the release light in the sample. Then, at each scanning position, the fluorescence intensity is detected in the same manner as above, and stored together with the information on each scanning position.

Here, the fluorescence is sequentially input through the objective lens, the beam scanner, and the beam splitter, and the first
It is possible to further include a second light restricting member that selectively allows the fluorescence that has entered the second pinhole located at a confocal position to the pinhole of FIG.

In this case, similarly to the case of using the confocal microscope, the generated light at the position in the sample where the release light is narrowed down can be detected with high S / N.

[0034]

BEST MODE FOR CARRYING OUT THE INVENTION Embodiments of a weak light detection method and a weak light detection device 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.

In the present embodiment, a change in the fluorescence characteristic due to the excitation light accompanying the change in the state in the sample due to the interaction between the decomposition product and the substance in the sample is detected. Note that the following description will be given by taking as an example the case of using caged glutamic acid as a photodegradable substance and measuring the change in membrane potential due to glutamic acid.

(First Embodiment) FIG. 1 is a block diagram of a weak light detection device of this embodiment. As shown in FIG. 1, this apparatus includes (a) an excimer laser (XeF) light source 100 that outputs decomposed light having a wavelength of 351 nm, (b) a mercury lamp light source 150 that outputs excitation light, and (c) an excimer laser. The decomposed light output from the light source 100 is input to the pinhole 5
A light restricting member 510 for selectively passing the decomposed light input to 11 and (d) an output direction instruction, and the pinhole 5
A beam scanner 400 that controls the output direction of the decomposed light toward the sample 900 via (11), and (e) the decomposed light through the beam scanner 400 is input and the decomposed light is output toward the sample 900, and the mercury lamp light source is used. A beam splitter 310 that inputs the excitation light output from 150 and outputs the excitation light in the direction toward the sample 900; and (f) a direction that inputs the decomposed light and the excitation light through the beam splitter 310 and proceeds toward the sample 900. A beam splitter 320 that outputs the incident light from the sample 900 side in a direction different from the input direction of the decomposed light and the excitation light,
(G) The decomposed light is input through the beam splitter 320, and the image of the pinhole 511 is displayed within the field of view of 1 of the sample 900.
An objective lens 200 that narrows the point to near the diffraction limit,
(H) The pinhole 6 is located at a confocal position with the pinhole 511.
11) and (i) as a result of irradiation with decomposed light, the fluorescence characteristics change according to the change in the membrane potential due to glutamic acid generated in the sample 900. Yes, the objective lens 200
And a photodetector 600 for detecting the amount of fluorescent light by inputting the generated light sequentially through the beam splitter 320, and (j)
The beam scanner 400 is provided with a processing unit 700 that notifies the output direction and collects the photodetection results output from the photodetector 600 to perform image reconstruction.

The beam splitter 310 has a wavelength of 400
Light having a wavelength of 500 to 550 nm is efficiently reflected.

The beam splitter 320 has a wavelength of 600.
Light having a wavelength of 600 nm or more is transmitted, and light having a wavelength of 600 nm or more is efficiently reflected.

The beam scanner 400 is constructed by combining two galvano mirrors that can be independently driven.

Further, when irradiating the sample 900 with the excitation light, the excitation light was coaxial with the decomposed light and was irradiated onto a specific portion of the sample.

In this embodiment, the apparatus of FIG. 1 is used to detect weak light as follows.

First, prior to the detection of weak light, a photodegradable substance is introduced into the sample 900 to be measured. For example, as the sample 900, a membrane potential sensitive dye (for example,
RH160) -acting Drosophila muscle cells are distributed and adjusted, and caged glutamate is introduced into these cells. Glutamic acid, which is a photodegradation product of caged glutamic acid, which is a photodegradable substance, acts on a glutamate receptor on the cell surface to change the membrane potential.
The fluorescence characteristic of the membrane potential sensitive dye changes according to the change of the membrane potential.

After the caged glutamic acid is introduced into the cells of the sample 900, the sample 900 is placed at the measuring position. Then, the decomposed light is output from the excimer laser light source 100 to irradiate the light limiting member 510 having the pinhole 511.
The decomposed light that has passed through the pinhole 511 is the beam scanner 400, the beam splitter 310, and the beam splitter 3.
The image of the pinhole 511 that has been carried is narrowed down to the initial position in the sample 900 through 20 and the objective lens 200 in order until the diffraction limit is reached in the field of view.

