JPH03125108A - Confocal scanning type microscope - Google Patents

Abstract

PURPOSE:To accomplish high-speed scanning and to prevent a sample from flying by integrally holding a light transmission optical system and a light reception optical system in a rotating member and rotating the rotating member so that the main scanning of a light point is performed. CONSTITUTION:Rotating members 15 and 16 which integrally hold the light transmission optical system and the light reception optical system and can be rotated on a plane in parallel with the sample placing surface of a sample table 22, and a driving means which rotates the rotating members 15 and 16 and performs the main scanning of the light point so that the locus of a circular arc is plotted on the sample 50 are provided. Then, a sub scanning means which relatively moves the rotating members 15 and 16 and the sample table 22 in a Y direction nearly orthogonally crossed with a chord obtained by combining both ends of the circular arc at the lower speed than the main scanning speed and performs the sub scanning of the light point on the sample 50 are provided. Since the sample table is not moved two-dimensionally, the sample is prevented from flying and the high-speed scanning is performed. On the other hand, the constitution is simplified and the microscope is manufactured 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 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, and at that time, the light that has passed through the sample or the light that has been reflected there is detected by a photodetector. Optical scanning microscopes are known that detect electrical signals that carry an enlarged image of a 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. It consists of a light receiving optical system that condenses the light beam from the sample and forms it into a point image, a photodetector that detects this point image, and a scanning mechanism that scans the light spot two-dimensionally on the sample. It is something that Furthermore, Japanese Patent Publication No. 132-2
No. 17218 and No. 82-218918 disclose examples 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 specimens are observed with microscopes, and if high-speed scanning is not possible when observing these biological specimens, problems may arise in which the specimen dies before an image of the specimen can be captured. obtain.

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, In a confocal scanning microscope equipped with a two-dimensional scanning mechanism for a light spot, the light transmitting optical system and the light receiving optical system are integrally held,
a rotating member rotatable in a plane parallel to the sample mounting surface of the sample stage; a driving means for rotating the rotating member to main scan the light spot so as to draw an arc locus on the sample; sub-scanning means for sub-scanning the light spot on the sample by relatively moving the rotating member and the sample stage at a speed lower than the main-scanning speed in a direction substantially orthogonal to a chord connecting both ends of the circular arc; This is characterized in that the scanning mechanism is configured by the following.

Note that the above-mentioned sub-scanning means may be one that moves a rotating member, or, on the contrary, may be one that moves a sample stage. Since the sub-scanning speed can be relatively low, 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.

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

FIG. 1 is a perspective view showing the main parts of a transmission type confocal scanning microscope according to a first embodiment of the present invention, and FIG. 2 shows its side profile. As shown, R.G.
The B laser 10 emits illumination light 11 consisting of red light, green light, and blue light. The beam diameter of this illumination light 11, which is parallel light, is expanded by a beam expander 12.
It is reflected by the mirror 13.

On the other hand, a rotating shaft I4 is disposed parallel to the traveling direction of the reflected illumination light 11, and two discs 15 and IB are fixed to the rotating shaft 14, facing each other with an interval between them. These discs 15 and te have a rotary shaft 14 connected to the pulley 1.
7. By being rotated by a motor 20 via a belt 18 and a pulley 19, it is rotated in the direction of arrow R in FIG. In addition, in the center of the disk 15, there are four reflective surfaces 21.
A rectangular pyramidal mirror 21 having mirrors A, 21B, 21G, and 21D is fixed. The mirror I3 mentioned above is arranged so that the illumination light 11 is incident on one of these reflecting surfaces 21A to 21D. The disk 15 has the four reflective surfaces 21A and 21B.

Four mirrors 2 facing each of 2IC and 21D
3A, 23B, 23C, 23D are fixed, and these mirrors 23A, 23B, 23C, 23
(7) Objective lens 24A,
24B, 24C.

24D is fixed. These objective lenses 24A~
24D is fixed with its optical axis parallel to the rotation axis 14.

On the other disk 16, there are four mirrors 41 and 43A which are completely equivalent to the above-mentioned square pyramid mirror 21, mirrors 23A to 23D1 and objective lenses 24A to 24D, respectively.
, 43B, 43C, 43D, four objective lenses 44A1
44B, 44C, and 44D are fixed. Note that, in FIG. 2, the mirrors 43B and 43D and the objective lenses 44B and 44D are omitted. The objective lenses 44A, 44B, 44C, and 44D are arranged so that their optical axes coincide with those of the objective lenses 24A, 24B, 24C, and 24D, respectively. A mirror 45 equivalent to the mirror 13 is disposed below the disk 16, facing toward the quadrangular pyramid mirror 41.

