JPH0440408A - Confocal scanning microscope - Google Patents

Abstract

PURPOSE:To enable a fast scanning, to simplify the constitution, and to form the microscope at low cost by moving a 1st pinhole plate reciprocally in a direction intersecting its optical axis and making a main scan of a light spot on a sample, and moving a 2nd pinhole plate integrally with the 1st pinhole plate and making them follow up the movement of the spot image. CONSTITUTION:The 1st pinhole plate 31 and 2nd pinhole plate 32 are united integrally through a holding member 33, which is coupled with a laminated piezoelectric element 34 for main scanning. The laminate piezoelectric element 34 for main scanning receives the driving electric power from a piezoelectric element driving circuit 36 and moves both pinhole plates 31 and 32 reciprocally at a high speed as shown by an arrow X. A laminate piezoelectric element 35 for subscanning, on the other hand, receives driving electric power from a piezoelectric element driving circuit 37 and moves both pinhole plates 31 and 32 reciprocally at a high speed as shown by an arrow Y. The position of the pinhole 32a of the 2nd pinhole plate 32 follows up the movement of the spot image Q and transmitted light 11' is made incident on a photodetector 24 through this pinhole 32a at all times. Consequently, the fast scanning is available and the microscope in simple structure can be formed at low cost.

Description

DETAILED DESCRIPTION OF THE INVENTION (Field of Industrial Application) The present invention relates to a confocal scanning microscope, and more particularly to a confocal scanning microscope with an improved illumination light spot scanning mechanism.

(Prior Art) Conventionally, illumination light is converged into a minute light spot, and this light spot is scanned two-dimensionally on a sample. At this time, the light that has passed through the sample or the light that has been reflected there, and also the sample Optical scanning microscopes are known in which the fluorescence emitted from the sample is detected by a photodetector to obtain an electrical signal carrying an enlarged image of the sample.

In particular, it is configured to generate illumination light from a light source and image it into a light spot on the sample, and then re-image the light flux from the sample into a point image, which is then detected by a photodetector. A confocal scanning microscope does not require a pinhole on the sample surface, making it easy to implement.

This confocal scanning microscope basically includes a light source that emits illumination light, a sample stage on which a sample is placed, a light transmission optical system that images this illumination light as a minute light spot on the sample, and the above-mentioned. A light-receiving optical system that collects the light flux (transmitted light, reflected light, or fluorescence) from the sample and forms it into a point image, and a photodetector that detects this point image; It is composed of a scanning mechanism that scans the image. Furthermore, Japanese Patent Application Publication No. 82-21
No. 7218 discloses an example of this confocal scanning microscope.

(Problems to be Solved by the Invention) In conventional confocal scanning microscopes, the above-mentioned scanning mechanism includes: ■ a mechanism that moves the sample stage two-dimensionally, or ■ a mechanism that moves the illumination light beam two-dimensionally using an optical deflector. A deflection mechanism was used.

However, when the mechanism (2) was adopted, there was a problem that the sample would fly away when high-speed scanning was performed. Many biological samples are observed with microscopes, and if high-speed scanning is not possible when observing these biological samples, it will be impossible to capture the subtle movements of the biological samples. In addition, there is a wide demand for capturing images of samples in near real time, not just for biological samples, and if high-speed scanning is not possible, it is natural that such a demand cannot be met. I can't.

On the other hand, the mechanism (■) allows sufficiently high-speed scanning, but in this mechanism, galvanometer mirrors and AOD
The disadvantage is that an expensive optical deflector such as an acousto-optic optical deflector is required. In addition, in this mechanism (2), since the illumination light beam is deflected by an optical deflector, this light beam enters the objective lens of the light transmission optical system at different angles from time to time, and the resulting aberrations are eliminated. It has also been recognized that the correction makes it difficult to design the objective lens. In particular, when an AOD is used, a special correction lens is required in addition to the objective lens because astigmatism occurs in the light beam emitted from the AOD, making the optical system more complicated.

SUMMARY OF THE INVENTION Therefore, an object of the present invention is to provide a confocal scanning microscope that is capable of high-speed scanning, yet has a simple configuration and can be manufactured at low cost.

