JPH03188408A - Scanning type optical microscope - Google Patents

Abstract

PURPOSE:To observe an image by light which is transmitted through a sample in spite of a system for scanning the fixed sample with a condensing point itself and forming the image by disposing a reflection optical system for receiving the light which is transmitted through the sample and returning it toward the sample so that the light may pass through the same path as an incident light path. CONSTITUTION:The luminous flux of the illuminating light projected from an objective lens 4 is condensed on the surface of the sample 5 so that scanning may be performed. The luminous flux is transmitted through the sample 5 so as to be made incident on the reflection optical system which is constituted of a convex lens 11 whose focusing position coincides with the surface of the sample 5 and a reflection mirror 12 such as a corner cube, etc., and then, the luminous flux is reflected so as to reverse through the very same optical path as that of the incident time. And the sample 5 is illuminated again from underneath with the luminous flux reflected on the optical system, and then, the condensing point is formed on the surface of the sample 5 and the scanning is performed. And then, the luminous flux is transmitted through the sample 5 and the very same path as the luminous flux of reflected light on the condensing point which is made on the sample 5 is traced by the luminous flux, at a final stage, it is received by a photodetector 8 so that the sample image may be formed. Thus, it is made possible to observe the image by the light which is transmitted through the sample in spite of the system for scanning the fixed sample with the condensing point itself and forming the image.

Description

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

[Conventional technology]

A scanning optical microscope focuses light emitted from a light source onto a specimen through an objective lens, and then reflects or transmits light from this focal point again through the same or different objective lens. This device collects light, places a pinhole at the focal point, and photoelectrically detects the light that passes through the pinhole to obtain a specimen image. However, in order to obtain a specimen image, it is necessary to two-dimensionally scan the focal point formed on the specimen, and there are roughly two scanning methods. One method is to fix the specimen and scan the focal point, and the other method fixes the focal point and scans the specimen itself, which essentially achieves the same effect as scanning the focal point. This is the way to obtain it.

First, the principle of a scanning optical microscope that scans the focal point itself will be briefly explained using FIG. 1θ.

In the figure, ■ is a laser light source, 2 is a beam splitter, 3 is an optical scanning optical system, 4 is an objective lens, 5 is a specimen, 6 is a collimator lens, 7 is a pinhole, and 8 is a photoelectric detector.

The light emitted from the light source 1 is reflected by the beam splitter 2, passes through the optical scanning optical system 3, and enters the objective lens 4, creating a condensing point that is scanned over the specimen 5. The light at the focal point is reflected by specimen 5,
The light travels backward through the objective lens 4 and the light scanning optical system 3 again, passes through the beam splitter 2, and heads toward the collimator lens 6. Since this light flux has traveled back and forth through the optical scanning optical system 3, its path is stable in terms of time, and therefore the collimator lens 6
The light is focused on one point on the optical axis. A pinhole 7 is placed at this focal point, and the light passing through the pinhole 7 is received by a photoelectric detector 8. A sample image is obtained by appropriately processing the electrical signal output from the photoelectric detector 8.

Next, the principle of a scanning optical microscope that scans the specimen itself will be briefly explained using FIG. 11. Compared to the one in FIG. 10, there is no optical device optical system 3, and instead, the specimen 5 is placed on a two-dimensional scanning stage 9, so that the specimen 5 itself is scanned. The other principles are the same as the scanning optical microscope of the type that scans the focal point itself as explained using FIG. 10. Furthermore, using FIG. 12, the principle of another scanning optical microscope that scans the specimen itself will be briefly explained.

A condensing point is created on the specimen 5 using the same principle as in FIG. 11, and the specimen 5 itself is scanned. However, the second objective lens lO receives the light that has passed through the specimen 5 instead of the light that has been reflected, thereby creating a focal point on the optical axis. A pinhole 7 is placed at this focal point,
After that, an image of the specimen is created using the same principle as the scanning optical microscope described above.

[Problems that the invention attempts to solve]

However, the scanning optical microscope of the type that scans the specimen itself as described using FIGS. 11 and 12 requires extremely highly accurate stage scanning in order to obtain high resolution. Furthermore, there are drawbacks such as limitations on the weight and size of the specimen 5 and a longer scanning time.

