JPH10104523A - Confocal microscope - Google Patents

Abstract

[PROBLEMS] Particularly in a confocal microscope using a low-magnification objective lens, it is possible to form a high-resolution image by using the NA inherent in the lens as effectively as possible and to form a slit. Also solves the problem of shading when using. SOLUTION: Light emitted from a light source 31 is transmitted to an incident end 32a.
And a fiber bundle in which the emission ends 32b are arranged in a line.
The sample is made incident on a slit 33 through a scanning mirror 18 that deflects the image of the slit in a direction orthogonal to the confocal optical systems 16, 19, 21, and 23 and the slit.
22 and project the reflected light from the sample through the scanning mirror to 1
The light is made incident on the two-dimensional linear image sensor array 24. Since the slit 33 is used, light is incident on the entire objective lens 21, so that the effective NA is not reduced, and a high-resolution image can be obtained over a wide field of view. Also, fiber bundle
Since the emission end 32b of the line 32 has a linear shape corresponding to the shape of the slit 33, the amount of light passing through the slit is uniform, and shading does not occur.

Description

DETAILED DESCRIPTION OF THE INVENTION

[0001]

BACKGROUND OF THE INVENTION 1. Field of the Invention The present invention relates to a confocal microscope, and more particularly to a confocal microscope suitable for using a wide-field, low-magnification objective lens.

[0002]

2. Description of the Related Art The present applicant has already proposed a laser microscope employing a confocal optical system, and its basic configuration is described, for example, in Japanese Patent Publication No. 4-38325.
This conventional confocal microscope employs a configuration as shown in FIG. That is, the laser beam emitted from the laser light source 11 is incident on the acousto-optic deflector 13 as a light beam having a large diameter by the beam expander 12. The acousto-optical deflecting device 13 is driven at a predetermined cycle related to the horizontal synchronizing signal of the television to deflect the laser beam in the main scanning direction (horizontal scanning direction). The laser beam deflected in this way is made incident on a cylindrical lens 14 having a refraction function only in the direction of deflection, and then made incident on a relay lens 15 to form a bright line A. The laser beam forming the bright line A is incident on the polarization beam splitter 17 via the relay lens 16, and the laser beam reflected on the polarization plane of the polarization beam splitter is incident on the scanning mirror 18 having a vibrating mirror. The scanning mirror 18 is driven in synchronization with the vertical scanning of the television to deflect the laser beam in a sub-scanning direction (vertical scanning direction) orthogonal to the main scanning direction.
Make it incident on 19. Therefore, a raster B by the laser beam is formed by the relay lens 19. The image of the raster B is formed on the sample surface 22 through the / 4λ plate 20 and the objective lens 21, and thereby the sample surface 22 is raster-scanned.

The laser beam reflected by the sample surface 22 is applied to an objective lens 21, a 1/4 λ plate 20, a relay lens 19, and a scanning mirror 18.
Then, the light enters the polarization beam splitter 17. here,
By passing through the scanning mirror 18, the deflection in the sub-scanning direction is canceled out. Further, the polarization plane of the laser beam is rotated 90 degrees by passing through the quarter λ plate 20 twice, so that the polarization plane of the polarization beam splitter 17 is transmitted. The laser beam transmitted through the polarizing beam splitter 17 in this manner is focused on the predetermined focal plane by the imaging lens 23.
It is converged on the dimensional image sensor array 24. After the image signal read from the one-dimensional image sensor array 24 is processed by the signal processing circuit 25, it is projected on a television monitor 26. Here, the bright line A and the raster B are conjugate, the raster B and the raster formed on the sample surface 22 are also conjugate, and the raster B and the image sensor array 24 are conjugate. Is configured.

[0004]

In the conventional confocal microscope shown in FIG. 1, an acousto-optical device 13 is used to deflect a laser beam in a horizontal direction.
In general, since the deflection angle of the acousto-optical device is not so large, the length of the bright line A is short, and accordingly, the horizontal dimension of the raster formed on the sample surface 22 is also short. That is, there is a problem in that the observation field of view is limited by the deflection angle of the acousto-optical device 13, and observation cannot be performed over a wide field of view.

