JPH03134607A - Confocal scanning type microscope - Google Patents

Abstract

PURPOSE:To obtain a bright and vivid microscopic image and simplify the constitution by detecting the reflected light or the transmitted light from a sample. CONSTITUTION:Deflected illuminating light 11 is converged as a minute light spot P on a sample 20, and reflected light 11' from the sample 20 is converted to parallel rays by a lens 18 and goes on the same optical path as the illuminating path in the opposite direction and passes a half mirror 13, a relay lens 24, and a lens 25 to form an image at a point Q. A columnar light transmission member 26 is arranged in the position irradiated with the reflected light 11', and the light reception face of a photodetector 27 is optically coupled to one end face of the member 26. The reflected light 11' goes in the member 26 while repeating total reflection and is detected by the photodetector 27. A signal S indicating the brightness of the spot image Q is inputted to a processing circuit 28 to obtain a time series signal Sp divided to picture elements bearing the two-dimensional image of the sample 20.

Description

DETAILED DESCRIPTION OF THE INVENTION (Industrial Application Field) The present invention relates to a confocal scanning microscope, and more particularly, to a confocal scanning microscope with an improved mechanism for detecting reflected light or transmitted light from a sample. It is something.

(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.

Among these, there is a system in which illumination light is generated from a light source and converged to a light spot on the sample, and the light flux from this sample is re-imaged into a point image, which is detected by a photodetector. A focal scanning microscope does not require a pinhole on the sample surface, making it easy to implement.

This confocal scanning microscope basically consists of a sample stage on which a sample is placed, a light source that emits illumination light, a light transmission optical system that converges this illumination light as a minute light spot on the sample, and this light. It is composed of a scanning mechanism that main and sub-scans a point on a sample, a light receiving optical system that focuses the light beam from the sample and forms it into a point image, and a photodetector that detects this point image. It is. Further, a pinhole plate is usually provided in front of the photodetector to cut out a point image halo and scattered light from the sample.

There are two types of confocal scanning microscopes: a reflective type that detects light reflected by a sample, and a transmission type that detects light that has passed through the sample.

(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.

On the other hand, although the mechanism (2) allows sufficiently high-speed scanning, another problem arises when this mechanism is applied to the above-mentioned transmission type confocal scanning microscope.

That is, in that case, since the illumination light is deflected, the imaging position of the light beam transmitted through the sample changes accordingly, so it is necessary to scan the photodetector two-dimensionally in synchronization with the deflection of the illumination light. Alternatively, if the photodetector is fixed, a means for deflecting the sample transmitted light in synchronization with illumination light scanning is required.

Providing such a two-dimensional scanning mechanism for the photodetector or a means for deflecting light transmitted through the sample would make the structure of the confocal scanning microscope extremely complicated.

In view of these circumstances, for example, Japanese Patent Application Laid-Open No. 62-20951
As shown in Publication No. 0, the light flux that has passed through the sample is deflected in synchronization with the illumination light sub-scanning so that it does not move in this direction, and the light flux that moves in the main scanning direction is deflected. There has also been a proposal to detect this using a line sensor extending in this direction.

Furthermore, even with a reflection-type confocal scanning microscope, if the main scanning of the illumination light is performed by an AOD (acousto-optic optical deflector), etc., and the sub-scanning is performed by a mechanical optical deflector, the wavefront may change. In order to prevent scanning unevenness from occurring due to the reflected light from the disturbed sample entering the AOD etc. again, or from reducing the detection efficiency of the reflected light,
After reflecting and deflecting this reflected light with a mechanical optical deflector, the AO
It has been considered to separate the illumination light from the optical path of the illumination light without making it re-enter the light beam D or the like. In this case as well, since the reflected light separated from the illumination light optical path moves in the main scanning direction as described above, it is convenient to detect it with a line sensor.

Transmission type or reflection type confocal scanning microscopes with the above structure detect the point image of the light beam from the sample using a point detector in the sub-scanning direction, but this is not the case in the main scanning direction. Although it cannot be called a perfect confocal scanning microscope, it has the advantage that the means for deflecting transmitted light or reflected light is simplified, and a scanning mechanism for a photodetector is not required.