From the mercury lamp light source 150, wavelength = 50
The excitation light of 0 to 550 nm is output, and the excitation light is irradiated to a specific portion in the sample 900 via the pinhole 161 of the light limiting member 160, the beam splitter 310, and the like.

When caged glutamic acid is present at one point in the sample 900 where the decomposition light has been narrowed down, the caged glutamic acid is irradiated with sufficient decomposition light, and photodecomposition is performed to develop glutamic acid which is a photodecomposition product. With the expression of glutamate, glutamate acts on the glutamate receptor on the cell membrane surface, causing a change in cell membrane potential. As a result, the fluorescence characteristic of the membrane potential sensitive dye changes due to the irradiation of the excitation light, and the amount of generated fluorescence changes.

Of the fluorescence, the objective lens 200, the beam splitter 320, and the pinhole 6 of the light limiting member 610.
The fluorescence sequentially passing through 11 is input to the photodetector 600, the light intensity is detected, and the processing unit 700 is notified.

The processing unit 700 collects the light amount detection result output from the photodetector 600 and stores it together with the initial position information in the sample 900.

Next, the processing section 700 causes the beam scanner 4
The output direction instruction is sent to 00, and the narrowing position of the release light is sequentially scanned in the sample 900. Then, at each scanning position, the fluorescence intensity is detected in the same manner as above, and stored together with the information on each scanning position.

By detecting the intensity of the fluorescence generated at each position in the sample 900, the membrane potential of glutamic acid in the sample 900 can be accurately measured in a non-contact manner.

(Second Embodiment) FIG. 2 is a block diagram of a weak light detection device according to this embodiment. As shown in FIG. 2, this apparatus includes (a) an excimer laser (XeF) light source 100 that outputs decomposed light having a wavelength of 351 nm, (b) a mercury lamp light source 150 that outputs excitation light, and (c) an excimer laser. The decomposed light output from the light source 100 is input to the pinhole 5
A light restricting member 510 for selectively passing the decomposed light input to 11 and (d) an output direction instruction, and the pinhole 5
A beam scanner 400 that controls the output direction of the decomposed light toward the sample 900 via (11), and (e) the decomposed light through the beam scanner 400 is input and the decomposed light is output toward the sample 900, and the mercury lamp light source is used. A beam that inputs the excitation light output from 150 and outputs the excitation light in the direction toward the sample 900, and outputs the incident light from the sample 900 side in a direction different from the input direction of the decomposed light and the excitation light. The splitter 310 and (f) the excitation light are input and output in the direction toward the sample 900, and the sample 900
Beam splitter 320 that outputs incident light from the side in a direction different from the input direction of excitation light, and (g) decomposed light that has passed through beam splitter 310 is input, and the image of pinhole 511 is sampled in the visual field. The objective lens 200 for narrowing down to one point in 900 close to the diffraction limit, (h) the light limiting member 610 having the pinhole 611 at the confocal position with the pinhole 511, and (i) the result of irradiation of the decomposition light, the sample 90
As a result of the change in the fluorescence characteristic according to the change in the membrane potential due to the glutamate generated in 0, the fluorescence generated by the irradiation of the excitation light is generated through the objective lens 200, the beam splitter 310, and the beam splitter 320 in sequence. Photodetector 600 that inputs light and detects the amount of fluorescent light
And (j) a processing unit 700 that notifies the beam scanner 400 of the output direction instruction, collects the photodetection results output from the photodetector 600, and performs image reconstruction.

Further, in the same way as in the first embodiment, the irradiation of the excitation light to the sample 900 was made to be coaxial with the decomposition light and irradiation to a specific portion in the sample.

In this embodiment, the apparatus of FIG. 2 is used to detect weak light as follows.

First, as in the first embodiment, a photodegradable substance is introduced into the sample 900 to be measured prior to the detection of weak light. For example, as the sample 900, Drosophila muscle cells that have been acted on by a membrane potential-sensitive dye (for example, RH160) are distributed and adjusted, and caged glutamate is introduced into these cells. Glutamic acid, which is a photodegradation product of caged glutamic acid, which is a photodegradable substance, acts on a glutamate receptor on the cell surface to change the membrane potential. The fluorescence characteristic of the membrane potential sensitive dye changes according to the change of the membrane potential.

