JPH09203862A - Scanning type optical microscope - Google Patents

Abstract

PROBLEM TO BE SOLVED: To provide a scanning type optical microscope which enables a sample to be observed excellently by increasing the S/N of the signal outputted by a photodetecting element by removing a peripheral diffracted wave generated by the edge of the pupil of an objective. SOLUTION: A projection lens 11 is arranged between a beam splitter 2 and the photodetecting element 4 and an image of the stop (pupil) 3a of the objective 3 is formed at a specific position PP. At the formation position PP of the projection image, an aperture plate 12 which has an aperture 13 having a smaller diameter than the projection image is arranged. The peripheral diffracted wave generated at the edge of the stop 3a of the objective 3 is cut off by the aperture plate 12 to prevent the peripheral diffracted wave BDW from being propagated to the photodetecting element 4.

Description

Detailed Description of the Invention

[0001]

BACKGROUND OF THE INVENTION 1. Field of the Invention The present invention relates to a so-called scanning optical microscope.

[0002]

2. Description of the Related Art A scanning optical microscope is configured to illuminate a sample with illumination light from a light source, while receiving light from the sample with a light receiving element via an objective lens.
In particular, while limiting the illumination site on the sample illuminated by the illumination light and / or the detection site detected by the light receiving element to a micro region on the sample, the illumination site and / or the detection site limited to the micro region are scanned. Thus, the images of a plurality of minute regions are sequentially detected to obtain the entire image of the sample.

FIG. 5 is a diagram showing an example of a conventional scanning optical microscope. The illumination site and the detection site are scanned by moving the sample two-dimensionally while limiting the illumination site and the detection site to a minute area. Then, the image of a plurality of minute regions is sequentially detected and a signal is output.

That is, in this scanning optical microscope, the illumination light is emitted from the point light source 1 in the Z direction, and is passed through the beam splitter 2 and the objective lens 3 at a predetermined position in the XY directions.
It is condensed in a spot shape on the sample S which is arranged so as to be scannable in a dimension, and is irradiated onto a minute region on the sample S. Reflected light from this illumination site (a minute area irradiated with illumination light) enters the beam splitter 2 through the objective lens 3, is guided to the light receiving element 4 side by this beam splitter 2, and is the same as the illumination site. The light receiving element 4 detects the image of the part. Therefore, by scanning the sample S two-dimensionally,
Light (reflected light) from each minute area of the sample S is sequentially incident on the light receiving element 4, and signals related to the image of each minute area are sequentially output from the light receiving element 4 to an image processing unit (not shown), and appropriate signals are output. After undergoing the image processing, the entire image of the sample S is displayed on the display (not shown).

Further, in this scanning optical microscope, in the vicinity of the light receiving surface (not shown) of the light receiving element 4, the focal plane FP
Since the pinhole plate 6 on which the pinhole 5 is formed is arranged at a position optically conjugate with, as shown by the solid line in the figure, a minute region which is an illuminated portion is on the focal plane FP. In this case, most of the light (reflected light) from the minute region is incident on the light receiving element 4 through the pinhole 5, and the amount of light (image density) detected by the light receiving element 4 increases. on the other hand,
When the minute area deviates from the focal plane FP, the amount of light incident on the light receiving element 4 is drastically reduced as shown by the broken line in FIG.
The contributions from other sources will be lost. Therefore, when the sample S is scanned, an image of only a specific surface in the three-dimensional sample S, that is, a so-called tomographic image is obtained. That is, the scanning optical microscope of FIG. 5 is a confocal microscope.

[0006]

By the way, in order to satisfactorily observe an image of a sample having a fine pattern, a sample in which only very weak light enters a light receiving element, or a sample of low contrast, in a scanning optical microscope. The light receiving element 4
It is necessary to increase the S / N of the signal from. Particularly, in a confocal microscope, the resolution in the optical axis direction, that is, the Z direction greatly depends on the S / N of the signal, and in order to improve the resolution, the S / N
High signal is required.

Here, the S / N of the signal largely depends on the image forming performance of the optical system and the presence of light such as ghost, flare and scattered light which is not necessary for image formation. As a result, it was found that the presence of the peripheral diffracted wave was significantly involved in the reduction of the S / N in addition to these factors. Hereinafter, the influence of the peripheral diffracted wave on the signal S / N will be described with reference to FIG.

For example, when observing the minute black spots 7 formed on the sample S by a conventional scanning optical microscope, FIG.
An image plane illuminance distribution as shown in is obtained on the light receiving element. It is assumed here that there is no ghost or scattered light.