Between the two discs 15.1B, there is a sample mounting surface 22a.
The sample stage 22 is arranged in such a manner that the sample stage 22 is parallel to these disks 15, 18 (that is, perpendicular to the rotation axis 14). This sample stage 22 is fixed to a two-dimensional movement stage 35. This two-dimensional movement stage 35 is reciprocated in the direction of arrow Y via a micrometer 38 by a pulse motor 37 that receives a drive current from a motor drive circuit 3B.

Hereinafter, as shown in FIG. 2, taking as an example the case where the illumination light 11 is incident on the reflective surface 2fA of the quadrangular pyramidal mirror 21,
The optical path of this illumination light 11 will be explained. When the illumination light 11 is reflected by the reflective surface 21A, it enters the mirror 23A, is reflected there, is then focused by the objective lens 24A, and is reflected onto the sample 5o placed on the sample stage 22 (on the surface or inside). The image is formed into a minute light spot P.

The beam of transmitted light 11' that has passed through the sample 50 is made into parallel light by an objective lens 44A, and then reflected one after another by a mirror 43A, a reflecting surface 41A of a quadrangular pyramid mirror 41, and a mirror 45. This transmitted light 11° is the dichroic mirror 2
6, and only the blue light 11B is reflected there. The blue light 11B is condensed by a condensing lens 51 and detected by the first photodetector 27 via a pinhole plate 52. Transmitted light 11' transmitted through the dichroic mirror 26
enters another dichroic mirror 28, and only the green light 11G is reflected there. This green light 11G is condensed by a condenser lens 53 and detected by a second photodetector 29 via a pinhole plate 54. The transmitted light 11' (that is, the red light 11R) transmitted through the dichroic mirror 28 is reflected by the mirror 30, condensed by the condensing lens 55, and passed through the pinhole plate 5B to the third
It is detected by the photodetector 31. Note that the photodetector 2
For example, a photodiode or the like is used as 7.29.31, and from these, signals SB, SG, and S carrying blue, green, and red components of an enlarged image of the sample 50 are respectively output.
R is output.

As is clear from the above description, in this embodiment, the mirror 13, the quadrangular pyramid mirror 21. A light transmission optical system is constituted by mirrors 23A to 23D and objective lenses 24A to 24D, while objective lenses 44A to 44D and mirrors 43A to
A light receiving optical system is composed of the 43D1 quadrangular pyramid mirror 41 and the mirror 45.

Next, two-dimensional scanning of the light spot P of the illumination light ll will be explained. When the motor 20 is driven by the motor drive circuit 57 and the disc 15.16 is rotated, the reflective surface 2LA rotates, so that the light point P focused by the objective lens 24A draws an arc on the sample 50. Main scan. Then, the disk 15.1B is
When rotates, another reflective surface 21B of the quadrangular pyramidal mirror 21 reaches a rotational position where the illumination light 11 is incident thereon, so that it is reflected by this reflective surface 21B and focused by the objective lens 24B. The light spot P also main scans over the sample 50. In the same manner, the objective lenses 24C, 24
The light point P focused at D, 24A... is sample 50.
Main scan the top one after another.

This main scanning speed is, for example, about 2 m/s.

As described above, the light spot P is main-scanned and the pulse motor 37 is driven, so that the sample stage 22 can move the sample 5
Arrow Y perpendicular to the chord connecting both ends of the arc locus of the light point P on 0
direction (direction away from the rotating shaft 14). As a result, each objective lens 24A, 124B, 24C, 24D
, 24A..., the scanning locus of the light point P (
(indicated by X in FIG. 1) is shifted little by little, and this light spot P is sub-scanned.

Note that this sub-scanning speed is, for example, about 2 mm/s, which is sufficiently lower than the main scanning speed. With this level of sub-scanning speed, even if the sample stage 22 is moved, the sample 50 can be prevented from flying away.

By scanning the light spot P two-dimensionally over the sample 5G as described above, time-series signals SB, SG, and SR carrying a two-dimensional image of the sample 50 are obtained.

These signals SB, SG, and SR are made into pixel-divided signals by, for example, integrating at predetermined intervals.

The image signals SB, SG, and SR thus obtained are subjected to conversion processing to correct the curvature of the main scanning line, if necessary.