However, since line sensors generally have lower sensitivity than photomultiplier tubes and the like, it is difficult for the confocal scanning microscope with the above configuration to obtain bright and clear microscopic images.

The present invention has been made in view of the above circumstances, and aims to provide a confocal scanning microscope that does not require a scanning photodetector and can employ a highly sensitive photodetector. This is the purpose.

(Means for Solving the Problems) A confocal scanning microscope according to the present invention includes the above-mentioned sample stage, light source, light transmission optical system, and illumination light spot on a sample for main and sub-scanning. a scanning mechanism for scanning, a light-receiving optical system,
In a confocal scanning microscope equipped with a photodetector and a deflection means that deflects the light beam from the sample in synchronization with the illumination light sub-scanning so that it does not move in the sub-scanning direction,
It is arranged at a position where it receives one-dimensional scanning of the point image of transmitted light or reflected light from the sample formed by the light receiving optical system, and the surface part receiving this scanning has a plurality of mask parts and a plurality of microscopic parts in the scanning direction. A transparent light guide member is provided in which light passing portions are arranged alternately, and a photodetector is optically connected to the end face of the light guide member to detect light incident on the light guide member. It is characterized by being arranged as follows.

(Function) In the above configuration, the light beam from the sample enters the light guide member from the light passing portion on the surface thereof, is totally reflected inside the member, travels to the end face side, and is detected by the photodetector. Ru. Therefore, as this photodetector, one having a wide light-receiving surface and high sensitivity, such as a photomultiplier tube, can be used.

Since the light beam from the sample alternately crosses the mask part and the light passage part of the light guide member in accordance with the main scanning of the illumination light spot, the output of the photodetector is periodically transmitted in synchronization with this main scanning. Modulated. In other words, when the point image formed by the above-mentioned light beam is on the mask part, the output of the photodetector is zero or at an extremely low constant level, whereas when the point image is on the part where the light passes between the mask members, the output of the photodetector is indicates the brightness of the point image. Therefore, pixel division can be performed based on periodic fluctuations in the output signal of this photodetector.

Further, when the point image is located in the light passing portion between the mask members, the point image is detected through a minute slit or pinhole.

This provides the effect of cutting off the point image halo and scattered light on the sample.

For example, in Japanese Patent Application Laid-open No. 59-81969, a light beam deflected in a one-dimensional direction is received by a surface portion, transmitted inside the surface portion to an end surface side, and is incident on a photodetector coupled to this end surface. On the other hand, a light guide member is shown in which an optical grating arranged in the light beam deflection direction is provided on the surface portion. However, the optical grating of this light guide member only modulates the output of the photodetector, and does not have the effect of cutting off the halo or scattered light described above.

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

FIG. 1 shows a reflection type confocal scanning microscope according to an embodiment of the present invention. As shown in the figure, a laser beam (illumination light) 11, which is parallel light emitted from a laser light source IO, has its beam diameter expanded by a beam expander 12 and enters an AOD (acousto-optic optical deflector) 14. The illumination light U is deflected by the AOD 14 in a direction substantially perpendicular to the plane of the paper, passes through a relay lens 15 for aberration correction, passes through a half mirror 13, and enters a vibrating mirror 16.

The vibrating mirror 16 swings in the direction of arrow A in the figure, thereby deflecting the illumination light 11 in a direction substantially perpendicular to the direction of deflection described above.

This deflected illumination light 11 passes through the relay lens 17 and enters the objective lens 18, and the objective lens 18
A minute light spot P on the sample 20 (on the surface or inside)
It is forced to converge. The sample stage 19 on which the sample 2° is placed is moved by a moving mechanism 21 in the direction of arrow Z, that is, in the optical axis direction of the objective lens 18.

The reflected light 11° reflected by the sample 20 is converted into parallel light by the objective lens 18. This reflected light 11 made into parallel light
' travels in the opposite direction along the optical path common to the relay lens 17, the vibrating mirror 16, and the illumination light 11, and reaches the half mirror 13.
incident on . The reflected light 11' is separated from the optical path of the illumination light 11 by this half mirror 13, and is passed through the relay lens 24.
, and is focused into a point image Q by the condenser lens 25.

