JPS61272714A - Scanning type optical microscope - Google Patents

Abstract

PURPOSE:To obtain a confocal type image for detecting transmission by arranging a peak value detection type photodetector array on an object image surface or near the image surface so that a peak value can be detected only when dot-like light reaches just above each photodetector element of the photodetector array. CONSTITUTION:An optical beam 10 from a laser is made incident upon the 1st light deflector 14 and the deflected optical beam 15 is made incident upon the 2nd light deflector 18 through pupil transmission lenses 16, 17. These light deflectors 14, 18 are used for horizontal and vertical scanning and a fine spot scans the surface of a sample 21 two-dimensionally as rasters. The optical beam transmitted through the sample 21 forms fine spots 28, 29 on an image surface 27 through a condenser lens 25 and an image forming lens 26. If the peak value detection type photodetector array 30 is arranged on the image surface 27, each photodetector element 32 detects only the quantity of light obtained when the fine spot 31 is located just above the element 32. Thus, the confocal type image can be obtained.

Description

[Detailed description of the invention] [Industrial application field] The present invention relates to a scanning optical microscope.

[Conventional technology]

Conventional general microscopes use a light source and an appropriate condenser lens to illuminate the entire observation area of a sample to be observed as uniformly as possible, and also use an objective lens to magnify the sample image and observe or photograph it through an eyepiece. However, illumination of the entire observation area causes a lot of flare, etc., making it impossible to achieve the theoretical resolution limit despite conventional efforts, and it is difficult to obtain low-contrast samples. It was hard to see.

Therefore, a point light projection type microscope was proposed in order to solve the drawback of the conventional optical microscope mentioned above, which is that the theoretical resolution limit cannot be achieved due to flare etc.
ed I+mage Microscopy, E, A
, Ash.

Acade+sic Prees 1980). Among these methods, the so-called confocal method is especially excellent. This method uses a point light source to irradiate the observation sample in a point-like manner, and then the transmitted light or reflected light from the irradiated sample is re-imaged into a point-like shape, and a detector with a pinhole aperture is used to obtain density information of the image. This is what I did. However, since this method only provides density information at the point irradiated with point light, mechanical raster scanning of the sample in two dimensions (X-Y) and synchronized CR
It was designed to form an image on T and observe it. This method of illuminating a sample with point-shaped light and detecting it with a point-shaped detector is called a confocal type.

This microscope will be explained based on an example described in US Pat. No. 3,013,467. FIG. 8 is a schematic diagram of the same, in which a point light source is formed by the light source 1 and the pinhole 2, and the point light source is imaged as a point on the sample 4 by the objective lens 3 whose aberrations are well corrected. Illuminate 4. Further, the point light on the sample 4 is again imaged as a point on the pinhole 6 by the condenser lens 5 whose aberrations are well corrected, and the formed point light is detected by the detector 7 through the pinhole 6. On the other hand, the drive circuit 8 mechanically scans the sample 4 in two dimensions of X-Y like raster scanning on a television.

By synchronizing the image signal from the detector 7 with the synchronizing signal from the drive circuit 8 and displaying it on the storage type CRT 9, the image of the sample 4 can be observed.

In this way, the sample is illuminated with point light and the signal is detected with a point detector, which results in better images with less flare and improved resolution compared to a normal detector.
Because it is a method that moves the sample mechanically and scans it, there are problems such as inconvenience.For example, the sample is limited to a limited size and is light, or a culture specimen in a petri dish etc. No unfixed samples could be observed. or,
It has also been difficult to apply this method to systems that continuously observe different samples (eg, flow cytometry).

Therefore, in order to solve the above-mentioned drawbacks caused by mechanically moving the sample, a method of scanning the point light itself in two dimensions was considered, but in this case, there is no need to detect the light reflected from the sample. Although there was no problem, it was difficult to detect the light transmitted through the sample using a confocal method.

Furthermore, in order to solve this problem, the mirror that scans the point light and the mirror that scans the light transmitted from the sample are synchronized so that the light that has passed through the sample is always re-imaged on the point detector. A method using a point light scanning mirror is proposed in U.S. Pat. No. 3,705,755, but a point light scanning mirror is used to detect the point light focused to the diffraction limit so that the point light does not move even slightly with a detector having a smaller pinhole. It was not possible to completely synchronize the scanning mirror.