On the other hand, the scanning optical microscope that scans the focal point on the specimen as explained using Figure 1O does not have these drawbacks, but the scanning optical microscope (transmitted illumination type) explained using Figure 12 does not have these drawbacks. The disadvantage is that unlike a microscope, it is not possible to create an image using light that passes through the specimen 5, but only with reflected light.

Furthermore, in the scanning optical microscope shown in FIG. 12, if the light scanning optical system 3 is introduced in front of the first objective lens 4 and after the second objective lens 10, in principle, the specimen 5 can be fixed and the light can be focused. Although it is possible to create a transmitted illumination type scanning optical microscope that scans the point itself, it requires extremely strict synchronization of the two light scanning optical systems, and two objective lenses. It is technically extremely difficult to match the magnification of the IO with extremely high accuracy.Therefore, when observing general biological specimens, there is a strong demand for viewing images using transmitted illumination (i.e., when observing general biological specimens, In many specimens, the specimen is inserted between two glass plates with a suitable mounting medium, and such specimens have poor light reflectance, so when performing microscopic observation, it is necessary to reflect the illumination light off the specimen. Since it is not possible to obtain a satisfactory image using the viewing method, we scan the focal point itself on the specimen, even though it is necessary to transmit the illumination light for observation.
A transmitted illumination type scanning optical microscope that can obtain an image using transmitted light is not currently in practical use.

In view of the above-mentioned problems, the present invention is a scanning type optical system that uses a method to create an image by scanning the condensing point itself over a fixed specimen, but which enables observation of the image using light that passes through the specimen. The aim is to provide microscopes.

[Means and effects for solving the problems] One of the scanning optical microscopes according to the present invention includes a light source, a scanning optical system that scans the light emitted from the light source, and a scanning optical system that scans the light emitted from the scanning optical system. an objective lens that focuses light on the sample; a reflective optical system that receives the light that has passed through the sample and returns the light to the sample through the same path as the incident path; and a reflection optical system that focuses the light on the sample again. The present invention is characterized in that it includes a photoelectric detector that receives the returned light via the objective lens and the scanning optical system.

That is, as shown in FIG. 1, this is the scanning optical microscope shown in FIG. 10 with a predetermined optical system added below the specimen 5. This predetermined optical system includes an appropriate lens 11 such as a positive lens whose front focal point coincides with the surface of the specimen 5.
and a reflecting mirror 12 such as a corner cube, and has the function of reflecting the incident light beam so that it travels backward along exactly the same optical path as the incident light beam.

Accordingly, the illumination light flux emitted from the objective lens 4 is focused on the surface of the specimen 5 and scanned using the same principle as that of the scanning optical microscope explained using FIG. This luminous flux is sample 5
and enters the predetermined optical system. The light beam reflected by this optical system illuminates the specimen 5 from below again, forms a condensing point on the surface of the specimen 5, and scans it. moreover,
This light beam passes through the specimen 5 and travels backward on the outward path. The light flux that travels backwards along the same optical path is as explained using Fig. 10 (
, follows exactly the same optical path as the reflected light beam from the condensing point formed on the specimen 5, and is ultimately received by the photoelectric detector 8, forming a specimen image. However, this specimen image is significantly different from the scanning optical microscope described in FIG. 1O in that it is created by light transmitted through the specimen 5, and this scanning optical microscope functions as a transmitted illumination type.

Another aspect of the scanning optical microscope according to the present invention includes a light source;
a scanning optical system that scans the light emitted from the light source; an objective lens that focuses the light emitted from the scanning optical system at a position shifted from the specimen; It is characterized by comprising a reflective optical system that focuses light on the specimen, and a photoelectric detector that receives the light that has been focused on the specimen and then returns through the objective lens and the scanning optical system. There is.

In other words, as long as the light reflected by the reflective optical system is focused on the specimen, the light emitted from the objective lens does not necessarily have to be focused on the specimen; on the contrary, the light emitted from the objective lens is not necessarily focused on the specimen. By shifting the light spot above the specimen, the reflected light on the specimen surface can be weakened and the contrast of the observed image can be improved.

〔Example〕

Hereinafter, the present invention will be described in detail based on the illustrated embodiments.

FIG. 2 shows an optical system of a first embodiment of a scanning optical microscope according to the present invention.

