JPH10232352A - Laser scan microscope - Google Patents

Abstract

PROBLEM TO BE SOLVED: To eliminate positioning operation whenever observation is performed and periodical positioning operation, to obtain the same brightness image with excellent handleability by light receiving excitation laser light separated with a second light separation means with a multi-division photodetector, driving an adjustment means so that outputs of respective photodetectors become the same size and making coincide the outgoing direction of the excitation laser light with an optical axis. SOLUTION: This microscope is provided with the second light separation means 5 arranged on an optical path between a light source 2 and a first light separation means 9 and the multi-division photodetector 20 detecting the excitation laser light separated by this second light separation means 5. The excitation laser light whose light quantity is detected by the multi-division photodetector 20, and respective photodetectors of the multi-division photodetector 20 output signals equivalent to light receiving amounts. A control means 7 inputted with these signals drives the adjustment means 4 after it performs prescribed processing, and moves a reflection means 3 to make coincide the outgoing direction of the excitation laser light with the optical axis. Then, the control means 7 drives the adjustment means 4 so that the outputs of respective photodetectors become the same size.

Description

DETAILED DESCRIPTION OF THE INVENTION

[0001]

BACKGROUND OF THE INVENTION 1. Field of the Invention The present invention relates to a laser scanning microscope used for measuring instantaneous changes and transient phenomena of chemical reactions in cells.

[0002]

2. Description of the Related Art A laser scanning microscope, for example, a confocal laser scanning microscope, includes a light source that emits an excitation laser beam, a reflection mirror that reflects the excitation laser beam in a predetermined direction, a reflection of the excitation laser beam, and a transmission of fluorescence. A beam splitter, a scanning device for two-dimensionally scanning the excitation laser beam on the sample, an objective lens disposed between the scanning device and the sample, and a pinhole provided at a position conjugate with the focal plane of the objective lens. And an observation device for observing the fluorescence that has passed through the pinhole.

According to this confocal laser scanning microscope, only fluorescent light emitted from the focal plane passes through the pinhole, so that a clear image can be obtained with the observation device.

[0004]

However, in the confocal laser scanning microscope, since only the fluorescence that has passed through the pinhole is observed, if the optical axis of the excitation laser light emitted from the light source is shifted, an image obtained by the observation device is obtained. Is very dark and difficult to see.

By the way, it is known that the deviation of the optical axis changes with the change of the environmental temperature of the confocal laser scanning microscope and with time.

[0006] Therefore, every time observation is performed with a confocal laser scanning microscope or at regular intervals, a positioning operation with the optical axis of the excitation laser light is performed. This positioning operation is performed, for example, by adjusting the position of the pinhole to a position where an observer can obtain an image of optimal brightness while viewing the image with the observation device.

[0007] However, this adjustment largely depends on the sense of the observer, and there is a problem that an image of the same brightness cannot always be obtained quantitatively.

The present invention has been made in view of such circumstances, and its object is to eliminate the need for an operation of aligning the excitation laser beam with the optical axis each time observation or periodic inspection is performed. Another object of the present invention is to provide an easy-to-use laser scanning microscope capable of obtaining an image of the same brightness.

[0009]

According to a first aspect of the present invention, there is provided a laser scanning microscope which emits excitation laser light, reflects the excitation laser light, and transmits fluorescence. In a laser scanning microscope provided with a first light separating means, a second light separating means disposed in an optical path between the light source and the first light separating means, and the second light separating means A multi-divided light receiving element that detects the excitation laser light separated by the light source, a deflecting unit that is disposed in an optical path between the light source and the second light separation unit, and deflects the excitation laser light in a predetermined direction; Adjusting means for adjusting the deflection direction of the excitation laser light by the deflection means; and control means for controlling the deflection direction of the excitation laser light by driving the adjustment means based on the output of each of the multi-divided light receiving elements. This The features.

The excitation laser light separated by the second light separating means disposed on the optical path between the reflecting means and the first light separating means is detected by a multi-divided light receiving element for the amount of light. Output a signal corresponding to the amount of received light. After inputting this signal, the control means performs predetermined processing and then drives the adjusting means to move the reflecting means so that the emission direction of the excitation laser light coincides with the optical axis.

According to a second aspect of the present invention, in the laser scanning microscope according to the first aspect, the control means drives the adjusting means so that the outputs of the respective light receiving elements have the same magnitude. It is characterized by doing.

The control means drives the adjusting mechanism so that the outputs of the respective light receiving elements have the same magnitude, and moves the reflection mirror so that the emission direction of the excitation laser light is on the optical axis.

[0013]

Embodiments of the present invention will be described below with reference to the accompanying drawings.

FIG. 1 is a block diagram of a laser scanning microscope according to the present invention.

The confocal laser scanning microscope 1 includes a laser light source 2, a total reflection mirror (deflecting means) 3, a beam splitter (second light separating means) 5, and a multi-segment sensor (multi-segment light receiving element) 20. , Microcomputer (control means) 7, beam expander 8, beam splitter (first light separating means) 9, pupil projection lens 10, XY
A scanner 11, an objective lens 12, and a condenser lens 13
And a detection-side pinhole 14 and a fluorescence detector 15.