Further, various objective lenses are prepared in a confocal microscope, and an optimum objective lens is selected and used according to the observation purpose. FIG. 2 is a diagram for explaining the NA (numerical aperture) of the objective lens. The NA is the sine of the angle α between the optical axis of the objective lens and the outermost light beam, and is an element that determines the resolution. . That is, the objective lens 21a having a large NA has a high resolution, while the objective lens 22b having a small NA has a low resolution.

As described above, in the conventional confocal microscope, the horizontal dimension of the raster is reduced, but this is an obstacle when a wide field of view is desired. In order to solve this problem, the field of view can be widened by appropriately selecting the focal length ratio of the relay lenses 15 and 16, but in that case, the light beam incident on the objective lens 21 becomes extremely small, and as a result, the objective lens 21 Only a small part of the center will be used. As shown in FIG.
When a is used, the light is incident on the entire area of the objective lens, so that there is no influence.
When b is used, only the central part of the lens is used, so that the effective NA is significantly reduced, and therefore the resolution is extremely low.

FIG. 4 shows the relationship between NA and magnification of a currently available microscope objective lens. The microscope objective lens includes an oil immersion system and a drying system. The oil immersion system generally has a high NA and a high magnification, and the drying system has a low NA and a low magnification. Therefore, when a low-NA, low-magnification dry-type objective lens is used in the above-mentioned confocal microscope, there is a problem that the resolution is reduced as described with reference to FIG. That is, in the conventional confocal microscope, when an objective lens having a low NA and a low magnification is used, the performance of the objective lens cannot be sufficiently exhibited, and a high-resolution image cannot be obtained. There is.

FIG. 5 explains how the NA of the objective lens is related to the observation of the inclined surface of the sample. In the high NA objective lens 21a, when a laser beam is irradiated on the inclined surface of the sample 27, a part of the reflected light on the inclined surface of the sample goes toward the objective lens.
Although a part of the reflected light enters the objective lens, when the low NA objective lens 21b is used, the reflected light on the inclined surface of the sample 27 does not go to the objective lens, and therefore does not enter the objective lens. As described above, in the confocal microscope, there is a problem that a sample with a rough surface cannot be observed over a wide field of view when a low-magnification objective lens is used.

Further, a conventional confocal microscope described in US Pat. No. 4,241,257 is also known. In this known confocal microscope, a laser light source 11, a beam expander 12, an acousto-optical device 13, and a cylindrical lens shown in FIG.
Instead of 14, a high-intensity lamp such as a high-pressure mercury vapor discharge tube is provided, and light emitted from the lamp is focused by a condenser lens on a slit extending in the main scanning direction. By arranging the slit at a position conjugate with the sample surface with respect to the objective lens, a confocal optical system is formed. In a confocal microscope using such a slit, a wider area of the objective lens can be used as compared with the confocal microscope shown in FIG. 1, so a wide-field, low-magnification, low-NA objective lens is used. In this case, the resolution does not decrease, the oblique surface of the sample can be observed, and the rough sample surface can be observed over a wide field of view. On the other hand, the light from the light source lamp is condensed using a condenser lens so that it is effectively incident on the slit. However, the amount of light from the end of the slit is lower than the amount of light from the center, and the display image However, there is a problem that so-called shading occurs and the quality of an image is deteriorated.
Furthermore, at the end of the slit, a light beam whose traveling direction is greatly inclined with respect to the optical axis also passes, and this has a problem that this adversely affects image formation. In order to solve such problems, experiments were conducted using light sources of various configurations.However, with currently available light sources, the problem of non-uniform light intensity and the problem of light rays traveling in the direction of a large inclination at the end of the slit was found. Could not be resolved.