A cylindrical light guide member 26 extending in a direction perpendicular to the plane of the paper in FIG. 1 is arranged at a position receiving the reflected light ll°. Note that this light guiding member 26 is shown in detail in FIG. The light guide member 26 is made of optical glass or the like, and is arranged so that its peripheral surface 26D is located at the point image Q formation position. On this circumferential surface 26D, a large number of linear masks 26A are formed from a light-shielding material and extend in the circumferential direction.
are arranged in parallel in the axial direction of the light guide member 26. These masks 2[iA each have a predetermined width and are provided at a predetermined interval from each other. Thereby, between these masks 2OA, there is a small light passage portion 26B with a predetermined width.
is formed. On the other hand, one end surface 26 of this light guide member 26
A light receiving surface 27A of the photodetector 27 is optically coupled to C. Note that as this photodetector 27, a highly sensitive one having a wide light-receiving surface 27A, such as a photomultiplier tube, can be used.

As mentioned earlier, the illumination light 11 that irradiates the sample 20 is A
Since it is deflected by the OD 14, the light spot P scans the sample 20 in the X direction, and at the same time, since the illumination light 11 is deflected by the vibrating mirror 16, the light spot P scans the sample 20 in the main direction. Sub-scanning is performed in the Y direction, which is approximately perpendicular to the scanning direction.

When the main and sub-scanning of the light point P is performed in this way, the reflected light 11' incident on the vibrating mirror 16 is reflected and deflected by the vibrating mirror 16 at the same angle as the illumination light 11, so that it is reflected and deflected by the half mirror 13. The incident reflected light ll° is
It does not move in the sub-scanning direction Y. Therefore, the light guiding member 26
On the circumferential surface 26D, the point image Q is in the main scanning direction X,
In other words, scanning is performed only in the axial direction of the light guide member 26. The point image Q that is scanned one-dimensionally in this way is the mask 26A.
and the light passing portion 26B alternately.

Under the condition that this point image Q is on the light passing portion 2i3B,
The reflected light 11' enters into the light guiding member 26 from the peripheral surface 26D. This reflected light 11' repeats total reflection within the light guiding member 2G, travels toward the end face 2BC, and is detected by the photodetector 27. The photodetector 27 detects a point image Q
A signal S indicating the brightness of the image is output.

Under the condition that the force point image Q is on the mask 2BA, the reflected light 1
1' is blocked by the mask 26A. Therefore, the time-series signal S changes as shown in FIG. That is, the value of the signal S (for example, the current value) is extremely low and constant when the reflected light 11' is blocked by the mask 28A, and is constant when the reflected light 11' passes through the light passing portion 28B. takes a value Is corresponding to the brightness of the point image Q.

In this way, the signal S is modulated in synchronization with the scanning of the light spot P. By processing this signal S in the signal processing circuit 28, the signal S is divided into pixels carrying a two-dimensional image of the sample 20. The sequence signal Sp can be easily obtained.

Note that the point image Q is the light passing portion 28B between the masks 26A.
, the point image Q is the same as being detected through a minute slit. Thereby, the effect of cutting off the halo of the point image Q and the scattered light on the sample 20 can be obtained. More precisely, the above effect is obtained only in the main scanning direction. However, compared to the case where there is no such mask part at all, in that case the halo and scattered light will enter the light guide member 2B two-dimensionally, whereas in this case they will be incident on the light guide member 2B in a two-dimensional manner. Since the light enters the light guide member 26 only dimensionally (in the sub-scanning direction), the resolution is significantly improved.

Further, in this embodiment, the sample stage 19 is moved by the moving mechanism 21 in the direction of arrow Z, which is perpendicular to the main and sub-scanning directions X1Y. If the light spot P is two-dimensionally scanned every time the sample 2o is moved a predetermined distance in the Z direction in this manner, only information on the focused plane is detected by the photodetector 27. Therefore, by importing the output signal Sp into the frame memory, it is possible to obtain a signal that represents an image in focus on all surfaces within the range in which the sample 20 is moved in two directions.

