JP2003084207A - Confocal microscope - Google Patents

Abstract

PROBLEM TO BE SOLVED: To provide a confocal microscope capable of forming a three- dimensional image which makes it possible to observe an oblique surface part of a sample and is more accurate. SOLUTION: The confocal microscope is equipped with light sources 1 and 13, a rotary disk 4 which has light transmission parts and light shading parts formed in specific pattern, 1st lighting optical systems 2 and 3 which guide the light from the light sources to the rotary disk 4, an objective 6 which irradiates a sample 7 with the light from the rotary disk 4, and 2nd lighting optical system 12 and 14 which light up the sample 7 along optical paths different from those of the 1st lighting optical system 2 and 3.

Description

Detailed Description of the Invention

[0001]

The present invention relates to a confocal microscope having a high sectioning effect.

[0002]

2. Description of the Related Art Generally, two types of confocal microscopes, a disk scan type and a laser scan type, are well known.
Among them, the disk scan confocal microscope not only has a high lateral resolution of the sample as compared with a normal microscope,
It has a great feature that it has a very high sectioning effect in the height direction (Z direction) of the sample. for that reason,
It has an excellent feature that it is possible to construct a three-dimensional image of a sample by combining a disk scan confocal microscope and image processing technology.

FIG. 11 shows a typical configuration example of a disk scan type confocal microscope. This disc scan type confocal microscope has two types of observation methods, that is, visual observation and observation by an image pickup device (CCD camera). Here, an example using a CCD camera is shown. As shown in the figure, the illumination light emitted from the light source 1 is reflected by the collimator lens 2
Then, the light enters the half mirror 3 and is reflected there to illuminate the rotating disk 4 as a mask pattern member.
The rotary disc 4 is, for example, a Nipkow disc having a plurality of pinholes formed in a spiral shape, but a disc having a slit pattern is also known. The rotating disk 4 is a rotating shaft 5a of the motor 5.
It is attached to and is rotated at a predetermined rotation speed.
The illumination light emitted to the rotating disc 4 is
Through the plurality of pinholes formed in the
An image is formed on the sample 7 by.

The reflected light from the sample 7 is returned to the objective lens 6 again.
Then, the light passes through the half mirror 3 through the pinhole of the rotating disk 4, passes through the condenser lens 8, and enters the CCD camera 9. The computer 10 captures the image signal output from the CCD camera 9, performs image processing to store desired image data, and displays the image on the monitor 11.

[0005]

By the way, in this disk scan type confocal microscope, the rotating disk 4 is used.
The illumination light that has passed through the pinhole illuminates the sample 7 through the objective lens 6, and the reflected light returns to the objective lens 6 again, and passes through the pinhole of the rotating disk 4 and the half mirror 3 by the condenser lens 8. Since the image is formed on the CCD camera 9, when the sample 7 has a three-dimensional structure as shown in FIG. 12 and the inclination angle of its outer surface is extremely large (steep), this inclination The reflected light on the surface is reflected outside the aperture of the objective lens 8 and does not return to the objective lens 8. Therefore, the image formed on the CCD camera 9 may be a very dark image due to lack of reflected light, and may not be an image. As a result, there is a problem that only a partial three-dimensional image can be constructed.

In this case, it is possible to take the reflected light into the aperture of the objective lens to some extent by switching to the objective lens with a large numerical aperture, but the objective lens with a large numerical aperture has a small working distance and is therefore applied. It is difficult to use it for various samples because the number of samples that can be produced is limited and the magnification is limited.

The present invention has been made in view of the problems of the prior art as described above. The purpose of the present invention is to observe a steep slope portion of a sample and to obtain a more accurate three-dimensional image. It is to provide a confocal microscope that can be constructed.

To achieve the above object, a confocal microscope according to the present invention comprises a light source, a mask pattern member having a light transmitting portion and a light shielding portion formed in a predetermined pattern, and the light from the light source to the mask. A confocal microscope including a first illumination optical system that guides a pattern member and an objective lens that irradiates a sample with light from the mask pattern member, and is different from the optical path of the first illumination optical system. A second illumination optical system that illuminates the sample in the optical path is provided. According to the invention,
The confocal microscope includes a switching mechanism that switches between illumination from the first illumination optical system and illumination from the second illumination optical system. Further, according to the present invention, the mask pattern member includes a light-shielding pattern area in which the predetermined pattern is formed and a light-transmitting area.