Furthermore, in the above-mentioned two types of known confocal microscopes, only a confocal observation with a very small depth of focus can be performed. In particular, in the case of FIG.
Since an objective lens with a high magnification is often used, it is difficult to find a desired portion of the sample to be observed, and there is a problem that it takes time.

Accordingly, an object of the present invention is to eliminate the above-mentioned disadvantages of the conventional confocal microscope, eliminate the disadvantages of the conventional confocal microscope shown in FIG. An object of the present invention is to provide a confocal microscope capable of solving the disadvantages and effectively using an objective lens having a wide field of view and a low magnification.

[0012]

A confocal microscope according to the present invention has a light source device and a large number of fibers, one end of which is bound to receive light emitted from the light source to form an incident end, and this light is used as an incident end. A fiber bundle having the other end arranged in a line shape so as to emit light as a line-like light and having an emission end, a slit for limiting light emitted from the emission end of the fiber bundle, and a position conjugate with the slit A confocal optical system including an objective lens that forms a line-shaped bright line on the placed sample, and forms reflected light or transmitted light from the sample on a predetermined image plane, and an optical path of the confocal optical system. A deflecting device inserted in the slit and deflecting the light restricted by the slit in a direction orthogonal to a longitudinal direction thereof, and the scheduler to receive a sample image formed by the confocal optical system. A one-dimensional image sensor array arranged on an image forming surface, an image processing circuit for processing an output signal from the one-dimensional image sensor array to output an image signal, and an image signal output from the image processing circuit. Display means for displaying a direction in which the slit extends as a main scanning direction and a direction of deflection by the deflecting device as a sub-scanning direction.

In a preferred embodiment of such a confocal microscope according to the present invention, the light source device comprises a high-pressure mercury vapor discharge lamp. Further, the slit is formed in an opaque plate-shaped member, an opening larger than the slit is formed in the plate-shaped member, and the slit and the opening formed in the plate-shaped member can be selectively inserted into the optical path. It is also preferable to perform a switching between confocal and non-confocal. If switching to non-confocal can be performed in this manner, focus adjustment can be performed very easily, a desired portion of the sample to be observed can be quickly searched, and then the confocal system can be switched to a confocal system. Detailed observations can be made.

[0014]

FIG. 6 is a diagram showing the configuration of one embodiment of a confocal microscope according to the present invention. The same components as those of the conventional confocal microscope shown in FIG. 1 are used in FIG. The same reference numerals as those shown above are used. Compared to the conventional confocal microscope shown in FIG. 1, a polarizing beam splitter 17, a scanning mirror 18,
The configuration of the relay lens 19, the 1/4 λ plate 20, the objective lens 21, the imaging lens 23, and the one-dimensional image sensor array 24 is the same as that of the conventional one.

In the present invention, a light source 31 having a high-intensity lamp, such as a high-pressure mercury vapor discharge tube with a reflecting mirror, is used.
And the light emitted from this light source is made incident on the binding end 32 a of the fiber bundle 32. Output end 32b of this fiber bundle 32
In FIG. 7, the fibers are arranged in a line as shown in FIG. Since such a fiber bundle 32 is used, the exit end 32
From b, a uniform amount of light is emitted at both the center and the end, and the traveling direction of the emitted light is also determined by the emission angle of the fiber. The inclination angle is small. The light emitted from the emission end 32 b of the fiber bundle 32 is made incident on the slit 33. The slit 33 is arranged at a position conjugate with the sample surface 22 with respect to the objective lens 21 to form a confocal optical system. Further, a polarizing plate 34 is disposed behind the slit 33.