Considering only the incident light (illumination light) propagating according to the law of geometrical optics, a part of the incident light is shielded by the minute black spots 7, while the rest is reflected by the original surface other than the minute black spots 7, Proceed to the light receiving element side. Therefore, the illuminance distribution in the light receiving element becomes zero, which corresponds completely to the minute black spot 7, as shown by the solid line in the same figure (b), while it has a high value at the position corresponding to the sample surface other than the minute black spot 7. Take
Therefore, the range from the lowest level to the highest level of concentration that can be detected by the light receiving element (corresponding to the dynamic range) is Rg, and a high S / N signal is obtained.

However, the image plane illuminance at the light receiving element is
In reality, the distribution shown by the alternate long and short dash line in FIG.
This is due to the influence of the peripheral diffractive wave (= Boundary Diffraction Wave), and a diffraction image that is the result of the combination of the geometrically incident light and the peripheral diffractive wave is formed on the light receiving element.

The "peripheral diffracted wave" is a diffracted wave generated from the end of light when the light enters the diffractive object. In the scanning optical microscope configured as described above, the diaphragm of the objective lens corresponds to a diffractive object, and peripheral diffracted waves are generated from the edge of the diaphragm. Here, when viewed from the light receiving element side, it seems that peripheral diffraction waves are generated from the edge of the pupil of the objective lens. Regarding this peripheral diffracted wave, for example, "Principle of Optics II (written by Max Born & Emil Wolf, translated by Toru Kusagawa and Eiji Yokota; Tokai University Press)", 67th
This is explained in detail on page 0. However, in "Optical principle II", "Boundary Diffraction Wave" is translated as "boundary diffraction wave", but it is the same as "peripheral diffraction wave".

It should be noted that in an actual objective lens for a microscope, the presence of a diaphragm is often unclear, but since there is a portion that virtually corresponds to the diaphragm, it will be referred to as a diaphragm here.

When a diffracted image is formed by the influence of the peripheral diffracted wave in this way, the image plane illuminance at the position corresponding to the minute black spot 7 does not become zero but increases by ΔR, and in this case the dynamic range Rg + d Is narrower by ΔR than the dynamic range Rg when the peripheral diffracted waves are ignored, and the S /
N decreases.

The present invention is intended to overcome the above-mentioned problems in the prior art, and enhances the S / N of the signal output from the light receiving element by removing the peripheral diffracted wave generated from the edge of the pupil of the objective lens. It is an object of the present invention to provide a scanning optical microscope capable of observing a sample well.

[0015]

According to a first aspect of the present invention, illumination light from a light source is directed toward a sample, while light from the sample is received by a light receiving element via an objective lens. Therefore, while limiting the illumination site of the sample illuminated by the illumination light and / or the detection site detected by the light receiving element to a micro region, the illumination site and / or the detection site limited to the micro region are scanned. A scanning optical microscope that sequentially detects images of the plurality of minute regions to obtain the entire image of the sample, and is arranged between the objective lens and the light receiving element to achieve the above object. A projection means for projecting the image of the pupil of the objective lens to a predetermined position on the side of the light receiving element, and an aperture having a diameter smaller than that of the projection image, the aperture being arranged at a position where the projection image is formed. It has a, and.

[0016]

FIG. 1 is a diagram showing an embodiment of a scanning optical microscope according to the present invention. This scanning optical microscope differs from the conventional example (FIG. 5) in that a projection lens 11 and an aperture plate 12 are arranged between the pinhole plate 6 and the light receiving element 4. Since the other configurations are the same, the same components are designated by the same reference numerals and the description thereof will be omitted.

Illumination light from the point light source 1 is condensed in a spot shape via the beam splitter 2 and the objective lens 3,
A small area on the sample S is irradiated. The light reflected by the minute area (illumination portion) is condensed by the objective lens 3 at the position P of the pinhole plate 6 to form a spatial image of the minute area positioned at the illumination portion.

Further, from this pinhole plate 6 to the light receiving element 4
A projection lens 11 is provided at a position separated by a certain distance on the side, and guides the light passing through the pinhole 5 formed in the pinhole plate 6 to the light receiving element 4 via the aperture 13 of the aperture plate 12. . In FIG. 1, the pinhole 5 is drawn large for convenience, but it is actually small, and in this embodiment, the detection site detected by the light receiving element 4 is limited to a minute region (illumination site). Has been done.

Further, the projection lens 11 projects the image of the diaphragm (pupil) 3a of the objective lens 3 to the position PP on the light receiving element 4 side. The aperture plate 12 is arranged at a position PP where this projected image is formed. As shown in FIG. 2, the aperture plate 12 is provided with an aperture 13 having a diameter D smaller than the projected image I of the diaphragm 3a of the objective lens 3. By setting the diameter D of the aperture 13 in this way, the peripheral diffracted wave BDW generated from the edge of the diaphragm 3a of the objective lens 3 can be blocked by the aperture plate 12. This will be described in detail with reference to FIG.