[0009]

BEST MODE FOR CARRYING OUT THE INVENTION Hereinafter, an embodiment of the present invention will be described based on illustrated examples. FIG. 1 is a configuration diagram of a first embodiment of a confocal microscope according to the present invention. In the figure, substantially the same members as those shown in the prior art are designated by the same reference numerals,
Descriptions thereof are omitted. In this embodiment, the illumination light emitted from the epi-illumination light source 1 passes through the collimator lens 2 and the half mirror 3 and illuminates the rotating disk 4 having a plurality of pinholes formed therein. The rotating disk 4 is rotated at a predetermined speed by a motor 5, and the illumination light that has passed through the plurality of pinholes formed in the rotating disk 4 is imaged on the sample 7 by the objective lens 6. The reflected light from the sample 7 is again reflected by the objective lens 6,
The light is transmitted through the half mirror 3 through the pinhole of the rotating disk 4 and is made incident on the CCD camera 9 by the condenser lens 8. The computer 10 takes in an image signal output from the CCD camera 9, performs image processing and the like as necessary, and displays an image or a calculation result on the monitor 11.

Further, a dome illumination device 12 is attached around the objective lens 6 with the optical axis of the objective lens 6 as the center. As shown in FIG. 2 (a), this dome lighting device 12 is, for example, an optical fiber bundle composed of a large number of optical fiber wires, and the tip ends of the optical fiber wires separated from each other are hemispherical. As shown in FIG. 2 (b), a plurality of light sources such as LEDs are attached to a hemispherical support, and their light emitting ends are substantially common on the sample surface. It is formed by being arranged so as to face one point. In the case of the illumination device shown in FIG. 2A, the illumination light from the oblique illumination light source 13 is guided to the dome illumination device 12 by the optical fiber 14,
In the case of the illumination device shown in FIG. 2 (b), the light sources are turned on all at once, so that the surface of the sample 7 cannot be obtained by epi-illumination (a wide angle up to an angle close to the horizontal direction).
It can be illuminated in a range.

Since the first embodiment is constructed as described above, when the sample 7 has a flat or gently inclined surface,
The reflected light from the sample 7 when illuminated by the epi-illumination light source 1 returns into the aperture of the objective lens 6, while the reflected light from the sample 7 when illuminated by the oblique illumination light source 13 is the aperture of the objective lens 6. Don't go back inside. Therefore, a good sectioning image can be obtained as in the conventional confocal microscope. On the other hand, as shown in FIGS. 3 (a) and 3 (b), when the sample 7 has a spherical or uneven structure and has a steep slope, the reflected light from the sample 7 when illuminated by the epi-illumination light source 1 Does not return into the aperture of the objective lens 6, so a sectioning image of this portion cannot be obtained. However, in this embodiment, the oblique illumination light source 1
Since the illumination is performed by the light source 3, the reflected light from the sample 7 when illuminated by the oblique illumination light source 13 returns to the inside of the aperture of the objective lens 6. As a result, it is possible to obtain an image of a portion that could not be obtained by the illumination using only the epi-illumination light source 1.

In the image obtained by the oblique illumination light source 13, the illumination light does not pass through the pinhole of the rotating disk 4. Therefore, the resolution in the plane perpendicular to the optical axis is lower than that of an image obtained by a conventional confocal microscope. However, since the reflected light from the sample 7 passes through the pinhole, the sectioning effect is not lost. However, the sectioning effect in this case in this embodiment cannot be said to be the same as the sectioning effect of the conventional confocal microscope.

As described above, according to the first embodiment, it is possible to observe a portion which cannot be observed by the conventional confocal microscope with a simple structure. Moreover, the sectioning effect is not impaired in the image obtained by any of the illuminations, although the degree varies.

