JPH0450911A - Cofocus scanning type microscope - Google Patents

Abstract

PURPOSE:To allow the cutting of the greater part of scattered light by selectively allowing the transmission or reflection of only the collimated beams of light after passage through a collimator lens. CONSTITUTION:The luminous flux of the respective transmitted light rays 11' transmitted through a sample 23 is collimated by the collimator lens 19 of a photodetecting optical system 21 to the collimated beams of light which are passed through and interference filter 8 and are then condensed by a condenser lens 20, by which the beams are imaged to an image point Q. This image point Q is detected by a photodetector 9. The interference filter 8 is disposed perpendicularly to the optical axis L of the collimator lens 19 in this case. Then, the transmitted light rays 11' progressing in parallel with the optical axis L well transmit the interference filter 8 and the scattered light 11d progresses in random directions even after the passage through the collimator lens 19; therefore, the greater part thereof do not pass the interference filter 8. The microscopic image of the high resolution is outputted in this way without being affected by the scattered light 11d.

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 that does not require a diaphragm member such as a pinhole plate in front of a photodetector. It relates to a focal scanning microscope.

(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 consists of 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 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.

(Problem to be Solved by the Invention) By the way, in the conventional confocal scanning microscope with the above configuration, in order to cut unnecessary light (mainly light scattered by the sample), the above point image is transferred to the photodetector. Detection is performed through a diaphragm member such as a pinhole plate placed in front.

This increases resolution, but on the other hand,
This diaphragm member needs to be positioned with extremely high precision so that its aperture is located at the point image formation position, which makes assembly of the confocal scanning microscope and optical axis adjustment extremely difficult. .

SUMMARY OF THE INVENTION Therefore, an object of the present invention is to provide a confocal scanning microscope capable of cutting off light scattered by a sample without using an aperture member as described above.

(Means 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, and a light spot. In a confocal scanning microscope comprising a dimensional scanning mechanism, the light receiving optical system includes a collimator lens that converts illumination light transmitted through or reflected from the sample into parallel light, and a transmittance or The device is characterized in that it is configured to include an optical element that has a uniquely high reflectance and is arranged so that the parallel light enters at this specific angle of incidence.

(Function) After passing through the collimator lens, the transmitted light and reflected light from the sample, which should originally be detected as light that carries a microscopic image, travel in parallel directions.

On the other hand, the light scattered by the sample continues in random directions even after passing through the collimator lens. Therefore, in the optical element described above, if only the parallel light that has passed through the collimator lens is selectively transmitted or reflected, most of the scattered light will be cut there.

Therefore, if the light that has passed through this optical element is detected by a photodetector, only the transmitted light or reflected light from the sample can be detected, excluding the scattered light, without using the pinhole plate described above. become.

Note that as the above-mentioned optical element, for example, an interference filter, a diffraction grating, etc. can be used.

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

FIG. 1 shows a transmission type confocal scanning microscope according to an embodiment of the present invention, and FIG. 2 shows in detail the scanning mechanism used therein. As shown in FIG. 1, a laser diode 5 that emits illumination light 11 is integrally held on a movable table 15. The moving table 15 also includes a light transmitting optical system 18 consisting of a collimator lens 16 and an objective lens 17, and a light receiving optical system 21 consisting of an objective lens (collimator lens) 19, an interference filter 8, and a condensing lens 20. They are fixed with their optical axes aligned with each other.

A sample stage 22, which is separate from the moving stage 15, is arranged between these optical systems 18, 21. A photodetector 9 is fixed to the movable table 15 below the light receiving optical system 21. As this photodetector 9, for example, a photodiode or the like is used.

Laser light (illumination light) emitted from laser diode 5
11 is made into parallel light by a collimator lens 16, and then condensed by an objective lens 17.
The light converges into a minute light spot P on the sample 23 placed on the sample 23 (on the surface or inside the sample 23).