The beam expander 8 has lenses 8a and 8b.
And an illumination side pinhole 8c disposed between the lens 8a and the lens 8b.

The total reflection mirror 3 for reflecting the excitation laser light emitted from the laser light source 2 has, for example, electromagnetic coils 4a and 4b for driving two rotation axes intersecting at right angles to each other.
And an adjusting mechanism (adjusting means) 4 such as a gimbal mechanism.

The beam splitter 5 is disposed between the total reflection mirror 3 and the beam expander 8, and reflects only a few% of the excitation laser light.

The multi-segment sensor 20 includes four light receiving elements 21 to
24 (see FIG. 2), and receives the excitation laser light reflected by the beam splitter 5. Each light receiving element 2
1 to 24 output signals having magnitudes corresponding to the amounts of light received by the respective light receiving elements 21 to 24.

The microcomputer 7 controls the adjusting mechanism 4 in accordance with a predetermined program based on signals from the respective light receiving elements 21 to 24.

In this embodiment, a program for controlling the adjusting mechanism 4 based on the result obtained by adding the outputs of two light receiving elements of a combination designated in advance and then subtracting the outputs. That is, assuming that the outputs of the respective light receiving elements 21 to 24 are 21A to 24A, respectively, the adjustment mechanism 4 is based on the result of performing two types of calculations of (21A + 22A)-(23A + 24A) and (21A + 24A)-(22A + 23A). Is controlling.

The beam expander 8 expands the excitation laser light so as to fill the pupil plane of the objective lens 12.

The beam splitter 9 does not transmit the excitation laser light but transmits the fluorescence excited by the sample 16.

The XY scanner 11 scans the excitation laser light two-dimensionally (XY directions).

The objective lens 12 transmits the excitation laser beam to the sample 1
The light is focused on 6.

The pinhole 14 is disposed at a position conjugate with the focal plane of the objective lens 12 and allows only the fluorescence collected by the condenser lens 13 to pass.

The fluorescence detector 14 detects the fluorescence passing through the pinhole 14, converts the fluorescence into an electric signal, and forms an image of the sample 16 using a monitor (not shown).

The operation of the confocal laser scanning microscope having the above configuration will be described with reference to FIGS.

The excitation laser light emitted from the light source 2 and guided on the optical path by the total reflection mirror 3 passes through the beam splitter 5 and then enters the beam expander 8, where the objective lens 12 Is expanded to a size that can satisfy the pupil plane of

The excitation laser light is applied to the beam splitter 9.
XY through the pupil projection lens 10
The objective lens 1 is swung two-dimensionally by the scanner 11.
The sample 2 is irradiated onto the sample 16 through the sample 2. The fluorescent substance in the sample 16 emits fluorescence when excited.

The fluorescent light travels backward along the optical path from the objective lens 12 to the XY scanner 11 together with the excitation laser light, and is separated from the excitation laser light by the beam splitter 9 via the pupil projection lens 10.

The fluorescence that has passed through the beam splitter 9 is condensed by the condenser lens 13. The pinhole 14 allows only the fluorescence of a predetermined slice surface of the sample 14 to pass therethrough (confocal effect). Thereafter, the fluorescence is received by the fluorescence detector 15 and converted into an electric signal, which is displayed as an image on a monitor.

A part of the excitation laser light reflected by the beam splitter 5 enters the multi-segment sensor 20.

FIG. 2 is a diagram showing a light receiving state of the multi-segment sensor when the excitation laser beam coincides with the optical axis, and FIGS.
(F) is a diagram for explaining an example of a control method by the microcomputer when the excitation laser beam coincides with the optical axis,
FIG. 4 is a diagram showing an example of a light receiving state of the multi-segment sensor when the excitation laser light does not coincide with the optical axis, and FIGS.
(F) is a diagram illustrating an example of a control method by the microcomputer when the excitation laser light does not coincide with the optical axis.

When the excitation laser light is emitted in the correct direction, each light receiving element 2
1 to 24 receive the same amount of light as shown in FIG.
The outputs 21A to 24A of the respective light receiving elements have the same magnitude.

Therefore, (21A + 22A) (FIG. 3)
(See (a)), (23A + 24A) (see FIG. 3 (b)), (21A + 24A) (see FIG. 3 (d)) and (2
2A + 23A) (see FIG. 3 (e)) are equal, so (21A + 22A)-(23A + 24A) (FIG. 3
(C)) and (21A + 24A)-(22A + 23)
A) (see FIG. 3 (f)) is zero, the microcomputer 7 does not drive the adjusting mechanism 4 and the total reflection mirror 3
Do not move.

On the other hand, when the environment (for example, temperature) of the microscope changes, the emission direction of the excitation laser light changes, or the reflection direction of the excitation laser light changes due to the misalignment of the adjusting mechanism 7 of the reflection mirror. When light is no longer emitted in the correct direction, each of the light receiving elements 21 to 24 of the multi-segment sensor 7 receives light, for example, as shown in FIG.