21 is a laser light source, 22 is a positive lens, 23 is a pinhole whose center is placed at the back focal position of the positive lens 22;
Reference numeral 24 denotes a positive lens whose front focal point is placed at the center of the pinhole, and these constitute an optical system that converts the light from the laser light source 21 into a parallel beam.

25 is a linear polarizing element that transmits only the S-polarized component of the parallel light beam, and 26 is a polarizing beam splitter that reflects the S-polarized component and transmits the P-polarized component. Reference numeral 27 denotes a deflection element such as a galvanometer mirror or an acousto-optic element that polarizes the direction of the incident light beam in the direction of the plane of the paper, and 28 and 29 are arranged so that the rear focal position of the former and the front focal position of the latter coincide. Two positive lenses make up the afocal optical system, 30 is a deflection element such as a galvanometer mirror or an acousto-optic element that deflects the direction of the incident light beam in a direction perpendicular to the plane of the paper, 31.32 is the rear side of the former Two positive lenses are arranged so that the focal position of the latter coincides with the front focal position of the latter, and constitute an afocal optical element. This constitutes a light scanning optical system that two-dimensionally scans the light beam while keeping it as a parallel light beam. Reference numeral 33 indicates an entrance pupil of a first objective lens to be described later, 34 indicates a first objective lens, and 35 indicates a specimen whose surface coincides with the rear focal plane of the first objective lens 34. Note that the two deflection elements 27.30 are in a conjugate relationship with each other with respect to the afocal optical system consisting of a positive lens 28.29, and furthermore, the entrance pupil 33 of the deflection element 30 and the first objective lens 34 is connected to the positive lens 31. They are in a conjugate relationship with each other with respect to the afocal optical system consisting of 32. That is,
The two deflection elements 27, 30 and the entrance pupil 33 of the first objective lens 34 are in a conjugate relationship with each other. Therefore, the parallel light beam emerging from the light scanning optical system is transmitted to the entrance pupil 33.
The direction changes two-dimensionally around the center of the specimen 35, and a focal point is created on the surface of the specimen 35 by the action of the first objective lens 34, and this is scanned two-dimensionally. Also, - Entrance pupil 33 for boat fishing
coincides with the front focal plane of the first objective lens 34, so the light ray passing through the center of the light beam forming the above focal point is:
parallel to the optical axis of the first objective lens 34, i.e. the specimen 35
perpendicular to the plane of

Reference numeral 36 denotes a second objective lens whose front focal plane is aligned with the surface of the specimen 35, and the light beam that has made a focal point on the surface of the specimen 35 passes through the specimen 35 and then passes through the second objective lens. 36, the light beam is again converted into a parallel light beam. 37 is a 1/4λ plate, and 38 is a corner arranged so that the position where the central ray of the light beam transmitted through the 1/4λ plate 37 crosses the optical axis, that is, the exit pupil position of the second objective lens 36 and the apex P coincide. These cubes constitute a reflective optical system. Furthermore, if the light beam incident on the corner cube 38 is incident perpendicularly to the surface of the specimen 35, the apex P of the corner cube 38 must coincide with the rear focal point of the second objective lens 36. Must be.

Therefore, the light flux that enters and is reflected by the corner cube 38 travels backward along exactly the same optical path as the incident optical path, as shown in FIG. 3(A). In addition, if the above arrangement is incorrect,
The outgoing path and the incoming path of the light beam incident on the corner cube 38 and reflected are shifted as shown in FIG. 3(B). Also, 1
The S-polarized linearly polarized light flux that passed through the /4λ plate 37 in a round trip is
Since a phase difference of 1/4λX2=1/2λ is given, P
The polarized light is converted into a linearly polarized light beam.

39 is a collimator lens that condenses the parallel light beam that has passed through the polarizing beam splitter 26; 40 is a pinhole placed as necessary at the back focal position (focusing point on the optical axis) of the collimator lens 39; 41; is a photoelectric conversion element that receives the light beam passing through the pinhole 40, and the electric signal of the photoelectric conversion element 41 is processed by a signal processing circuit (not shown) and visualized as a specimen image.