The light beams of each transmitted light 11' that passed through the sample 23 are made into parallel lights by the collimator lens 19 of the light receiving optical system 21, pass through the interference filter 8, and are then condensed by the condensing lens 20 to form a point image Q. image is formed. This point image Q is detected by the photodetector 9. From this photodetector 9,
A signal S indicating the brightness of each part of the sample 23 irradiated with the light spot P is output. Note that the operation of the interference filter 8 will be explained in detail later.

Next, regarding the two-dimensional scanning of the light point P of the illumination light 11, the second
This will be explained with reference to the drawings. The movable table 15 is supported by the pedestal 32 so as to be movable in the direction of arrow X.

That is, one end portions of two guide rods 40.40 are fixed to the pedestal 32, and these guide rods 40.40 are loosely fitted into two guide holes 41.41 provided in the movable table 15. A laminated piezo element 33 is interposed between the movable table 15 and the pedestal 32. This laminated piezo element 33 receives driving power from a piezo element drive circuit 34 and causes the movable table 15 to reciprocate at high speed in the direction of arrow X. The frequency of this reciprocating movement is, for example, 10kHz. In that case, if the main scanning width is 100 μm, the main scanning speed is 10XIO3X100XIO'X2-2 m/s.

On the other hand, the sample stand 22 is supported by the pedestal 32 so as to be movable in the direction of the arrow Y, which is perpendicular to the direction of the arrow X. That is, one ends of two guide rods 45.45 are fixed to the pedestal 32, and these guide rods 45.45 are loosely fitted into two guide holes 46.46 provided in the sample stage 22. . A laminated piezo element 47 is interposed between the sample stage 22 and the pedestal 32. This laminated piezo element 47 receives drive power from a piezo element drive circuit 48 and moves the sample stage 22 back and forth in the direction of arrow Y at high speed. Thereby, the sample stage 22 is moved relative to the moving stage 15, and the light spot P sub-scans the sample 23 in the Y direction perpendicular to the main scanning direction X.

Note that the time required for this sub-scanning is, for example, 1/20 second,
In that case, if the sub-scan width is 100 μm, the sub-scan speed is: 20X100 XIO bow - 0,002 m/s 2 mm
/s, which is sufficiently lower than the main scanning speed. With this level of sub-scanning speed, it is possible to prevent the sample 23 from flying away even if the sample stage 22 is moved.

By scanning the light spot P two-dimensionally over the sample 23 as described above, an analog signal S carrying a two-dimensional image of the sample 23 is obtained. This signal S is periodically sampled and made into pixel-divided digital image data. Generation of this image data will be explained in detail later.

Note that a synchronization signal is inputted to the piezo element drive circuits 34 and 48 from a control circuit (not shown), whereby main and sub-scanning of the light spot P is synchronized.

Although not particularly shown in the figure, the main and sub-scanning directions
The sample stage 22 can also be moved in the direction of arrow Z (see FIG. 1) perpendicular to Y, that is, in the direction of the optical axis of the optical system 18.21. If the light spot P is two-dimensionally scanned each time the sample stage 22 is moved a predetermined distance in the Z direction in this manner, only information on the focused plane is detected by the photodetector 9. Therefore, by importing the image data obtained from the output S of the photodetector 9 into the frame memory, the image data that is responsible for the image that is in focus on all surfaces within the range in which the sample 23 is moved in two directions. It becomes possible to obtain.

Here, as the interference filter 8, one that mainly transmits light in the wavelength range of transmitted light 11' (that is, illumination light 11) is used. Moreover, this interference filter 8 has a third
It has the incident angle versus transmittance characteristics as shown in the figure. In other words, this interference filter 8 has a specific angle of incidence θ. (in this example, θ, -0°) is transmitted with particularly high transmittance.