At this time, each of the light receiving elements 21 to 24
Receive different light amounts, (21A + 22A) (FIG. 5)
(See (a)), (23A + 24A) (see FIG. 5 (b)), (21A + 24A) (see FIG. 5 (d)), and (2)
2A + 23A) (see FIG. 5 (e)).

Therefore, (21A + 22A)-(23
A + 24A) (see FIG. 5C) and (21A + 24).
A)-(22A + 23A) (see FIG. 5 (f)) do not become zero.

The microcomputer 7 calculates (21A + 2
2A)-(23A + 24A) and (21A + 24A)-
The adjusting mechanism 4 is driven so that all of (22A + 23A) become zero, and the total reflection mirror 3 is moved so that the excitation laser light is emitted in a correct direction.

According to this embodiment, the excitation laser light separated by the beam splitter 5 disposed in the optical path between the total reflection mirror 3 and the beam expander 8 is received by the multi-split sensor 20, and each of them is received. By driving the adjusting mechanism 4 to move the total reflection mirror 3 so that the outputs of the light receiving elements 21 to 24 have the same magnitude, the excitation laser light can be emitted in the correct direction.

Therefore, it is not necessary to perform an alignment operation with the optical axis of the excitation laser beam every time an observation or a periodic inspection is performed, and an image with the same brightness can always be obtained quantitatively. The usability of the laser scanning microscope is improved.

Since the beam splitter 5 is disposed on the optical path between the total reflection mirror 3 and the beam expander 8, it has the following advantages.

As a multi-split sensor, a small diameter (1 m
m) which can receive the excitation laser light (for example, the diameter of the excitation laser light is about 10 mm after the beam expander).

For example, even when a multi-type light source capable of emitting ultraviolet laser light and visible excitation laser light is used, it is necessary to use different beam expanders for each excitation laser light. Is fine.

In the above-described embodiment, the control unit performs the software processing using the microcomputer. However, the control unit may control the driving of the adjustment mechanism using a random logic circuit. In addition, the confocal laser scanning microscope of the present embodiment performs the fluorescence observation, but the present invention is not limited to this, and for example, may simply observe the reflected light of the sample. At this time, the beam splitter 9 shown in FIG. 1 is replaced with a half mirror. Further, the member that polarizes the excitation laser light is not limited to the total reflection mirror, and may be, for example, an acousto-optic deflector (AOD) or a plane-parallel plate.

[0047]

As described above, according to the laser scanning microscope of the first or second aspect of the present invention, the second light source disposed in the optical path between the polarizing means and the first light separating means. The excitation laser light separated by the light separating means is received by the multi-divided light receiving element, and the adjusting means is driven so that the output of each light receiving element has the same magnitude, and the emission direction of the excitation laser light through the deflecting means. Coincides with the optical axis, which eliminates the need for each observation and periodic alignment operation with the excitation laser light's optical axis.Also, images with the same brightness can always be obtained quantitatively. The scanning microscope can be made easy to use.

[Brief description of the drawings]

FIG. 1 is a block diagram of a confocal laser scanning microscope according to the present invention.

FIG. 2 is a diagram illustrating a light receiving state of a multi-segment sensor when an excitation laser beam coincides with an optical axis.

FIGS. 3A to 3F are diagrams illustrating an example of a control method by a microcomputer when an excitation laser beam coincides with an optical axis.

FIG. 4 is a diagram illustrating an example of a light receiving state of the multi-segment sensor when the excitation laser light does not coincide with the optical axis.

FIGS. 5A to 5F are diagrams illustrating an example of a control method by a microcomputer when the excitation laser light does not coincide with the optical axis.

[Explanation of symbols]

 Reference Signs List 1 confocal laser scanning microscope 2 laser light source 3 total reflection mirror (deflecting means) 4 adjusting mechanism (adjusting means) 5 beam splitter (second light separating means) 7 microcomputer (control means) 9 beam splitter (first light) Separation unit) 20 Multi-segment sensor (multi-segment light receiving element) 21 to 24 light receiving element

Claims (2)

[Claims] 1. A laser scanning microscope comprising: a light source that emits excitation laser light; and a first light separating unit that reflects the excitation laser light and transmits fluorescence. A second light separating unit disposed on an optical path between the light source and the second light separating unit; a multi-divided light receiving element for detecting an excitation laser beam separated by the second light separating unit; A deflecting unit disposed on an optical path between the light separating unit and deflecting the excitation laser light in a predetermined direction; an adjusting unit for adjusting a deflection direction of the excitation laser light by the deflecting unit; A laser scanning microscope comprising: a control unit that drives the adjustment unit based on each output to control a deflection direction of the excitation laser light. 2. The laser scanning microscope according to claim 1, wherein said control means drives said adjusting means so that outputs of said respective light receiving elements have the same magnitude.