FIG. 4 is a block diagram of a second embodiment of the confocal microscope according to the present invention. In the drawing, substantially the same members as those shown in the prior art and the first embodiment are designated by the same reference numerals, and the description thereof will be omitted. In this embodiment, the rotary disk 15 is formed with a pinhole pattern as shown in FIG. 5, for example. That is, a plurality of pinholes are formed as a mask pattern on one semi-circular portion of the rotating disk 15, and no mask pattern is formed on the other semi-circular portion, so that light passes through. Further, a pattern 15a for identifying which semicircular portion passes through the optical path is formed on the peripheral portion of the rotating disk 15. A photo sensor 16 arranged so as to cooperate with the pattern 15a is installed in the peripheral portion of the rotating disk 15 to generate a signal (see FIG. 6A) synchronized with the rotating operation of the rotating disk 15. It is supposed to be done.

The output end of the photosensor 16 is connected to the power supply circuit of the epi-illumination light source 1 and the power supply circuit of the oblique illumination light source 13 via an inverter 17, and the photosensor 16 is connected.
Emits an ON signal (see Fig. 6 (b)) to the epi-illumination light source 1 only while the semi-circular part where a plurality of pinholes as mask patterns are formed is passing through the optical path, and conversely An OFF signal (see FIG. 6C) is output to the illumination light source 13 by the inverter 17. On the contrary, during a period in which the semicircular portion where the mask turn is not formed is passing through the optical path, an OFF signal is output to the epi-illumination light source 1 and an ON signal is output to the oblique illumination light source 13 by the inverter 17. Is output. The rotation speed of the rotary disk 15 is CC
The D camera 9 is set to rotate at a speed half that of the frame rate. That is, the rotating disk 15
It is set so that one image is obtained every half rotation of.

Since the second embodiment is constructed as described above, when the sample 7 has a flat or gently inclined surface, it is illuminated by the epi-illumination light source 1 as in the case of the first embodiment. The reflected light from the sample 7 at that time returns to the inside of the aperture of the objective lens 6. On the other hand, the sample 7 when illuminated by the oblique illumination light source 13
The reflected light from does not return into the aperture of the objective lens. Therefore, a good sectioning image can be obtained as in the conventional confocal microscope. On the other hand, when the sample 7 is spherical or has an uneven structure and has a steep slope, the reflected light from the sample 7 when illuminated by the epi-illumination light source 1 does not return into the aperture of the objective lens 6. On the other hand, the reflected light from the sample 7 when illuminated by the oblique illumination light source 13 returns to the inside of the aperture of the objective lens 6.

However, the second embodiment differs from the first embodiment in the following points. That is, at the first point, in addition to the illumination light, the reflected light from the sample 7 does not pass through the pinhole of the rotating disk 15, so that in the plane perpendicular to the optical axis as compared with the image obtained by the conventional confocal microscope. The image has low resolution and no sectioning effect. That is, the image is the same as the bright field image of the conventional optical microscope (non-confocal type). However, since the reflected light hardly returns in the conventional confocal microscope, it becomes possible to observe the portion that cannot be observed. Further, the image obtained by the oblique illumination light source 13 is taken during the period in which the semicircle on which the mask pattern of the rotating disk 15 is not formed passes through the optical path, and therefore the light amount loss is smaller than that in the first embodiment. A few bright images are obtained.

The second point is that the rotating disk 15 has a transparent region through which light passes. In this case, the light from the epi-illumination light source 1 performs two different illuminations by the pattern area where the pinhole is formed and the transparent area. Therefore, for example, considering the case where the sample 7 is flat, both the light passing through the pattern region and the light passing through the transparent region are reflected by the sample 7 and pass through the aperture of the objective lens.
As a result, the sectioning image is darker than the bright field image.

By the way, the three-dimensional image is obtained from the sectioning image. That is, the sectioning image is captured while changing the distance between the sample 7 and the objective lens 6, and signal processing is performed to hold the brightest value in each pixel. Therefore, if this processing is performed in a state in which a bright field image is obtained, the bright field image is brighter than the sectioning image, and thus the sectioning image is always subjected to signal processing based on the bright field image. However, since the bright field image is not a so-called slice image, an accurate three-dimensional image cannot be obtained even if the above signal processing is performed. Therefore, in the second embodiment, the light source is turned ON /
The OFF control is performed so that a bright-field image cannot be obtained in a portion where a sectioning image can be obtained. When a three-dimensional image is constructed by the computer 10, the luminance values are compared pixel by pixel for the two types of images obtained alternately as described above, and the one having the larger luminance is used to obtain an accurate cubic image. I am trying to get the original image.