In this example, corresponding to the value of θ = Q +1 above,
The interference filter 8 is arranged perpendicular to the optical axis of the collimator lens 19. Therefore, the transmitted light 11' traveling parallel to the optical axis can be well transmitted through the interference filter 8 and detected by the photodetector 9 as described above.

On the other hand, since the scattered light lid scattered by the sample 23 travels in random directions even after passing through the collimator lens 19, most of it cannot pass through the interference filter 8-. Therefore, this scattered light lid is almost never detected by the photodetector 9 even if no aperture member such as a pinhole plate is particularly arranged in front of the photodetector 9. Therefore, based on the output S of the photodetector 9, it is possible to output a high-resolution microscope image that is not affected by the scattered light lid.

Of course, it is preferable to use an interference filter 8 having a steeper incident angle versus transmittance characteristic as shown in FIG. 3. In addition, as shown in FIG.
You may arrange the lens 7 which has. By doing so, the scattered light lid, which would be incident on the interference filter 8 at an incident angle θ when the lens 7 is not provided, will be incident on the interference filter 8 at a larger incident angle φ, so that it will be more reliably cut by the interference filter 8.

Further, in place of the interference filter 8, other optical elements such as the above-mentioned diffraction grating can also be used. An example using this diffraction grating is shown in FIG. Furthermore, this fifth
In the figures, elements that are equivalent to those in FIG. 1 are given the same numbers, and explanations thereof will be omitted. In this confocal scanning microscope, transmitted light 11° incident on the diffraction grating 6 at an incident angle Oo is well diffracted and travels toward the photodetector 9. On the other hand, the scattered light 11d that is incident on the diffraction grating 6 at an angle far different from the incident angle θ0 can be diffracted there, and is therefore not detected by the photodetector 9.

In addition, the embodiments described above apply the present invention to a transmission type confocal scanning microscope. It is equally applicable. Furthermore, the present invention is also applicable to confocal scanning microscopes that output color images.

(Effects of the Invention) As explained in detail above, in the confocal scanning microscope of the present invention, the light receiving optical system includes a collimator lens and an optical element that mainly transmits or reflects light incident at a specific angle of incidence. By providing this optical element, it is possible to cut off the light scattered by the sample.

Therefore, the confocal scanning microscope of the present invention can eliminate the influence of the scattered light without placing an aperture member such as a pinhole plate in front of the photodetector, so the optical adjustment of this aperture member is possible. This makes assembly and adjustment much easier than before.

[Brief explanation of the drawing]

FIG. 1 is a schematic front view showing a confocal scanning microscope according to an embodiment of the present invention, FIG. 2 is a perspective view showing main parts of the confocal scanning microscope, and FIG. A graph showing the light incident angle versus transmittance characteristics of an interference filter used in a focal scanning microscope; FIG. 4 is a front view showing another example of the light receiving optical system used in the confocal scanning microscope of the present invention; FIG. 5 is a front view showing a confocal scanning microscope according to another embodiment of the present invention. 5... Laser diode 6... Diffraction grating 7...
Lens 8...Interference filter 9...Photodetector 11...Illuminating light 11'...Transmitted light 15...Moving table 16.19...Collimator lens 17...Objective lens 18. ...Light transmission optical system 2
0... Condensing lens 21... Light receiving optical system 22
... Sample stand 23 ... Sample 32 ... Mount 33.47 ... Laminated piezo element 34
．． 48... Piezo element drive circuit diagram θ (within human interest)

Claims (1)

[Claims] A sample stage on which a sample is placed; a light source that emits illumination light; a light transmission optical system that images the illumination light as a minute light spot on the sample; The light-receiving optical system includes a light-receiving optical system that focuses light and forms a point image, a photodetector that detects the point image, and a scanning mechanism that scans the light spot two-dimensionally on the sample. The system includes a collimator lens that converts illumination light transmitted through or reflected from the sample into parallel light; A confocal scanning microscope characterized in that it is configured to include an optical element arranged so that the light is incident on the microscope.