[Problem that the invention seeks to solve]

As described above, with the conventional method of scanning point light, it has been impossible to realize confocal transmission detection.

SUMMARY OF THE INVENTION In view of the above-mentioned problems, it is an object of the present invention to provide a scanning optical microscope capable of realizing confocal transmission detection using a point light scanning method.

[Means and effects for solving the problems] The scanning optical microscope according to the present invention has a peak value detection type photodetector array arranged at or near the object image plane, so that point light is detected by the photodetector array. Detection is performed only when the photodetector element is exactly above the photodetector element.

〔Example〕

The present invention will be described in detail below based on an embodiment shown in FIGS. 1 to 3. Figure 1 shows an optical system that scans a light beam as point light.This is an optical system that takes the pupil into consideration, and even when the light beam is scanned by an optical deflector, the optical axis is not aligned in the scanning system. It is kept constant.

A light beam 10 from a laser, which can be equivalently considered as a point □ light source, passes through a beam splitter 11 and enters a first optical deflector 14 placed at a position conjugate with a pupil 13 of an objective lens 12 . The light beam 15 deflected by this optical deflector 14 passes through the pupil transmission laser 16.17 and enters the second optical deflector 18 placed at the pupil position. Used for horizontal and vertical scanning. Next, the pupil projection lens 19. The light passes through the imaging lens 20 and enters the pupil 13 of the objective lens 12 . The off-axis light beam formed by the optical deflector 14.degree. 18 also accurately enters the pupil 13 of the objective lens 12 because its direction and center are in the same direction as the off-axis chief ray of the objective lens 12. These light beams produce minute spots on the sample 21 that are limited by diffraction by the objective lens 12. By deflecting the light beam with the light deflectors 14 and 18, the minute spot raster-scans the sample 21 two-dimensionally. The light reflected from the sample 21 is transmitted through the objective lens 12. The imaging lens 20 etc. return in the opposite direction along the same path and reach the beam splitter 11.
A condensing lens 23 condenses the light onto a pinhole 24 .

Since this reflected light (detection beam) returns through the optical deflector 14, 18, it does not move even when scanning off-axis, and is always focused on the pinhole 24. In this way, even with the light beam scanning method, a confocal type can be easily realized in reflection detection.

In addition, the light beam that has passed through the sample 21 forms a minute spora on the image plane 27 by the condenser lens 25 and the imaging lens 26.
yields 28.29. 28 is an on-axis spot, and 29 is a spot when scanning off-axis. As described above, in transmission detection, since the spot position moves along with scanning, it is difficult to detect with a pinhole, and confocal detection is therefore impossible. However, if a peak value detection type photodetector array 30 is placed on the image plane 27, confocal type detection can be realized as described in detail below.

FIG. 2 shows how the minute spot 31 of the detection beam moves on the photodetector array 30 according to off-axis scanning. When the photodetector array 30 is of the peak value detection type, the photodetector element 32 detects only the amount of light when the minute spot 31 is exactly above the photodetector element 32 as shown by the dotted line, that is, when it is at its brightest. It will be detected. This is exactly the same as confocal detection in which only the central portion of a minute spot 33 is detected using the pinhole 34 as shown in FIG. Therefore, when the photodetector array 30 is of the peak value detection type, all the photodetector elements 32 of the photodetector array 30 perform pinhole detection, and a confocal type image can be obtained. If the photodetector array 30 is a commonly used so-called CCD type image sensor.

In the case of an accumulation type image sensor such as an MO3 type image sensor, the entire amount of light while the minute spot 31 passes through the photodetector element 32' is accumulated.

Therefore, this is the same detection as in FIG. 3 except for the pinhole 34, and is no longer confocal detection.

As described above, if a peak value detection type photodetector array is provided at the reimaging position in transmission detection of a light beam scanning type scanning optical microscope, a confocal type can be realized.

FIG. 4 shows, as a specific example of the above-mentioned embodiment, an optical system of a scanning type optical W4 microscope which can also be used for observation using an ordinary microscope.

The laser beam from the laser light source 36 passes through the condensing lens 3
7. Spatial filter 38, collimator lens 39
．． The beam passes through a beam splitter 40 and enters a galvanometer mirror 42 placed at a position conjugate with the pupil position of an objective lens 41 .