As shown by the broken line in FIG. 1, the peripheral diffracted wave BDW generated from the edge of the diaphragm 11 propagates to the aperture plate 12 side through the projection lens 11. Where the aperture plate 1
Observing the image I of the aperture 3a formed on the second aperture 2, the aperture 3a
The edges of the image look shiny. The reason why the edge of the diaphragm 3a looks shining in this way is that the peripheral diffracted wave BDW generated from the edge of the diaphragm 3a of the objective lens 3 is apparent from the above description. In this embodiment, since the diameter D of the aperture 13 is smaller than the projected image I of the diaphragm 3a as described above, the peripheral diffracted wave BDW is blocked by the aperture plate 12 and the light receiving element 4 for the peripheral diffracted wave BDW is obtained. Propagation to the side is prevented. Therefore, the peripheral diffracted wave BDW can be prevented from passing through the aperture 13 of the aperture plate 12 and entering the light receiving element 4, and the influence of the peripheral diffracted wave BDW can be eliminated.

Further, the light which has passed through the aperture 13, that is, the light which does not include the peripheral diffracted wave BDW, is incident on the light receiving element 4, and an image signal corresponding to the image of the minute area which coincides with the illuminated portion is output from the light receiving element 4. To be done.

In the above description, the light receiving element 4 detects an image of a minute area existing in the illuminated area and outputs a corresponding image signal, but the sample S is moved by a sample moving means (not shown). Because of the movement, the illumination portion and the detection portion simultaneously and two-dimensionally scan by this movement, sequentially detect the images of the minute regions of the sample S, and output corresponding image signals from the light receiving element 4. Then, the image signal thus obtained is given to an image processing unit (not shown), and after being subjected to predetermined image processing, displayed on a display (not shown).

As described above, according to the scanning optical microscope of this embodiment, the diaphragm (pupil) 3a of the objective lens 3 is formed.
The peripheral diffracted wave BDW generated from the edge of the aperture plate 12
Since the influence of the peripheral diffracted wave BDW is removed by blocking with, the S / N ratio of the image signal output from the light receiving element 4 can be increased, and the sample can be displayed well on the display and observed.

FIG. 3 is a diagram showing another embodiment of the scanning optical microscope according to the present invention. In this scanning optical microscope, illumination light from a planar light source (not shown) enters a Nipkow disk 23 via a polarizer 21 and a beam splitter 22. The Nipkow disk 23 has a plurality of through holes 23a formed in a spiral shape. When illumination light from a light source enters a part of the Nipkow disk 23, the Nipkow disk 23 emits a plurality of lights. Focusing on one of the plurality of emitted lights, the emitted light is
After passing through the / 4 wavelength plate 24, it is condensed in a spot shape by the objective lens 25 and irradiated onto a minute area on the sample S.
Although not shown in the drawing, other emitted lights can similarly enter different small areas and illuminate a plurality of small areas simultaneously and independently of each other.

The Nipkow disk 23 is a motor 26.
Is configured to rotate about a rotary shaft 26a extending in the optical axis direction (Z direction) by a motor 26, and this rotational movement causes a plurality of light beams emitted from the Nipkow disk 23 to move in a horizontal plane (XY direction). ), And minute areas on the sample S are sequentially illuminated.

When illumination light (light emitted from the Nipkow disk 23) is incident on each minute area, it is reflected by the minute area and travels in the same direction as the illumination light in the opposite direction to enter the beam splitter 22. After being reflected by the splitter 22, the light is guided to the two-dimensional light receiving element 30 via the analyzer 27, the imaging lens 28, and the aperture plate 29.
In this way, the light receiving element 30 sequentially detects the images of the minute regions, the image signal related to the images of the minute regions is given to the image processing unit (not shown), and the predetermined image processing is performed, and then the sample S is displayed on the display. View the big picture.

By the way, also in this embodiment, the aperture plate 29 is arranged at the position PP where the projection image of the diaphragm (pupil) of the objective lens 25 is formed by the imaging lens 28, and is formed on the aperture plate 29. The diameter of the aperture 31 is smaller than the projected image. That is, in this embodiment, the imaging lens 28 functions as a projection unit, and as in the scanning optical microscope according to the above-described embodiments, the peripheral diffraction generated from the edge of the diaphragm of the objective lens 25. Waves can be prevented from entering the light receiving element 30, the S / N of the image signal output from the light receiving element 30 can be increased, and the sample can be displayed well on the display and observed.