As described above, according to the second embodiment, it becomes possible to observe a portion which cannot be observed by the conventional confocal microscope. Further, it is possible to obtain a bright image with less light amount loss than in the first embodiment. In the second embodiment, the epi-illumination light source and the oblique illumination light source are controlled in synchronism with the rotation of the rotary disk 15 in order to avoid the adverse effect of each illumination light. However, such control is performed. In some cases, the same effect may be obtained even without the use. Further, although the relationship between the frame rate of the rotary disk 15 and the CCD camera 9 is set to a fixed value, a method of giving a signal generated by the rotary operation of the rotary disk 15 to the CCD camera 9 to achieve synchronization, or There is also a method of obtaining a more stable image by controlling the rotation of the rotating disk 15 and controlling the number of rotations of the rotating disk 15 with respect to the frame rate of the CCD camera 9.

FIG. 7 is a block diagram of the third embodiment of the confocal microscope according to the present invention. In the figure, substantially the same members as those shown in the prior art and the embodiments described above are designated by the same reference numerals,
Descriptions thereof are omitted. In this embodiment, ON / OFF control is performed for the epi-illumination light source 1 and the oblique illumination light source 13 as in the second embodiment, but this control is performed by a computer using, for example, an I / O port. It was done. That is, in this embodiment, when the shape (height or structure) of the sample 7 is known in advance, it is possible to determine which light source (or both light sources) should be used for illumination for each height at which a sectioning image is obtained. By programming in advance, the computer 10 can control the epi-illumination light source 1 and the oblique illumination light source 13 to construct a three-dimensional image.

Further, instead of the rotating disk 4 on which the mask pattern is uniformly formed, a rotating disk on which a different pattern is formed in accordance with the radial direction of the rotating disk is used, and the motor 5 is moved so that the optical path is formed in the optical path. Different mask patterns may be arranged, and the mask patterns may be switched according to the illuminating light source. For example, as shown in FIG. 8, the conventional pinholes are arranged in the first region, and the rotating disk 4 having no mask pattern is used in the second region. Is illuminated by the epi-illumination light source 1 and the first region of the rotating disc is placed in the optical path to obtain an image with a sectioning effect. The inclined surface of the sample 7 is illuminated by the oblique illumination light source 13 and the second region of the rotating disk 4 is arranged in the optical path, so that a bright image of the inclined portion can be obtained.

The three embodiments have been described above.
The present invention is not limited to this. For example, in each of the first, second and third embodiments described above, a rotary disk having a plurality of pinholes formed therein is used as a mask pattern member, but the present invention is not limited to this, and FIGS.
As shown in 0, a rotating disk 18 having a pattern in which linear light-transmitting portions 18a and light-shielding portions 18b are alternately arranged may be used. Alternatively, for example, the liquid crystal display device may be realized by rotating and displaying the pinhole as if the rotating disk is rotating, or by swinging the pinhole within a predetermined range. Although the dome illumination device is used as the oblique illumination device, the same effect can be obtained by arranging a plurality of ring illuminations concentrically.

In the first embodiment shown in FIG. 1, the light source 13 is provided for illumination by the dome illumination device 12, but instead of the light source 13, the half mirror 3 is transmitted to pass through the condenser lens. The light condensed at 2'may be used as a light source (illustrated by a chain line in FIG. 1). Since a light source for a confocal microscope is often used with high brightness, light transmitted through the half mirror 3 can be sufficiently used as illumination light. Further, the transmittance characteristic of the half mirror 3 may be set so that an image having the same brightness can be obtained by the epi-illumination and the oblique illumination. It is also possible to provide the shutters 19 on the light emitting sides of the light sources 1 and 13 and switch the shutters 19 to obtain the same effect as described in the second and third embodiments. In this case, the shutter 19 may be provided in either one of the light sources 1 and 13 and operated.