Here, the laser beam is deflected and horizontally scanned.

Next, through pupil transmission lenses 43 and 44, the light enters a galvanometer mirror 45, which is also provided at a position conjugate with the pupil position of the objective lens. Here the laser beam is deflected and scanned in the vertical direction.

Although galvanometer mirrors 42 and 45 are shown in the drawings as if they both deflect the laser beam in the same direction, they actually perform horizontal and vertical scanning, respectively. The two-dimensionally scanned laser beam passes through the pupil projection lens 46.
The light passes through the imaging lens 47 and enters the pupil of the objective lens 41. Then, a minute spot limited by diffraction is generated on the sample 48, and the sample 48 is scanned two-dimensionally. Here, when performing scanning observation, the prism 49 for visual observation and the beam splitter 50 for epi-illumination are removed from the optical path. Otherwise, the laser beam will hit your eyes, which can be dangerous and cause flares.

In the case of reflection detection, the light from the sample 48 returns through the same path as when it entered the sample 48, is reflected by the beam splinter 40, is focused on the pinhole 52 by the condensing lens 51, and is detected by the detector 53,
Confocal images can be obtained.

In the case of transmission detection, the collector lens 54. A scanning image of the sample 48 is projected by the imaging lens 55 onto a peak value detection type photodetector array 56 .

That is, the minute spot moves in a raster pattern on the photodetector array 56 in accordance with the scanning of the galvanometer mirrors 42 and 45.

FIG. 5 shows an example of a peak value detection type photodiode array, which is shown here in one dimension for simplicity. When light hits the photodiode element 57, a current is generated, and its peak value is connected to the diode 58 and capacitor 5.
The capacitor 59 is
is memorized.

Then, the scanning circuit 60 switches the switching FET 6
1 is turned on, the peak value is output through the output amplifier 62.

Peak value detection type photodetector array 5 shown in FIG.
6 is a two-dimensional element. Note that the size of the elements of the photodetector array is preferably smaller than a minute spot in order to perform confocal detection.For example, in FIG. 2, the size of the minute spot 31 is preferably larger than the photodetector element 32. If NA' is the actual numerical aperture on the image side of the imaging lens 55 in FIG. It is desirable that the spacing be smaller than 4NA. If the spacing is larger than this, even if you try to make full use of the resolving power of the optical system by making it confocal, the spacing will be too large and the ability of the optical system will not be fully used. It is from.

The video signal from the peak value detection type photodetector array is
The image is input to an image memory or the like in synchronization with the scanning of the galvanometer mirror and displayed on the CRT.

Also, as a matter of course, the two-dimensional scanning position of the laser beam and the photodetector elements of the peak value detection type photodetector array must be accurately aligned.

Next, as another specific example, an example in which an acousto-optical deflection element is used as the optical deflection element will be described. The galvanometer mirror used in the above specific example can only speed up scanning synchronization to about several hundred Hz, and it takes time on the order of seconds to obtain a two-dimensional image, making real-time observation impossible. In that respect, the acousto-optical deflection element is the same as that of a general NTSC TV.
Since it is also possible to scan at 5 KHz, real-time observation is possible.

FIGS. 6A and 6B show a front view and a side view of the optical system. A laser beam 70 from a light source (not shown) is transmitted to an acousto-optic deflection element 71 placed at the pupil position of the optical system.
incident on . The beam 72 diffracted by the acousto-optical deflection element 71 is reflected by the adjustment mirror 73 and becomes the beam 7.
4 and enters the pupil transmission lens 75. Beam 74 reflected by mirror 76 passes through pupil transmission lens 77 and becomes beam 78 . The laser beam 78 is diffracted into a beam 80 by an acousto-optic deflection element 79 placed at the pupil position. Beam 80 is reflected by adjustment mirror 81 to become beam 82 and enters pupil projection lens 83 . The beam that has passed through the pupil projection lens 83 enters the pupil of an objective lens (not shown) and forms a spot on the sample. Here, the laser beams 70.72.74, 78.80 in the figure
゜82 represents the center of the beam deflected as axial light, and 1.
b゛h#4311[fit46(7)'? 'fb4・
.