Since the present embodiment is constructed as described above, the light emitted from the laser light source 21 is focused at the center of the pinhole 23 by the positive lens 22, and the light beam passing through this becomes a parallel light beam by the positive lens 24. Only the S-polarized light component passes through the linear polarizing element 25 and enters the polarizing beam splitter 26. Next, the parallel light beam consisting only of the S-polarized light component is reflected by the polarizing beam splitter 26, and is two-dimensionally scanned as a parallel light beam by a light scanning optical system consisting of a deflection element 27 to a positive lens 32, and then After passing through the beam, the light is focused by a positive lens 34 and scanned two-dimensionally while creating a focusing point on the surface of the specimen 35. - The first condensed light beam passes through the specimen 35, is converted into a parallel light beam again by the second objective lens 36, and is reflected by the corner cube 38 after passing through the 1/4λ plate 37. The light beam reflected by the corner cube 38 travels backward along exactly the same optical path as the incident light beam passed through, and when it passes through the 1/4λ plate 37 again, it becomes a parallel light beam consisting only of the P-polarized component, and passes through the second objective lens 36. The light is focused from behind the specimen 35 and is made to enter the specimen 35 to form a focal point on the surface of the specimen 35. Of course, at this time, the focal point is two-dimensionally scanned. Then, the light flux is transmitted from the first objective lens 34 to the polarizing beam splitter 26.
Follow the optical path in the opposite direction to polarizing beam splitter 2.
6, the light beam enters the collimator lens 39 and is focused at the rear focal position, that is, the center of the pinhole 40, and the light beam passing through the center is received by the photoelectric conversion element 41. Thereafter, the electrical signal from the photoelectric conversion element 41 is processed by a signal processing circuit (not shown) and visualized as a specimen image.

Thus, according to this embodiment, although the condensing point itself scans the fixed specimen 35 to create an image, it is possible to observe the image using the light transmitted through the specimen 35.

Incidentally, a scanning optical microscope in which the pinhole 40 is arranged is generally called a confocal scanning optical microscope. Although the features of this microscope are well known, only the effects will be briefly explained. - In a fishing optical microscope, the depth of focus is extremely shallow because observation is performed using an objective lens with a large numerical aperture, and if the specimen is thick, the observed image will be a blurred image of the specimen before and after focusing on the focused image of the specimen that you really want to observe. Since the images overlap, the effective resolution deteriorates. However, with this confocal scanning optical microscope, only the in-focus plane can be observed brightly, and the images in front and behind it are dark, so extremely good resolution can be obtained not only for thin specimens but also for thick specimens. , is very effective in observing biological specimens.

By the way, the rear focal point position of the second objective lens 36 must be aligned with the apex (point P) of the corner cube 38, so it must be relatively far downward from the lower end surface of the second objective lens 36. There must be. Therefore, if necessary, the configuration of the second objective lens 36 may be adjusted to be positive as shown in FIG.

It is preferable to use a second group configuration consisting of a first group positive lens 36a and a second group positive lens 36b having a positive focal length. The front focal position of the first group positive lens 36a is placed slightly below the surface of the specimen 35, and a conjugate position (point Q) with the surface of the specimen 35 is created below the first group positive lens 36a. Second group positive lens 36b
The front focal position of is made to coincide with this conjugate position. As a result, the front focal position of the entire system of the two positive lenses 36a and 36b coincides with the surface of the specimen 35. That is, one condition required for the second objective lens 36 is satisfied.

Furthermore, due to this arrangement, the rear focal position (point R) of the first group positive lens 36a is naturally closer to the first group positive lens 36a than the conjugate position (point Q), so the second group positive lens 36b
Therefore, this conjugate position (point S) is created below the rear focal position (point Q') of the second group positive lens 36b. That is, this position is the rear focal position (point P) of the entire system of the two positive lenses 36a and 36b, and this position is the position of the second group positive lens 36.
By increasing the focal length of b, two positive lenses 3
6a and 36b can be easily separated from the entire system. In this way, by using a lens with a two-group configuration,
Both of the two conditions required for the second objective lens 36 can be satisfied. Furthermore, as shown in FIG. 5, the second group positive lens 36b is composed of a negative lens 36b' and a positive lens 36b', and by moving the rear focal point further backward, the entire system of these lenses can be improved. Further separation of the back focus position can be achieved. Moreover, if the rear focal positions of all these lens systems are the same, the entire lens system can be constructed compactly.

