JP3361003B2 - Scanning optical microscope - Google Patents

Description

Description: BACKGROUND OF THE INVENTION 1. Field of the Invention The present invention relates to a so-called scanning optical microscope. 2. Description of the Related Art A scanning optical microscope is configured to irradiate illumination light from a light source toward a sample while receiving light from the sample by a light receiving element via an objective lens.
In particular, the illumination part and / or the detection part limited to the minute area are scanned while the illumination part irradiated with the illumination light and / or the detection part detected by the light receiving element is limited to the minute area on the specimen. Thus, images of a plurality of minute regions are sequentially detected to obtain an entire image of the sample. FIG. 5 is a view showing an example of a conventional scanning optical microscope, in which the illumination part and the detection part are limited to a small area, and the illumination part and the detection part are scanned by moving the sample two-dimensionally. Then, signals of a plurality of minute areas are sequentially detected and a signal is output. That is, in this scanning optical microscope, illumination light is emitted from a point light source 1 in the Z direction, and is passed through a beam splitter 2 and an objective lens 3 at a predetermined position in the X and Y directions.
The light is condensed in the form of a spot on a sample S which is arranged so as to be scannable in a three-dimensional manner, and is irradiated on a minute area on the sample S. The reflected light from the illumination site (the minute area irradiated with the illumination light) enters the beam splitter 2 via the objective lens 3 and is guided to the light receiving element 4 side by the beam splitter 2, and is the same as the illumination site. The image of the part is detected by the light receiving element 4. Therefore, by scanning the sample S two-dimensionally,
Light (reflected light) from each minute area of the sample S sequentially enters the light receiving element 4, and a signal related to the image of each minute area is sequentially output from the light receiving element 4 to an image processing unit (not shown). After receiving the image processing, the entire image of the sample S is displayed on a display (not shown). In this scanning optical microscope, the focal plane FP is located near a light receiving surface (not shown) of the light receiving element 4.
Since the pinhole plate 6 in which the pinhole 5 is formed is disposed at a position optically conjugate with the above, as shown by the solid line in FIG. In this case, most of the light (reflected light) from the minute region enters the light receiving element 4 via 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 region deviates from the focal plane FP, as shown by the broken line in FIG.
There is no contribution from other sources. 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 a scanning optical microscope, images of a sample having a fine pattern, a sample in which only very weak light is incident on the light receiving element, and a sample having a low contrast can be satisfactorily reproduced. In order to observe, the light receiving element 4
It is necessary to increase the S / N of the signal from In particular, in a confocal microscope, the resolution in the optical axis direction, that is, the Z direction largely depends on the S / N of a signal.
High signal is required. Here, the S / N of the signal greatly depends on the imaging performance of the optical system and the existence of light unnecessary for imaging such as ghost, flare and scattered light. As a result, it was found that, besides these factors, the presence of peripheral diffracted waves greatly contributed to the reduction of S / N. Hereinafter, the influence of the peripheral diffraction wave on the S / N of the signal will be described with reference to FIG. For example, when a minute black spot 7 formed on a sample S is observed by a conventional scanning optical microscope, FIG.
The image plane illuminance distribution as shown in FIG. Here, it is assumed that there is no ghost or scattered light. When only incident light (illumination light) propagating in accordance with the law of geometric optics is considered, a part of the incident light is shielded by the minute black spots 7, and the rest is reflected by the document surface other than the minute black points 7. Proceed to the light receiving element side. Therefore, the illuminance distribution in the light receiving element becomes zero completely corresponding to the minute black spot 7 as shown by the solid line in FIG. Take.
Therefore, the range from the lowest level to the highest level (corresponding to the dynamic range) of the density that can be detected by the light receiving element is Rg, and a high S / N signal can be obtained. However, the image plane illuminance at the light receiving element is
In practice, the distribution is as shown by the one-dot chain line in FIG.
This is due to the influence of a peripheral diffraction wave (= Boundary Diffraction Wave), because a diffraction image is formed on the light receiving element as a result of the combination of the geometrical optical incident light and the peripheral diffraction wave. [0011] The "peripheral diffracted wave" is a diffracted wave generated from an end when light enters a diffractive object. In the scanning optical microscope configured as described above, the stop of the objective lens corresponds to a diffractive object, and a peripheral diffracted wave is generated from the edge of the stop. Here, when viewed from the light receiving element side, it appears that a peripheral diffraction wave is generated from the edge of the pupil of the objective lens. The peripheral diffraction wave is described in, for example, No. 67 of “Principle of Optics II (by Max Born & Emil Wolff, translated by Toru Kusakawa and Eiji Yokota; Tokai University Press)”.
This is described in detail on page 0. However, although "Boundary Diffraction Wave" is translated as "boundary diffraction wave" in "Principle of Optics II", it is the same as "peripheral diffraction wave". In an actual microscope objective lens, the existence of the stop is often unclear, but since there is a virtual portion corresponding to the stop, this is used as the stop here. When a diffraction image is formed under the influence of the peripheral diffracted waves, the illuminance of the image plane at the position corresponding to the minute black spot 7 does not become zero, but increases by ΔR, and the dynamic range Rg + d in this case is obtained. Is smaller by ΔR than the dynamic range Rg when the peripheral diffracted waves are ignored, and the signal S /
N decreases. The present invention intends to overcome the above-mentioned problems in the prior art, and removes peripheral diffracted waves generated from the edge of the pupil of the objective lens to increase the S / N of the signal output from the light receiving element. It is an object of the present invention to provide a scanning optical microscope capable of satisfactorily observing a sample. According to a first aspect of the present invention, while illuminating light from a light source is irradiated toward a sample, light from the sample is received by a light receiving element via an objective lens. The illumination part and / or the detection part which is limited to the minute area while limiting the illumination part irradiated with the illumination light and / or the detection part detected by the light receiving element to the minute area in the sample. A scanning optical microscope that scans a site to sequentially detect images of the plurality of minute regions to obtain an overall image of the sample, and to achieve the above object, includes the objective lens and the light receiving element. A projection unit disposed between the projection unit and the projection unit for projecting an image of a pupil of the objective lens to a predetermined position on the light receiving element side; and an aperture having a smaller diameter than the projection image and disposed at a position where the projection image is formed. Aperture plate. FIG. 1 is a view showing one 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 a pinhole plate 6 and a light receiving element 4. Since other configurations are the same, the same components are denoted by the same reference numerals and description thereof is omitted. The illumination light from the point light source 1 is condensed into a spot through the beam splitter 2 and the objective lens 3,