(Means and Effects for Solving the Problems) A confocal scanning microscope according to the present invention includes a sample stage as described above, a light source, a light transmitting optical system, a light receiving optical system, a photodetector, A confocal scanning microscope equipped with a two-dimensional scanning mechanism for a light spot includes a first pinhole plate disposed between the light source and the light transmission optical system; A scanning mechanism is constituted by a pinhole plate moving means for main-scanning the light spot on the sample by reciprocating in a direction intersecting the axis, and a sub-scanning means for sub-scanning the light spot on the sample; In order to correspond to the movement of the point image due to main scanning of the light spot, a second pinhole plate is provided at the image formation position, and this second pinhole plate is moved integrally with the first pinhole plate. , is characterized in that it follows the movement of the point image.

Note that if the pinhole plate moving means is configured to also move the first and second pinhole plates in a direction substantially perpendicular to the direction of movement for main scanning, the pinhole plate movement Depending on the means, it can also serve as a sub-scanning means.

Further, this sub-scanning means may also be a means for moving the sample stage, for example. Since the sub-scanning speed can be relatively low compared to the main-scanning speed, even if the sample stage is moved as described above, it is possible to prevent the sample from flying away.

In the above configuration, since the illumination light beam is not swayed for light point scanning, the design of the optical system only needs to be done considering the light rays on the optical axis. It becomes very easy.

Furthermore, in the above configuration, what is moved for main scanning of the illumination light spot are two pinhole plates, and since such pinhole plates can generally be made extremely lightweight, these It becomes possible to move the pinhole plate and, in turn, to perform main scanning of the illumination light at extremely high speed.

(Example) Hereinafter, the present invention will be described in detail based on an example shown in the drawings.

FIG. 1 shows a monochrome transmission confocal scanning microscope according to a first embodiment of the present invention.

As shown in the figure, a light source 12 that emits illumination light 11 is fixed to the movable table IO. As this light source 12,
For example, a semiconductor laser or the like is used. Furthermore, the movable table 10 is held movably in the vertical direction relative to the pedestal 13, and a laminated piezo element 14 is interposed between the movable table 10 and the pedestal 13 to move the movable table 10 in the above-mentioned direction. ing.

The illumination light 11, which is diverging light, is converted into parallel light with a relatively large beam diameter by the collimator lens 15.

A first pinhole plate 31 is located at a position that blocks this illumination light 11.
is installed. The minute pinhole 31 of this first pinhole plate 31. The illumination light 11 that has passed through a is incident on the collimator lens 16 in a diverging state to become parallel light, and is then condensed by the objective lens 17 and sent to the sample stage 22.
The image is formed into a minute light spot P on the sample 23 placed on the surface.

The beam of transmitted light 11' that has passed through the sample 23 is made into parallel light by the objective lens 19 of the light receiving optical system 21, and then condensed by the condensing lens 20 to form a point image Q. A second pinhole plate 32 is disposed at this imaging position, and the amount of transmitted light of 11° passing through the pinhole 32a of the second pinhole plate 32 is detected by the photodetector 24. As this photodetector 24, for example, a photomultiplier tube or the like having a relatively large light-receiving surface is used, and from there,
A signal S indicating the brightness of the part of the sample 23 irradiated with the illumination light is output.

Next, two-dimensional scanning of the light spot P of the illumination light 11 will be explained. The first pinhole plate 31 and the second pinhole plate 32 described above are integrated via a holding member 33, and this holding member 33 is connected to the main scanning laminated piezo element 34. This main scanning laminated piezo element 34 is held on the movable table 10 via a sub-scanning laminated piezo element 35, and receives driving power from a piezo element drive circuit 36 to support the holding member 33, that is, both pinhole plates 31 .32 is moved back and forth at high speed in the direction of arrow X. At the same time, the sub-scanning laminated piezo element 35 receives drive power from the piezo element drive circuit 37, and moves the main-scanning laminated piezo element 34, that is, both pinhole plates 3 and 32, back and forth in the Y direction at high speed.

Note that the moving speed in the Y direction is slower than the moving speed in the X direction.

By moving the first pinhole plate 31 in the X and Y directions as described above, the position of the pinhole 31a (
In other words, since the emission position of the illumination light 11 changes, the illumination light spot P comes to scan the sample 23 two-dimensionally. Thereby, a continuous signal S carrying a two-dimensional image of the sample 23 is obtained from the photodetector 24. This signal S is
For example, by sampling at predetermined intervals, the signal is divided into pixels.