As shown in FIG. 7, the acousto-optical deflection elements 71 and 79 consist of a medium 88 for transmitting sound waves and a piezoelectric element 89. When a high frequency voltage (approximately 100 MH) is applied to the piezoelectric element 89, sound waves are generated in the medium 8B. A diffraction grating is generated, and when a laser beam 90 is incident, a 0th-order diffraction light 91 and a 1st-order diffraction light 92 are generated.By changing the frequency of the high frequency wave applied to the piezoelectric element, the direction of the -order light can be changed from direction 93 to direction 93. 94. This is the optical deflection method in the acousto-optical deflection element.Therefore, the direction corresponding to the optical axis is set as 92, and the off-axis direction is set as 93 or 94.Therefore, in FIG. The external light is deflected in directions 84, 85 above and below the optical axis 72 by the acousto-optical deflection element 71.The pupil transmission lenses 75, 77, and 83 are similar to the pupil transmission lenses 16, 17, and 19 in FIG. The two acousto-optical deflection elements 71 and 79 placed at the pupil position correspond to the first
X corresponds to optical deflectors 14 and 18 in the figure, respectively.

Scan the laser beam in the Y direction. As a result, the laser beam is scanned in a raster pattern over the trial soil.

From the viewpoint of adjustment of the optical system, when the optical system is arranged three-dimensionally, it is desirable that the optical axes of the optical system are perpendicular or parallel. However, the acousto-optic deflection element 7
1 has an angle θ that is not 90° with respect to the incident light 70. For example, the angle θ is about 4°. Then, the laser beam is scanned while changing the diffraction angle by about ±2° before and after this. Therefore, in order to keep the optical axis of the optical system perpendicular, it is preferable to provide an adjustment mirror 73 to reflect the diffracted beam 72 and make it enter the pupil transmission lens 75 as a laser beam 74 perpendicular to the incident laser beam 70. . This is the relationship between the acousto-optical deflection element 79 and the adjustment roller 81,
The same holds true for the relationship between laser beam 78 and laser beam 82.

The lens 86 is a cylindrical lens that corrects the lens effect of the acousto-optical deflection element 79.

The configuration other than the scanning system is the same as that in FIG. 4.

Further, it is desirable that the signal readout from the peak value detection type photodetector array is synchronized with beam scanning, and the signals from each element of the peak value detection type photodetector array are read out a little later than the scanning beam. Moreover, if a microchannel plate (manufactured by Hamamatsu Photonics Co., Ltd., for example) is provided for optical amplification in front of the peak value detection type photodetector array, the energy of the scanning beam can be reduced.

〔Effect of the invention〕

As described above, the scanning optical microscope according to the present invention can realize a confocal type not only in the case of reflection detection but also in the case of transmission detection in the point light scanning method.

Therefore, high-resolution microscopic images can be obtained without limiting the sample.

[Brief explanation of the drawing]

FIG. 1 is a diagram showing the optical system of an embodiment of a scanning optical microscope according to the present invention, and FIG. 2 is a diagram showing the movement of a minute spot on the peak value detection type photodetector array of the above embodiment.
FIG. 3 is a diagram showing an example of detection using a pinhole of a minute spot, FIG. 4 is a diagram showing a specific example of the above embodiment, and FIG. 5 is a diagram showing an example of a peak value detection type photodiode array. FIG. 6 is a diagram showing another specific example of the above embodiment, FIG. 7 is a sectional view of an acousto-optic deflection element, and FIG. 8 is a diagram showing a conventional optical system. 10... Light beam, 11... Beam splitter, 12... Objective lens, 13... Pupil, 140.
・, optical deflector, 15191. Light beam, 16.17...
0. Pupil transmission lens, 180. ...Light deflector, 19...
- Pupil projection lens, 20...imaging lens, 21...
・Sample, 23. . ...Condenser lens, 24...Pinhole, 25...0
．． Condenser lens, 26...Imaging lens, 27
-1...image plane, 28.29...spot, 30...
・Peak value detection type photodetector array. Figure 4 Off diagram O S diagram

Claims (1)

[Claims] a light source; an objective lens for condensing light emitted from the light source onto an object; and an objective lens disposed between the light source and the objective lens for focusing light on the object by changing the incident angle of the light entering the objective lens. 1. A scanning optical microscope comprising: a scanning optical deflection member; and a peak value detection photodetector array disposed at or near an object image plane.