After the caged glutamic acid is introduced into the cells of the sample 900, the sample 900 is placed at the measurement position. Then, the decomposed light is output from the excimer laser light source 100 to irradiate the light limiting member 510 having the pinhole 511.
The decomposed light that has passed through the pinhole 511 is the beam scanner 400, the beam splitter 310, and the objective lens 2.
00 sequentially, the image of the pinhole 511 carried thereby is narrowed down to the initial position in the sample 900 until the diffraction limit is reached in the visual field.

From the mercury lamp light source 150, wavelength = 50
Outputs 0 to 550 nm excitation light and outputs the beam splitter 3
20, a specific portion of the sample 900 is irradiated with excitation light through the pinhole 161, the beam splitter 310, and the objective lens 200 of the light limiting member 160.

When caged glutamic acid is present at one point in the sample 900 where the decomposition light is narrowed down, the caged glutamic acid is irradiated with sufficient decomposition light, and photodecomposition is carried out to express glutamic acid which is a photodecomposition product. With the expression of glutamate, glutamate acts on the glutamate receptor on the cell membrane surface, causing a change in cell membrane potential. As a result, the fluorescence characteristic of the membrane potential sensitive dye changes due to the irradiation of the excitation light, and the amount of generated fluorescence changes.

Of the fluorescence, the objective lens 200, the beam splitter 310, the pinhole 611 of the light limiting member 610,
The fluorescence that has sequentially passed through the beam splitter 320 and the beam splitter 320 is input to the photodetector 600, and the light intensity is detected.
Will be notified.

The processing section 700 collects the light amount detection result output from the photodetector 600 and stores it together with the initial position information in the sample 900.

Thereafter, similarly to the first embodiment, the processing section 700 notifies the beam scanner 400 of the output direction instruction, and sequentially scans the sample 900 with the stop position of the release light. Then, at each scanning position, the fluorescence intensity is detected in the same manner as above, and stored together with the information on each scanning position.

Thus, by detecting the intensity of the fluorescence generated at each position in the sample 900, the membrane potential of glutamic acid in the sample 900 can be accurately measured in a non-contact manner.

(Third Embodiment) FIG. 3 is a block diagram of a weak light detection device according to this embodiment. As shown in FIG. 3, this apparatus includes (a) an excimer laser (XeF) light source 100 that outputs decomposed light having a wavelength of 351 nm, (b) a mercury lamp light source 150 that outputs excitation light, and (c) an excimer laser. The decomposed light output from the light source 100 is input to the pinhole 5
The light restricting member 510 that selectively passes the decomposed light input to 11 and (d) the decomposed light that has passed through the light restricting member 510 are input and the decomposed light is output in the direction toward the sample 900. Sample 90 by inputting the output excitation light
Beam splitter 3 that outputs excitation light in the direction toward 0
10 and (e) the decomposed light and the excitation light via the beam splitter 310 are input and output in the direction toward the sample 900, and the incident light from the sample 900 side is different from the input direction of the decomposed light and the excitation light. Beam splitter 320 for outputting in the direction, and (f) a beam scanner 400 for controlling the output direction of the decomposed light toward the sample 900 via the beam splitter 310 according to the output direction instruction.
And (g) the decomposed light is input through the beam scanner 400, and the image of the pinhole 511 is displayed in the field of view of the sample 1
An objective lens 200 that narrows the point to near the diffraction limit,
(H) The pinhole 6 is located at a confocal position with the pinhole 511.
11) and (i) as a result of irradiation with decomposed light, the fluorescence characteristics change according to the change in the membrane potential due to glutamic acid generated in the sample 900. Yes, the objective lens 20
0, beam scanner 400, and beam splitter 3
The light detector 600 that sequentially inputs the generated light through 20 to detect the light amount of fluorescence, and (j) the beam scanner 400 are notified of the output direction, and the light detection result output from the light detector 600 is displayed. And a processing unit 700 that collects and reconstructs an image.

Further, in the same way as in the first embodiment, when the excitation light is applied to the sample 900, the excitation light is made coaxial with the decomposition light and is applied to a specific portion in the sample.

In this embodiment, the apparatus shown in FIG. 3 is used to detect weak light as follows.