When the light spot P moves as described above, the imaging position of the point image Q also moves two-dimensionally accordingly.

However, since the second pinhole plate 32 moves integrally with the first pinhole plate 31, the position of the pinhole 32a of the second pinhole plate 32 follows the movement of the point image Q, and the position of the pinhole 32a of the second pinhole plate 32 always follows the movement of the point image Q. The transmitted light 11' passes through the photodetector 2
4.

In order to do this, the collimator lens 16, the objective lens 17.19 and the condensing lens 20 are used to set the lateral magnification β=-1, the angular magnification γ=-1, and the objective lens 17.19
An optical system is constructed in which a beam convergence point exists between the two. As for the vertical magnification α, it is desirable to set α=1.

More specifically, for example, when the focal lengths of the objective lenses 1.7.19 are fl and fz, and the distance between them is d, then fl
-f2, d=fl +f2, an optical system equivalent to an afocal system is constructed, and the collimator lens IB and the condensing lens 20 are equivalent to each other, and the distance between the collimator lens 16 and the objective lens 17 is The distance and the distance between the objective lens 19 and the condensing lens 20 are made equal.

Note that by detecting the point image Q through the second pinhole plate 32, light scattered by the halo and the sample 23 can be cut.

Further, in this embodiment, the laminated piezo element 14 receives drive power from the piezo element drive circuit 38 to move the movable table 10 in the direction of the arrow Z perpendicular to the main and sub-scanning directions XSY (that is, the light of the optical system 18. axial direction). If the light spot P is two-dimensionally scanned each time the movable table 1o is moved a predetermined distance in the Z direction in this manner, only information on the focused plane is detected by the photodetector 24. Therefore, by taking the output S of this photodetector 24 into the frame memory,
Within the range in which the sample 23 is moved in the Z direction, it is possible to obtain a signal that represents an image that is in focus on all surfaces.

Note that instead of moving the moving stage 1o in the Z direction as described above, the sample stage 23 may be moved in two directions. Furthermore, if a slight deviation of the confocal point is acceptable, the first pinhole plate 31 may be moved in the Z direction.

Furthermore, a synchronization signal is inputted to the piezo element drive circuits 36, 37 and 38 from a control circuit (not shown), thereby synchronizing the main and sub-scanning of the light spot P and the movement of the moving table 1o in the Z direction.

Next, a second embodiment of the present invention will be described with reference to FIG. Note that in FIG. 2, parts common to those in FIG. 1 are given the same numbers, and explanations thereof will be omitted unless particularly necessary (the same applies hereinafter).

The confocal scanning microscope of this embodiment is of a monochrome reflection type, and a beam splitter 40 is disposed between the collimator lens 16 and the objective lens 17. Sample 2
3, the reflected light 11'' is converted into parallel light by the objective lens 17, which is shared by the light transmitting and light receiving optical systems, and is then reflected by the beam splitter 40 and separated from the illumination light 11.This reflected light 11'' is then reflected by the mirror 41 and focused into a point image Q by the condenser lens 20. The brightness of this point image Q is measured by the photodetector 2 through the second pinhole plate 32.
Detected by 4.

In this embodiment, the photodetector 24 is fixed to the second pinhole plate 32 and moves together with the second pinhole plate 32. In this way, a small device with a small light-receiving area, such as a photodiode, can be used as the photodetector 24.

Also in this second embodiment device, the first pinhole plate 31
The second pinhole plate 32 is moved in the X1Y direction by the main scanning laminated piezo element 34 and the sub-scanning laminated piezo element 35 via the holding member 33. As a result, the illumination light spot P is two-dimensionally scanned, and the pinhole 32a of the second pinhole plate 32 follows the movement of the point image Q due to this scanning.

Next, a third embodiment of the present invention will be described with reference to FIG. The confocal scanning microscope of this embodiment is of a color reflection type, and an RGB laser is used as the light source 12. The reflected light 11'' from the sample 23 is reflected by the beam splitter 40 and sent to the dichroic mirror 42.
, and only the blue light 11B is reflected there. The blue light ILB is detected by the first photodetector 44 through one pinhole 32a of the second pinhole plate 32. Reflected light 11 transmitted through the dichroic mirror 42
" enters another dichroic mirror 43, and only the green light JIG is reflected there. This green light 11G is
It is detected by the second photodetector 45 through another pinhole 32b of the second pinhole plate 32. The reflected light 11", that is, the red light +1R, which has passed through the dichroic mirror 43 is reflected by the mirror 41, passes through another pinhole 32c of the second pinhole plate 32, and passes through the third pinhole plate 32.
It is detected by a photodetector 46.