Note that the light deflection means for deflecting the illumination light 11 is not limited to the AOD 14 and the vibrating mirror 1G described above, but other known means such as an EOD (electro-optic light deflector) and a polygon mirror may be used. Good too.

Moreover, two photodetectors can be installed so as to be connected to both end surfaces of the light guide member 26, respectively, to further improve the detection efficiency of the reflected light 11°.

Furthermore, instead of the light guide member 26 described above, a light guide member 30 as shown in FIG. 4 may be used.

This light guide member 30 has a mask 30A widely formed on a circumferential surface 30D, and this mask 30A is provided with a large number of dot-shaped light passing portions 30B lined up in the main scanning direction X. . When such a light guide member 30 is used, the point image Q is detected through a pinhole, so compared to the case where the light guide member 26 is used, the effect of cutting the halo and scattered light is lower. is obtained more clearly.

Furthermore, as described above, the present invention provides a reflective confocal scanning microscope that eliminates movement of the transmitted light in the sub-scanning direction by guiding the transmitted light from the sample to the back surface of the sub-scanning vibrating mirror. It is also possible to apply it to

Furthermore, although the confocal scanning microscope of the embodiments described above is for obtaining monochrome microscopic images, the present invention is applicable to conventionally known confocal scanning microscopes for obtaining color images. Of course, this is also possible.

(Effects of the Invention) As explained in detail above, the confocal scanning microscope of the present invention allows reflected light or transmitted light from a sample to enter a transparent light guide member, and connects to the end face of this light guide member. Since this light is detected by a photodetector, a highly sensitive photodetector with a wide light-receiving surface can be used, thereby making it possible to obtain a bright and clear microscope image.

Further, in the confocal scanning microscope of the present invention, a plurality of mask portions and a plurality of minute light passing portions are alternately arranged on the surface portion of the light guiding member that is irradiated with a point image of reflected light or transmitted light from the sample. By arranging them in the same direction, it is possible to cut out the point image halo and scattered light from the sample, thereby increasing the resolution, and furthermore, it is possible to easily perform pixel division by modulating the output signal of the photodetector. It becomes possible.

Since the confocal scanning microscope of the present invention is configured to detect reflected light or transmitted light from the sample as described above, there is no need to scan the photodetector in synchronization with illumination light scanning. Therefore, the structure is simple and can be formed at low cost.

[Brief explanation of the drawing]

FIG. 1 is a schematic side view showing a confocal scanning microscope according to an embodiment of the present invention, FIG. 2 is a perspective view showing a light guide member used in the confocal scanning microscope, and FIG. , a graph showing the output signal waveform of the photodetector in the confocal scanning microscope; FIG. 4 is a perspective view showing another example of the light guide member used in the confocal scanning microscope of the present invention. 10... Laser light 1111X 11... Illumination light 11'... Reflected light 13... Half mirror 14... AOD 15.17.24.
・・Relay lens 1G・・Vibration mirror 18・・
・Objective lens 19...Sample stage 20...Sample 25...Condensing lens 26.30...Light guiding member 26A, 30A...Mask 26B, 30B...
Light passing portion 28C... End faces 28D, 30D of light guide member... Circumferential surface 27 of light guide member... Photodetector 27A... Light receiving surface 28... Signal processing circuit

Claims (1)

[Scope of Claims] A sample stage on which a sample is placed; a light source that emits illumination light; a light transmission optical system that converges the illumination light as a minute light spot on the sample; a scanning mechanism for main and sub-scanning; a light-receiving optical system for condensing the light beam from the sample and forming a point image; and a light receiving optical system for deflecting the light beam in synchronization with the sub-scanning direction. a deflection means that does not move; and a deflection means arranged at a position that receives the one-dimensional scanning of the point image, and a plurality of mask portions and a plurality of minute light passage portions on the surface portion receiving the scanning in the direction of the scanning. A confocal scanning microscope comprising: transparent light guide members arranged alternately; and a photodetector that is optically connected to the end face of the light guide member and detects light incident on the light guide member.