First, as in the first embodiment, a photodegradable substance is introduced into the sample 900 to be measured prior to the detection of weak light. For example, as the sample 900, Drosophila muscle cells that have been acted on by a membrane potential-sensitive dye (for example, RH160) are distributed and adjusted, and caged glutamate is introduced into these cells. Glutamic acid, which is a photodegradation product of caged glutamic acid, which is a photodegradable substance, acts on a glutamate receptor on the cell surface to change the membrane potential. The fluorescence characteristic of the membrane potential sensitive dye changes according to the change of the membrane potential.

After the caged glutamic acid is introduced into the cells of the sample 900, the sample 900 is placed at the measurement position. Then, the decomposed light is output from the excimer laser light source 100 to irradiate the light limiting member 510 having the pinhole 511.
The decomposed light that has passed through the pinhole 511 is the beam splitter 310, the beam splitter 320, and the beam scanner 4.
00 and the objective lens 200 in this order, the image of the pinhole 511 carried thereby is narrowed down to the initial position in the sample 900 up to the diffraction limit in the field of view.

From the mercury lamp light source 150, wavelength = 50
The excitation light of 0 to 550 nm is output, and the excitation light is irradiated to a specific portion in the sample 900 via the pinhole 161 of the light limiting member 160, the beam splitter 310, and the like.

When caged glutamic acid is present at one point in the sample 900 where the decomposition light has been narrowed down, the caged glutamic acid is irradiated with sufficient decomposition light, and photodecomposition is performed to develop glutamic acid which is a photodecomposition product. With the expression of glutamate, glutamate acts on the glutamate receptor on the cell membrane surface, causing a change in cell membrane potential. As a result, the fluorescence characteristic of the membrane potential sensitive dye changes due to the irradiation of the excitation light, and the amount of generated fluorescence changes.

Of the fluorescent light, the fluorescent light that has passed through the objective lens 200, the beam scanner 400, the beam splitter 320, and the pinhole 611 of the light limiting member 610 in order is input to the photodetector 600, the light intensity is detected, and the processing unit is processed. 700 is notified.

The processing section 700 collects the light amount detection result output from the photodetector 600 and stores it together with the initial position information in the sample 900.

Thereafter, as in the first embodiment, the processing section 700 notifies the beam scanner 400 of the output direction instruction, and sequentially scans the sample 900 for the stop position of the release light. Then, at each scanning position, the fluorescence intensity is detected in the same manner as above, and stored together with the information on each scanning position.

Thus, by detecting the intensity of the fluorescence generated at each position in the sample 900, the membrane potential of glutamic acid in the sample 900 can be accurately measured in a non-contact manner.

(Fourth Embodiment) FIG. 4 is a block diagram of the weak light detection device of the present embodiment. As shown in FIG. 4, this apparatus includes (a) an excimer laser (XeF) light source 100 that outputs decomposed light having a wavelength of 351 nm, (b) a mercury lamp light source 150 that outputs excitation light, and (c) an excimer laser. The decomposed light output from the light source 100 is input to the pinhole 5
The light restricting member 510 that selectively passes the decomposed light input to 11 and (d) the decomposed light that has passed through the light restricting member 510 are input and the decomposed light is output in the direction toward the sample 900. Sample 90 by inputting the output excitation light
The excitation light is output in the direction toward 0 and the sample 90
Beam splitter 310 that outputs incident light from the 0 side in a direction different from the input direction of decomposed light and excitation light
And (f) a beam splitter 320 that inputs the excitation light and outputs it in a direction toward the sample 900, and outputs incident light from the sample 900 side in a direction different from the input direction of the excitation light, ) A beam scanner 400 that controls the output direction of the decomposed light toward the sample 900 through the pinhole 511 according to the output direction instruction, and (g) the decomposed light through the beam scanner 400 are input, and the pinhole 51 is input.
The objective lens 200 for narrowing the image of No. 1 to one point in the sample 900 within the field of view to near the diffraction limit, and (h) the pinhole 51.
1 and a light limiting member 610 having a pinhole 611 at a confocal position, and (i) as a result of irradiation with decomposed light, as a result of a change in fluorescence characteristics according to a change in membrane potential due to glutamic acid generated in the sample 900, Fluorescence generated by irradiation of excitation light, which is the objective lens 200 and the beam scanner 40.
0, the beam splitter 310, and the beam splitter 320 are sequentially input, and a photodetector 600 that detects the amount of fluorescence light and (j) the beam scanner 400 are notified of the output direction and the photodetector. A processing unit 700 that collects the light detection results output from 600 and performs image reconstruction.