Irradiation is performed on a minute area on the sample S. The light reflected on the minute area (illumination site) is condensed by the objective lens 3 at the position P where the pinhole plate 6 is provided, and forms a spatial image of the minute area located at the illumination area. Further, the light receiving element 4 is
A projection lens 11 is provided at a position separated by a certain distance on the side, and guides light passing through the pinhole 5 formed in the pinhole plate 6 to the light receiving element 4 through the aperture 13 of the aperture plate 12. . In FIG. 1, the pinhole 5 is drawn large for the sake of convenience, but is actually sufficiently small. In this embodiment, the detection region detected by the light receiving element 4 is limited to a minute region (illumination region). Have been. The projection lens 11 projects an image of the aperture (pupil) 3a of the objective lens 3 at a 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, an aperture 13 having a smaller diameter D than the projected image I of the stop 3a of the objective lens 3 is formed on the aperture plate 12. By setting the diameter D of the aperture 13 in this manner, the peripheral diffracted wave BDW generated from the edge of the stop 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 stop 11 propagates through the projection lens 11 to the aperture plate 12 side. Here, the aperture plate 1
Observing the image I of the aperture 3a formed on the aperture 2, the aperture 3a
The edge of appears to shine. The reason why the edge of the stop 3a looks shiny is due to the peripheral diffracted wave BDW generated from the edge of the stop 3a of the objective lens 3 as 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 stop 3a as described above, the peripheral diffracted wave BDW is cut off by the aperture plate 12, and the light receiving element 4 of the peripheral diffracted wave BDW Side propagation is prevented. Therefore, it is possible to prevent the peripheral diffracted wave BDW from passing through the aperture 13 of the aperture plate 12 and to enter the light receiving element 4, thereby eliminating the influence of the peripheral diffracted wave BDW. Further, the light that has passed through the aperture 13, that is, the light that 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 coinciding with the illumination site is output from the light receiving element 4. Is done. In the above description, the description has been given of the case where the image of the minute region existing in the illumination site is detected by the light receiving element 4 and the corresponding image signal is output. In order to move, the illuminated part and the detected part simultaneously and two-dimensionally scan by this movement, sequentially detect the image of the minute area of the sample S, and output the corresponding image signal from the light receiving element 4. The image signal thus obtained is provided to an image processing unit (not shown), and after a predetermined image processing is executed, is displayed on a display (not shown). As described above, according to the scanning optical microscope of this embodiment, the aperture (pupil) 3a of the objective lens 3
Of the peripheral diffracted wave BDW generated from the edge of the aperture plate 12
, The influence of the peripheral diffraction wave BDW is removed, so that the S / N of the image signal output from the light receiving element 4 can be increased, and the sample can be displayed and observed well on the display. 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. A plurality of through holes 23 a are formed in the Nippow disk 23 in a spiral shape. When illumination light from a light source enters a part of the Nippow disk 23, a plurality of light beams are emitted from the Nippow disk 23. The following description focuses on one of the plurality of emitted lights. As shown in FIG.
After passing through the 波長 wavelength plate 24, the light is condensed into a spot by the objective lens 25, and is irradiated on a minute area on the sample S.
Although not shown, other outgoing light can similarly be incident on different minute regions, respectively, and simultaneously illuminate a plurality of minute regions independently of each other. The Nippou disk 23 is a motor 26
, And is configured to rotate around a rotation axis 26 a extending in the optical axis direction (Z direction) by a motor 26, so that a plurality of outgoing lights from the Nippow disk 23 are converted into a horizontal plane (XY directions) by this rotational movement. ), And minute areas on the sample S are sequentially illuminated. In each minute area, when illumination light (light emitted from the Nippow disk 23) enters, the light is reflected by the minute area, travels in the same direction as the illumination light in the opposite direction, and enters the beam splitter 22, where After being reflected by the splitter 22, the light is guided to a two-dimensional light receiving element 30 via an analyzer 27, an imaging lens 28, and an aperture plate 29.
In this manner, the image of the minute area is sequentially detected by the light receiving element 30, and an image signal related to the image of the minute area is given to an image processing unit (not shown) to execute predetermined image processing. Display the whole picture. By the way, also in this embodiment, the aperture plate 29 is arranged at the position PP where the projection image of the stop (pupil) of the objective lens 25 is formed by the imaging lens 28, and the aperture plate 29 is formed on the aperture plate 29. The diameter of the aperture 31 is made smaller than the projected image. That is, in this embodiment, the imaging lens 28 functions as a projection unit, and similarly to the scanning optical microscope according to the above-described embodiment, the peripheral diffraction generated from the edge of the stop of the objective lens 25. Waves can be prevented from being incident on 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 function as projection means, and the imaging lens 2
An aperture plate 29 for blocking peripheral diffracted waves is arranged between the light receiving element 8 and the light receiving element 30.
Instead, as shown in FIG. 4, as shown in FIG. 4, the micro lenses 41 are brought into close contact with the plurality of through holes 23 a formed in the Nippow disk 23, and the micro apertures 42 respectively corresponding to the through holes 23 a of the Nippow disk 23. May be provided, and the Nippou disk 23 and the micro aperture array plate 43 may be integrally rotated by the motor 26. However, in this modified example,
Micro lens 41 and micro aperture array plate 43
Must be arranged so as to satisfy the following relationship. That is, the microlens 41 functions as a projection unit, and forms a projection image of the aperture (pupil) of the objective lens 25 at the position PP. It is necessary to dispose the microaperture array plate 43 at this position PP. Further, the diameter of each micro aperture 42 needs to be smaller than the projected image. By satisfying such a request, the same effect as in the above-described embodiment can be obtained. In this modification, instead of bringing the microlens 41 into close contact with the Nippow disk 23,
A microlens array in which the microlenses 41 corresponding to the through holes 23a of the Nippow disk 23 may be arranged so as to overlap the Nippow disk 23. Also, the lens aperture of the micro lens array is
It is also possible to make the same function as the through hole 23a of the Nippow disk 23. In this case,
Can be omitted. In some cases, the microlens 41 can be omitted by providing the Nippou disk 23 with an imaging function based on the principle of a pinhole camera. In the scanning optical microscope shown in 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 to which the present invention is applied 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
The scanning optical microscope of FIGS. 3 and 4 scans the illumination site in the sample S by shaking the illumination light using the Nippou disk 23 by moving However, the means for scanning the illumination site and / or the detection site is not limited to these. That is, the application object of the present invention is a scanning optical microscope in general. 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 image is projected at the position of the projection image. Since an aperture plate with an aperture smaller than the image is arranged, peripheral diffraction waves generated from the edge of the aperture (pupil) of the objective lens can be blocked by the aperture plate to eliminate the effects of the peripheral diffraction waves. By increasing the S / N of the signal output from the light receiving element, the sample can be displayed and observed satisfactorily.