As described above, the confocal microscope of the present invention has the following features in addition to the features described in the claims. (1) The confocal microscope according to claim 3, wherein a boundary line between the pattern region and the transmissive region is formed from the center of the mask pattern member toward the periphery thereof. (2) The confocal microscope according to claim 3, wherein a boundary line between the pattern region and the transmissive region is concentrically formed. (3) The confocal microscope according to claim 1, further comprising a second light source that supplies light to the second illumination optical system. (4) The confocal microscope according to claim 3, wherein the light transmission region of the pattern region is formed in a slit shape. (5) The confocal microscope according to claim 1, further comprising an optical element that supplies light from the light source to the second illumination optical system. (6) The confocal microscope according to claim 1, wherein the second illumination optical system illuminates the sample from a direction inclined with respect to the optical axis. (7) The second illumination optical system has a plurality of light emitting means,
The confocal microscope according to (6) above, characterized in that the light emitting means in at least a part of the region can be selectively made to emit light. (8) The confocal microscope according to (3), wherein the second light source is a light emitting element. (9) The first illumination optical system and the second illumination optical system are switched by operating a shutter provided in at least one of the first illumination optical system and the second illumination optical system. The confocal microscope according to claim 1, wherein:

[0026]

As described above, according to the present invention, it becomes possible to observe the slope portion of the sample which cannot be observed by the conventional confocal microscope, so that it is possible to construct a more accurate three-dimensional image. At the same time, it is possible to provide a confocal microscope applicable to various applications.

[Brief description of drawings]

FIG. 1 is a configuration diagram of a first embodiment of a confocal microscope according to the present invention.

FIG. 2A is a partially cutaway perspective view of an example of a dome lighting device used in the first embodiment, and FIG. 2B is a partially cutaway perspective view of another example.

FIG. 3A is an explanatory view showing a state of reflected light in the first embodiment when the sample is spherical and FIG. 3B is a case where the sample has an uneven structure and a steep slope.

FIG. 4 is a configuration diagram of a second embodiment of the confocal microscope according to the present invention.

FIG. 5 is a plan view showing a pinhole pattern of a rotary disk used in a second embodiment.

FIG. 6A is a diagram showing a change in photosensor output in synchronism with the rotating operation of the rotating disk in the second embodiment;
(B) is a diagram showing ON / OFF signals for the epi-illumination light source,
(C) is a diagram showing ON / OFF signals for the oblique illumination light source.

FIG. 7 is a configuration diagram of a third embodiment of the confocal microscope according to the present invention.

FIG. 8 is a plan view showing a pinhole pattern of a rotary disk used in a third embodiment.

FIG. 9 is a plan view showing another embodiment of the rotating disk.

FIG. 10 is a plan view showing still another embodiment of the rotating disk.

FIG. 11 is a configuration diagram of a conventional confocal microscope.

12A and 12B are explanatory views showing a state of reflected light in a conventional confocal microscope. FIG. 12A is a case where the sample is spherical, and FIG. 12B is a case where the sample has an uneven structure and has a steep slope.

[Explanation of symbols]

1 Epi-illumination light source 2 Collimator lens 2 ', 8 Condensing lens 3 Half mirrors 4, 15, 18 Rotating disk (mask pattern member) 5 Motor 6 Objective lens 7 Sample 9 CCD camera 10 Computer 11 Monitor 12 Dome illumination device 13 Oblique illumination Light source 14 Fiber 16 Photo sensor 17 Inverter 19 Shutter

Claims (3)

[Claims] 1. A light source, a mask pattern member having a light transmitting portion and a light shielding portion formed in a predetermined pattern, a first illumination optical system for guiding light from the light source to the mask pattern member, and the mask. A confocal microscope provided with an objective lens for irradiating a sample with light from a pattern member, comprising a second illumination optical system for illuminating the sample in an optical path different from the optical path of the first illumination optical system. A confocal microscope characterized in that 2. The confocal microscope according to claim 1, further comprising a switching mechanism that switches between illumination from the first illumination optical system and illumination from the second illumination optical system. 3. The confocal microscope according to claim 1, wherein the mask pattern member includes a light-shielding pattern region in which the predetermined pattern is formed and a light transmitting region.