Further, in the same manner as in the first embodiment, when the excitation light is irradiated to the sample 900, it is coaxial with the decomposed light and is irradiated to a specific portion in the sample.

In this embodiment, the apparatus shown in FIG. 4 is used to detect weak light as follows.

First, as in the first embodiment, a photodegradable substance is introduced into the sample 900 to be measured prior to the detection of weak light. For example, as the sample 900, Drosophila muscle cells that have been acted on by a membrane potential-sensitive dye (for example, RH160) are distributed and adjusted, and caged glutamate is introduced into these cells. Glutamic acid, which is a photodegradation product of caged glutamic acid, which is a photodegradable substance, acts on a glutamate receptor on the cell surface to change the membrane potential. The fluorescence characteristic of the membrane potential sensitive dye changes according to the change of the membrane potential.

After the caged glutamic acid is introduced into the cells of the sample 900, the sample 900 is placed at the measuring position. Then, the decomposed light is output from the excimer laser light source 100 to irradiate the light limiting member 510 having the pinhole 511.
The decomposed light that has passed through the pinhole 511 is the beam splitter 310, the beam scanner 400, and the objective lens 2.
00 sequentially, the image of the pinhole 511 carried thereby is narrowed down to the initial position in the sample 900 until the diffraction limit is reached in the visual field.

From the mercury lamp light source 150, wavelength = 50
Outputs 0 to 550 nm excitation light and outputs the beam splitter 3
20, a specific portion in the sample 900 is irradiated with the excitation light via the pinhole 161, the beam splitter 310, the beam scanner 400, and the objective lens 200 of the light limiting member 160.

When caged glutamic acid is present at one point in the sample 900 where the decomposition light has been narrowed down, the caged glutamic acid is irradiated with sufficient decomposition light, and photodecomposition is performed to express glutamic acid which is a photodecomposition product. With the expression of glutamate, glutamate acts on the glutamate receptor on the cell membrane surface, causing a change in cell membrane potential. As a result, the fluorescence characteristic of the membrane potential sensitive dye changes due to the irradiation of the excitation light, and the amount of generated fluorescence changes.

Of the fluorescent light, the objective lens 200, the beam scanner 400, the beam splitter 310, and the light limiting member 61.
0 pinhole 611 and beam splitter 320
The fluorescence that has sequentially passed through is input to the photodetector 600, the light intensity is detected, and the processing unit 700 is notified.

The processing section 700 collects the light amount detection result output from the photodetector 600 and stores it together with the initial position information in the sample 900.

Thereafter, similarly to the first embodiment, the processing section 700 notifies the beam scanner 400 of the output direction instruction, and sequentially scans the sample 900 for the stop position of the release light. Then, at each scanning position, the fluorescence intensity is detected in the same manner as above, and stored together with the information on each scanning position.

Thus, by detecting the intensity of the fluorescence generated at each position in the sample 900, the membrane potential of glutamic acid in the sample 900 can be accurately measured in a non-contact manner.

The present invention is not limited to the above embodiment, but can be modified. For example, instead of a membrane potential sensitive dye, a Ca ²⁺ sensitive dye (eg, Fluo
(3 etc.) is introduced into the cell and the change in intracellular Ca ²⁺ induced in response to the change in the membrane potential is also measured, and similarly, the change in the membrane potential can be accurately measured in a non-contact manner. it can.

[0084]

As described above in detail, according to the weak light detection method and the weak light detection device of the present invention, the release light is narrowed down to the diffraction limit and irradiated on the sample into which the photodegradable substance is introduced. While scanning the release light narrowing irradiation position in the sample, the change in the amount of fluorescent weak light due to the action reaction in the sample due to the expression of the decomposition product at each scanning position is measured, so that the sample can be accurately measured in a non-contact manner. The state of can be measured.

[Brief description of the drawings]

FIG. 1 is a configuration diagram of a first embodiment of a weak light detection device of the present invention.

FIG. 2 is a configuration diagram of a second embodiment of a weak light detection device of the present invention.