BRIEF DESCRIPTION OF THE DRAWINGS FIG. 1 is a diagram showing one embodiment of a scanning optical microscope according to the present invention. FIG. 2 is a diagram illustrating a relationship between an aperture and a projected image of a stop 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 a minute black spot is observed by the scanning optical microscope of FIG. 5; [Description of Signs] One-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 reception element 41 Micro Lens (projection means) 42 Micro aperture 43 Micro aperture array plate BDW Peripheral diffraction wave D Diameter (of aperture) I Projection image (of stop of objective lens) PP (formation of projection image) Position S Sample

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Claims (1)

(57) [Claim 1] Irradiation light from a light source is directed toward a sample, and light from the sample is received by a light receiving element via an objective lens. Scanning the illumination site and / or the detection site limited to the minute region while limiting the illumination region of the sample irradiated with the illumination light and / or the detection region detected by the light receiving element to the minute region. In a scanning optical microscope that sequentially detects images of the plurality of minute regions to obtain an entire image of the sample, the scanning optical microscope is disposed between the objective lens and the light receiving element, and the image of a pupil of the objective lens is A scanning optical microscope, comprising: a projection unit for projecting a predetermined position on a light receiving element side; and an aperture plate disposed at a position where the projection image is formed and having an aperture having a smaller diameter than the projection image.