In the above embodiment, the imaging lens 2
8 as a projection means, and the imaging lens 2
An aperture plate 29 for blocking peripheral diffracted waves is arranged between the light receiving element 30 and the light receiving element 30.
Instead of providing the microlenses 41, as shown in FIG. 4, the microlenses 41 are closely attached to the plurality of through holes 23a formed in the Nippon disc 23, and the microapertures 42 respectively corresponding to the through holes 23a of the Nippon disc 23 are provided. It is also possible to provide a micro-aperture array plate 43 having the above, and rotate the Nippon disk 23 and the micro-aperture array plate 43 integrally by the motor 26. However, even in this modification,
Micro lens 41 and micro aperture array plate 43
And must be arranged so as to satisfy the following relationship. That is, the microlens 41 functions as a projection means and forms a projection image of the diaphragm (pupil) of the objective lens 25 at the position PP, but it is necessary to dispose the microaperture array plate 43 at this position PP. Furthermore, it is necessary to make the diameter of each micro aperture 42 smaller than the projected image. By satisfying such a requirement, the same effect as that of the above-described embodiment can be obtained.

In this modified example, instead of bringing the microlens 41 into close contact with the Nippon disc 23,
A microlens array in which the microlenses 41 corresponding to the through holes 23a of the Nippon disc 23 are arranged may be superposed on the Nippon disc 23. In addition, the lens aperture of the micro lens array,
It is also possible to make the same function as the through hole 23a of the Nippon disc 23. In this case, the Nippon disc 23
Can be omitted.

In some cases, the microlens 41 can be omitted by providing the Nipkow disk 23 with an image forming function based on the principle of a pinhole camera.

In the scanning optical microscope of FIGS. 3 and 4, the polarizer 21, the quarter-wave plate 24 and the analyzer 27 are used.
However, these components are not essential components for the scanning optical microscope and may be omitted.

Although the microscope described above is a confocal microscope, the object of application to the present invention is not limited to the confocal microscope. In the scanning optical microscope of FIG. 1, the point light source 1 and the light receiving element 4 are fixed and the sample S
Is moved to scan the illuminated portion and the detected portion on the sample S, and the scanning optical microscope of FIGS. 3 and 4 scans the illuminated portion on the sample S by shaking the illumination light using the Nipkow disk 23. However, the means for scanning the illumination portion and / or the detection portion is not limited to these. That is, the object of application of the present invention is all scanning optical microscopes.

[0033]

As described above, according to the scanning optical microscope of the present invention, the image of the pupil of the objective lens is formed at a predetermined position by the projection means, and the projected image is formed at the position of this projected image rather than the projected image. Since an aperture plate having an aperture with a small diameter is arranged, the peripheral diffraction wave generated from the edge of the aperture (pupil) of the objective lens can be blocked by the aperture plate and the influence of the peripheral diffraction wave can be removed. The S / N ratio of the signal output from the sample can be increased to display and observe the sample well.

[Brief description of drawings]

FIG. 1 is a diagram showing an embodiment of a scanning optical microscope according to the present invention.

FIG. 2 is a diagram showing a relationship between an aperture and a projected image of a diaphragm of an objective lens formed on an aperture plate.

FIG. 3 is a diagram showing another embodiment of the scanning optical microscope according to the present invention.

FIG. 4 is a diagram showing another embodiment of the scanning optical microscope according to the present invention.

FIG. 5 is a diagram showing an example of a conventional scanning optical microscope.

6 is a diagram showing an image plane illuminance distribution obtained when observing minute black dots with the scanning optical microscope of FIG.

[Explanation of symbols]

 1 point light source 3,25 objective lens 4,30 light receiving element 11 projection lens (projection means) 12,29 aperture plate 13,31 aperture 28 imaging lens (projection means) 29 aperture plate 30 light receiving element 41 microlens (projection means) 42 Micro Aperture 43 Micro Aperture Array Plate BDW Peripheral Diffraction Wave D Diameter (of Aperture) I Projected Image (of Objective Lens Aperture) PP (Formation of Projected Image) Position S Sample

Claims (1)

[Claims] 1. The illumination light from the light source is directed toward the sample while the light from the sample is received by the light receiving element via the objective lens, and the illumination light is irradiated onto the sample. Image of the plurality of minute regions by scanning the illuminated region and / or the sensing region limited to the minute region while limiting the illuminated region and / or the sensing region detected by the light receiving element to the minute region. In the scanning optical microscope, which sequentially detects the light beam and obtains the entire image of the sample, it is arranged between the objective lens and the light receiving element and projects the image of the pupil of the objective lens to a predetermined position on the light receiving element side. A scanning optical microscope comprising: a projection unit; and an aperture plate that is arranged at a position where the projection image is formed and that has an aperture having a diameter smaller than that of the projection image.