FIG. 3 is a configuration diagram of a weak light detection device according to a third embodiment of the present invention.

FIG. 4 is a configuration diagram of a weak light detection device according to a fourth embodiment of the present invention.

FIG. 5 is an explanatory diagram of a weak light detection method of Conventional Example 1.

FIG. 6 is an explanatory diagram of a weak light detection method of Conventional Example 2.

[Explanation of symbols]

100 ... Excimer laser light source, 150 ... Mercury lamp light source, 2
00 ... Objective lens, 310, 320 ... Beam splitter, 400 ... Beam scanner, 160, 510, 610
... Light restricting member, 161, 511, 611 ... Pinhole,
600 ... Photodetector, 700 ... Processing part, 900 ... Sample to be measured.

Claims (6)

[Claims] 1. A first step of introducing a photodegradable substance into a sample, and a first light restricting member having a first pinhole with the decomposed light of the first wavelength output from the first light source. A second step of irradiating, and narrowing down the image of the first pinhole with respect to the decomposed light that has passed through the first pinhole to a substantially diffraction limit within the field of view,
A third step of scanning the narrowed-down image of the first pinhole in the sample and irradiating the sample with excitation light of a second wavelength output from a second light source; A substance is irradiated with the decomposition light, and the intensity of fluorescence of a third wavelength generated by the irradiation of the excitation light according to the change in the state in the sample accompanying the expression of the decomposition product is detected for each scanning position. A weak light detection method, comprising: 2. In the fourth step, generated light passing through a second light limiting member having a second pinhole at a confocal position with the first pinhole is detected. The weak light detection method according to claim 1. 3. A first light source that outputs decomposed light having a first wavelength, a second light source that outputs excitation light having a second wavelength, and a decomposed light that is output from the first light source. And a first light restricting member for selectively passing the decomposed light input to the first pinhole, and an output of the decomposed light toward the sample through the first pinhole according to an output direction instruction. A beam scanner that controls the direction, and inputs the decomposed light that has passed through the beam scanner and the excitation light that is output from the second light source to output in a direction toward the sample, and from the sample side. A beam splitter that outputs the incident light in a direction different from the input direction of the decomposed light and the excitation light, and the decomposed light that has passed through the beam splitter, and the image of the first pinhole is viewed. Within the trial An objective lens that narrows down to a diffraction limit near one point, fluorescence that is generated by irradiation of the decomposition light and irradiation of the excitation light, and fluorescence that is sequentially passed through the objective lens and the beam splitter is input, A photodetector for detecting the light amount of the fluorescence, and notifying an output direction instruction to the beam scanner, and collecting the photodetection result output from the photodetector,
A weak light detection device, comprising: a processing unit that performs image reconstruction. 4. The fluorescence is sequentially inputted through the objective lens and the beam splitter, and the fluorescence inputted through a second pinhole located at a confocal position with the first pinhole is selectively passed therethrough. The weak light detection device according to claim 3, further comprising a second light restricting member. 5. A first light source that outputs decomposed light having a first wavelength, a second light source that outputs excitation light having a second wavelength, and a decomposed light that is output from the first light source. A first light restricting member that selectively transmits the decomposed light input to the first pinhole, and the decomposed light that has passed through the first light restricting member and the second light source. A beam splitter that inputs the generated excitation light and outputs it in a direction toward the sample, and outputs incident light from the sample side in a direction different from the input direction of the decomposed light and the excitation light. A beam scanner for inputting light through the beam splitter and controlling an output direction of the decomposed light toward a sample through a first pinhole in accordance with an output direction instruction; and the decomposition through the beam scanner. Enter the light, An objective lens that narrows down the image of the first pinhole to one point in the sample in the field of view to near the diffraction limit; fluorescence generated by irradiation of the decomposition light and irradiation of the excitation light, A photodetector for inputting fluorescence through the lens and the beam splitter in order to detect the light amount of the fluorescence, and an output direction instruction to the beam scanner, and a photodetection result output from the photodetector. Collect
A weak light detection device, comprising: a processing unit that performs image reconstruction. 6. The objective lens, the beam scanner,
And the second fluorescence at the position confocal with the first pinhole by inputting the fluorescence through the beam splitter.
The weak light detection device according to claim 5, further comprising a second light limiting member that selectively allows the fluorescence that has entered the pinhole to pass therethrough.