FIG. 6 shows the main parts of the optical system of the second embodiment.
In the embodiment, the corner cube 38 was used as a reflecting member for reversing the parallel beam transmitted through the specimen 35 and produced by the second objective lens 36, whereas in this embodiment, a collimator lens 42 and a plane mirror 43 were used. The difference is that they are used in combination. The collimator lens 42 is arranged so that the front focal position of the collimator lens 42 coincides with the position where the central ray of the light beam exiting the second objective lens 36 crosses the optical axis. This makes the central ray parallel to the optical axis, but in other words, it can be said that the exit pupil of the second objective lens 36 is projected by the collimator lens 42 to an infinite distance. Further, the plane mirror 43 is arranged so that the opposite slope of the plane mirror 43 is located at a position that is conjugate with the surface of the specimen 35 with respect to the second objective lens 36 and the collimator lens 42 . As a result, the light flux that forms a point image on the specimen 35 and passes through it forms a point image again on the surface of the plane mirror 43 and is reflected. Moreover, since the central ray of the light beam is perpendicularly incident on the surface of the plane mirror 43, as shown in FIG.
The reflected light beam travels in the opposite direction along exactly the same optical path that the incident light beam has passed through, and transmits and illuminates the specimen 35 from behind as in the first embodiment. If the central ray of the light beam exiting the first objective lens 34 passes through the surface of the specimen 35 perpendicularly, the front focal position of the collimator lens 42 will be the rear focal point of the second objective lens 36. Just place it so that it matches the position. Note that the function of the 1/4λ plate 37 is exactly the same as in the first embodiment.

In addition, the collimator lens 42 and the plane mirror 4 in this embodiment
It is also possible to construct lens 3 and 3 with a single plano-convex lens 43' as shown in FIG. That is, in the plano-convex lens 43' shown in FIG. 7, the first surface 43'a, which is a convex surface, has positive refractive power, that is, has the same effect as the collimator lens 42 shown in FIG. The second surface 43'b is coated with a thin aluminum film and has the same function as the plane mirror 43 in FIG.
This will serve both of the functions of 3. Therefore, it is possible to perform transmitted illumination in the same manner as in FIG. 6 with a simpler configuration.

FIG. 8 shows the main parts of the optical system of the third embodiment.
In the embodiment, the corner cube 38 was used as a reflection member for reversing the parallel light beam transmitted through the specimen 35 and produced by the second objective lens 36, whereas in this embodiment, a concave mirror 44 was used to reflect the parallel light beam. The difference is that the convergence point of the light by the second objective lens 36 is made to coincide with the surface. The concave mirror 44 is disposed at a position where its reflective surface is conjugate with the surface of the specimen 35 with respect to the second objective lens 36, and the curvature of the reflective surface is such that the central ray of the light beam exiting the second objective lens 36 aligns with the optical axis. The center of the radius of curvature R is selected to coincide with the crossing position, that is, the exit pupil position 36a of the second objective lens 36. As a result, the light flux that forms a point image on the specimen 35 and passes through it forms a point image again on the surface of the concave mirror 44 and is reflected.

Furthermore, since the central ray of the light beam is perpendicularly incident on the surface of the concave mirror 44, the reflected light beam travels backward along the same optical path as the incident light beam passed through, as shown in FIG. Similarly, the specimen 35 is illuminated from behind. If the first
The central ray of the light beam exiting the objective lens 34 is the specimen 35.
When the light is perpendicularly transmitted through the surface of the concave mirror 44, the curvature may be selected so that the center of curvature of the concave mirror 44 coincides with the rear focal position of the second objective lens 36. In addition, 1/4λ plate 3
The function of 7 is exactly the same as in the first embodiment.

In the three embodiments described above, in order to prevent the problem of the reflected light from the surface of the specimen 35 entering the photoelectric conversion element 41 and reducing the contrast of the transmitted observation image of the specimen 35, the linear polarizing element 25. Polarizing beam splitter 26. Although a 1/4λ plate 37 is used, there is a configuration that can prevent a decrease in contrast without using these. In either embodiment, this is done by moving the optical system beyond the surface of the specimen 35 somewhat along the optical axis or away from the optical axis.