In this way, each of the photodetectors 44, 45, 48
Signals SB, SG, and SR carrying the blue, green, and red components of the enlarged image of the sample 23 are output.

In this embodiment as well, the photodetectors 44, 45, and 48 are fixed to the second pinhole plate 32 and move together with the second pinhole plate 32.

Also in this third embodiment device, the first pinhole plate 31
The second pinhole plate 32 is moved in the X and Y directions by the main scanning laminated piezo element 34 and the sub-scanning laminated piezo element 35 via the holding member 33. As a result, the illumination light spot P is two-dimensionally scanned, and the pinholes 32a, 32b, and 32C of the second pinhole plate 32 follow the movement of the point image Q due to this scanning.

Incidentally, it is of course possible to form a transmission type confocal scanning microscope as shown in FIG. 1 in a manner similar to the third embodiment described above so as to be capable of capturing color images.

Furthermore, in the confocal scanning microscope of the present invention, instead of performing sub-scanning of the light spot P by moving the first pinhole plate 31, sub-scanning of the light spot P is performed by moving the sample stage 23. You can do it like this. Furthermore, the movement of the pinhole plate 31.32 is caused by the laminated piezo element 34.3.
5, it is also possible to use, for example, a tuning fork, a voice coil, or a scanning method that utilizes the natural vibration of a solid body caused by ultrasonic waves.

(Effects of the Invention) As explained in detail above, in the confocal scanning microscope of the present invention, the pinhole plate is moved back and forth to perform main scanning of the light spot, so the sample stage is moved at high speed. Therefore, it is possible to prevent the sample from flying away. Furthermore, since the pinhole plate to be moved as described above can be made lightweight, high-speed scanning is also possible.

In the confocal scanning microscope of the present invention, since the illumination light beam is not deflected, the optical system can be easily designed, and an expensive optical deflector such as a galvanometer mirror or AOD is not required, resulting in a simple structure. Therefore, this device can be manufactured at a lower cost than conventional confocal scanning microscopes.

Furthermore, the pinhole plate described above has an extremely simple structure compared to a lens optical system, etc., so even if it is moved at high speed, problems such as optical axis deviation and breakage will not occur. Therefore, the confocal scanning microscope of the present invention can be sufficiently reliable and durable.

[Brief explanation of the drawing]

1.2 and 3 are schematic front views showing confocal scanning microscopes according to embodiments 1.2 and 3 of the present invention, respectively. 10... Moving table 11... Illumination light 11'
...Transmitted light 11"...Reflected light 11B.
...Blue light 11G...Green light 11R...
Red light 12...Light source 14...Laminated piezo element 15.16...Collimator lens 17.19...Objective lens 18...Light transmission optical system 20...Condensing lens 21... Light receiving optical system 2
2... Sample stand 23... Sample 24.44
．． 45.46...Photodetector 31...First pinhole plate 31a % 32a % 32b s 32cm...Pinhole 32...Second pinhole plate 33...Holding member 34...Main scanning Laminated piezo element 35...Laminated piezo element for sub-scanning

Claims (2)

[Claims] (1) A sample stage on which a sample is placed, a light source that emits illumination light, a light transmission optical system that images this illumination light as a minute light spot on the sample, and a light transmission system that focuses the light flux from the sample. a light-receiving optical system that forms a point image using a light-receiving optical system; a photodetector that detects this point image; a first pinhole plate disposed between the light source and the light-transmitting optical system; A second pinhole plate disposed at the image position and the first pinhole plate are moved back and forth in a direction intersecting the optical axis thereof to main scan the light spot on the sample, and the second pinhole plate is A system comprising a pinhole plate moving means for moving the hole plate integrally with the first pinhole plate to follow the movement of the point image, and a sub-scanning means for sub-scanning the light spot on the sample. Focal scanning microscope. (2) The pinhole plate moving means is configured to also move the first and second pinhole plates in a direction substantially perpendicular to the direction of movement for the main scanning, and the sub-scanning means The confocal scanning microscope according to claim 1, characterized in that it also serves as a confocal scanning microscope.