Further, in this embodiment, the two-dimensional moving stage 35 is driven by a pulse motor 4 that receives a drive current from a motor drive circuit 39.
0, it is moved in the direction of arrow Z parallel to the rotation axis 14. If the light spot P is two-dimensionally scanned every time the two-dimensional moving stage 35 is moved a predetermined distance in two directions in this manner, only information on the focused plane is detected by the photodetectors 27, 29, and 31. Therefore, these photodetectors 27.29.3
By importing the outputs SB, SG, and SR of 1 into the frame memory, within the range in which the sample 50 is moved in two directions,
It becomes possible to obtain a signal that carries an image that is in focus on all planes.

Note that a synchronization signal is inputted to the motor drive circuit 3B=39 and 57 from a control circuit (not shown), and as a result,
Main and sub-scanning of the light spot P and movement of the sample stage 22 in the Z direction are synchronized.

Next, a second embodiment of the present invention will be described with reference to FIG. The embodiment shown in FIG. 3 is a reflection type confocal scanning microscope in which the light transmitting optical system also functions as a light receiving optical system. Note that in FIG. 3, parts common to those in FIG. 2 are given the same numbers, and detailed explanation thereof will be omitted.

For example, the illumination light 11 that is focused by the objective lens 24A and focused on a light spot P on the surface of the sample 50 is reflected there. This reflected light 11'' travels in the same optical path as the illumination light 11 in the opposite direction, and is separated from the optical path of the illumination light 11 by a half mirror 7o arranged between the quadrangular pyramidal mirror 21 and the mirror 13. The blue light 11B, the green light 11G, and the red light 11R of the light 11'' are respectively detected by photodetectors 27, 29, and 31 in exactly the same manner as in the apparatus of the first embodiment.
detected by. Furthermore, the two-dimensional scanning of the light spot P is performed in exactly the same manner as in the apparatus of the first embodiment.

Various modifications can be made to the embodiments described above. For example, if a lightweight semiconductor laser or the like is used as the illumination light source, the light source may be mounted on the disk 15. If so, each objective lens 24A to 24D
It is preferable to mount four light sources coaxially.

Also, when the illumination light source is mounted on the disk 15 as described above, or as in the above embodiment, the illumination light source is mounted on the disk 15.
5, only one objective lens for forming an image of the illumination light 11 on the sample 50 may be provided. However, if the objective lenses are provided in multiple stages, there will be no long waiting time between each main scanning period, which is more advantageous in terms of shortening the imaging time.

(Effects of the Invention) As explained in detail above, in the confocal scanning microscope of the present invention, the light transmitting optical system and the light receiving optical system are integrally held by a rotating member, and the rotating member is rotated to emit light. Since the structure is configured to perform main scanning of points, there is no need to move the sample stage at high speed, so it is possible to prevent the sample from flying away, and 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.

[Brief explanation of the drawing]

1 and 2 are respectively a perspective view and a partially cutaway side view showing a confocal scanning microscope according to a first embodiment of the present invention, and FIG. FIG. 2 is a partially cutaway side view showing a focal scanning microscope. 10...RGB laser 11...Illumination light 11°
...Transmitted light 11''...Reflected light 13.23
A, 23B, 23C, 23D. 30.43A, 43B, 43C, 43D, 45... Mirror 14... Rotating shaft 15.16... Disk 17.19... Pulley 18... Belt 2
0.37.40...Motor 21, 41...Square pyramid mirror 22 River sample stage 24A,
24B, 24C, 24D. 44A, 44B, 44C, 44D... Objective lens 2B, 28... Guicroic mirror 27.29.3
1 River optical detector 35...2-dimensional movement stage 3G, 3
9.57...Motor drive circuit 38...Micrometer 5o...Sample 51.53.55...Condenser lens 52.54.58...Pinhole plate 7o...Half mirror-1st figure

Claims (1)

[Claims] A sample stage on which a sample is placed, a light source that emits illumination light, a rotating member that is rotatable in a plane parallel to the sample mounting surface of the sample stage, and a rotating member that is held by the rotating member. , a light transmission optical system that images the illumination light as a minute light spot on the sample; a drive unit that rotates the rotating member to main scan the light spot so as to draw a circular arc trajectory on the sample; a light-receiving optical system held by the rotating member and condensing a light beam from the sample to form a point image; a photodetector detecting the point image; and a photodetector that detects the point image; sub-scanning means for sub-scanning the light spot on the sample by relatively moving it at a speed lower than the main-scanning speed in a direction substantially orthogonal to a string connecting both ends of the confocal scanning microscope.