FIG. 9 shows, as a fourth embodiment, the main parts of an optical system in which the same thing is implemented in the second embodiment. That is, the position of the point image created by exiting the first objective lens 36 is not at the surface of the specimen 35, but at a position shifted vertically from there, and is reflected by the plane mirror 43 to illuminate the specimen 35 from behind. A point image is formed on the surface of the specimen 35. By doing this, the luminous flux density of the illumination light flux that passes through the surface of the specimen 35 from top to bottom becomes thicker (reduced), so that the contrast caused by the reflected light on the surface of the specimen 35 does not substantially become a problem. This can be understood from the characteristics of a confocal scanning optical microscope.

Note that the point image position of the light beam exiting the first objective lens 34 and the pinhole 23 must be conjugate with respect to the optical system between them, and the surface of the specimen 35 and the pinhole 40 must also be conjugate with respect to the optical system between them. Therefore, when the position of the plane mirror 43 is shifted as in this embodiment, the position of the pinhole 23 or 40 must also be shifted somewhat along the optical axis accordingly.

The transmitted illumination type scanning optical microscope according to the present invention has been described above, but the one thing that can be said in common with the transmitted illumination type scanning optical microscope according to the present invention is that it can easily be used as a reflected illumination type scanning optical microscope. It is. For example, if the second objective lens 36 is pushed away or its position is significantly moved downward or upward, the reflected light of the corner cube 38 will not be able to travel backwards correctly, and the 1/2 A 1/4λ plate having the same function as the 4λ plate 37 may be inserted so that the reflected light beam from the surface of the specimen 35 can be efficiently transmitted through the polarizing beam splitter 26 as a P-polarized light beam.

〔Effect of the invention〕

The scanning optical microscope according to the present invention has a practical importance in that it is a system in which an image is created by scanning a fixed specimen with a condensing point itself, but it is possible to observe an image using light that passes through the specimen. It has many advantages.

[Brief explanation of drawings]

Fig. 1 is a conceptual diagram of the optical system of the scanning optical microscope according to the present invention, Fig. 2 is a diagram showing the optical system of the first embodiment, and Fig. 3 is a diagram illustrating the principle of operation of the corner cube of the first embodiment. , 4th
5 and 5 respectively show the main parts of the optical system of a modification of the first embodiment, FIG. 6 shows the main parts of the optical system of the second embodiment, and FIG. 7 shows the main part of the optical system of the second embodiment. 8 is a diagram showing the main parts of the optical system of the third embodiment. FIG. 9 is a diagram showing the main parts of the optical system of the fourth embodiment. 10 to 12 are conceptual diagrams of conventional optical systems. 1.21...Laser light source, 2...Beam splitter, 3...Light scanning optical system, 4...Objective lens, 5.35...Specimen, 11... Lens, 12.
...Reflector, 22...Positive lens, 23.40...
...Pinhole, 24.28.29.31.32...
・Positive lens, 25...Linear polarizing element, 26...
Polarizing beam splitter, 27.30... deflection element,
33... Entrance pupil, 34... First objective lens,
36... Second objective lens, 37... Bow/4λ plate, 38... Corner cube, 39.42...
Collimator lens, 41... Photoelectric conversion element, 43...
...Plane mirror, 43'...Plano-convex lens. 1'P3 Figure (A) (B) Figure 4 Figure O8 Figure 1P12 Figure

Claims (2)

[Claims] (1) A light source, a scanning optical system that scans the light emitted from the light source, an objective lens that focuses the light emitted from the scanning optical system onto a specimen, and a light source that receives the light transmitted through the specimen and collects the light. a reflective optical system that returns the light to the specimen through the same path as the incident path; and a photoelectric detection system that receives the returned light after condensing it again on the specimen through the objective lens and the scanning optical system. A scanning optical microscope equipped with an instrument and a (2) A light source, a scanning optical system that scans the light emitted from the light source, an objective lens that focuses the light emitted from the scanning optical system at a position offset from the specimen, and a lens that receives the light that has passed through the specimen. A reflective optical system that focuses the lever light onto the specimen, and a photoelectric detector that receives the light that has been focused on the specimen and then returns through the objective lens and the scanning optical system. scanning optical microscope.