In the confocal microscope of the present invention, a raster 33 is formed by forming an image of the slit 33 by the relay lenses 16 and 19 using the slit 33, and a reduced image of the raster is formed on the sample surface 22 by the objective lens 22. Then, the sample surface is raster-scanned, and the raster image is formed on the light receiving surface of the one-dimensional image sensor array 24 by the objective lens 22, the relay lens 19 and the imaging lens 23. In this case, since the light incident on the objective lens 21 covers not only the central portion of the objective lens but also a wide range, the effective NA of the objective lens increases as described with reference to FIG. This is particularly advantageous when an object lens having a wide field of view and a low magnification is used. In addition, when the effective NA increases, FIG.
As described in relation to the above, the inclined surface of the sample 22 can also be observed. Further, according to the present invention, the light exiting end 32b of the fiber bundle 32 in which the light incident on the slit 33 is linearly arranged
, The light quantity becomes uniform and the inclination angle with respect to the optical axis becomes small, so that a high-quality image can be displayed.

FIG. 8 is a diagram showing the configuration of another embodiment of a confocal microscope according to the present invention, including an image processing portion for processing in accordance with a high definition television standard. The description will be omitted.
In this example, the slit 33 is formed in an opaque plate 35, and an opening 36 having a width larger than that of the slit 33 is also formed in the opaque plate. It is configured to be able to move up and down as shown.
When used as a confocal microscope, the slit 33 is inserted into the optical path, and when used as a non-confocal microscope, the opening 36 is inserted into the optical path. In this way, by moving the opaque plate 35, it is possible to easily switch between confocal observation and non-confocal observation. Therefore, when observing a wide range of the sample, coarse adjustment of the focus can be easily performed by performing non-confocal observation with a large depth of focus, and as a result, a desired portion of the sample can be easily and quickly found. be able to.

As shown in FIG. 8, in order to adjust the focus of the objective lens 21, a drive device 41 for driving the objective lens in the optical axis direction is provided, and the position is detected by a laser holoscale 42 and an encoder 43. . The driving device 41 and the encoder 43 are connected to a computer 45 via a system controller 44, and a console 46 is further connected to the system controller 44. The image signal read from the one-dimensional image sensor array 24 is supplied to an amplifier 47.
After the amplification, the image data is supplied to the image processing circuit 48. The image processing circuit 48 includes an A / D conversion circuit, a focus shift memory, a D /
An A-conversion circuit and the like are provided to process image signals in accordance with high-definition television standards. This processing is, for example, 1125
/ 60 HDTV rate (33.75KHz horizontal, 60Hz vertical, 2: 1
Interlaced). The image signal thus obtained is supplied to the monitor 49, so that a high-quality image can be displayed.

The focus movement memory provided in the image processing circuit 48 stores the amount of movement in the Z-axis direction supplied from the computer 45 and the image signal from the image sensor array 24 in association with each other. Thus, extremely accurate three-dimensional measurement can be performed. That is, position (height) information in the Z-axis direction at which the maximum luminance is obtained is stored for each pixel, and when performing three-dimensional measurement, the position information in the Z-axis direction thus stored is read out. This is processed by the computer 45. In this way, it is possible to accurately measure the distance in the Z-axis direction of the sample surface by utilizing the characteristic of the confocal microscope having a very shallow depth of focus.

As described above, in this embodiment, since the image processing is performed in accordance with the HDTV standard, the monitor 49 is also assumed to comply with the standard. Therefore, the number of display pixels on the monitor 49 reaches 2,000,000, and it is possible to display a much higher definition image than a conventional confocal microscope (about 400,000 pixels) according to a general television standard. Therefore, when measuring the dimensions based on the image displayed on the monitor 49, the accuracy can be significantly increased.

In the embodiment shown in FIG. 8, when various objective lenses 21 are used, the magnification, NA,
Table 1 shows the magnification on the monitor, the size of the field of view, the pixel size, and the position measurement accuracy (σ) in the Z-axis direction.

[0022]

[Table 1]

FIGS. 9 and 10 show that the magnification shown in Table 1 is 10%.
The relationship between the spatial frequency (lines / mm) and the degree of modulation (%) when using × and 20 × objective lenses, respectively, is the width of the slit 33.
(mμ) is a graph shown as a parameter, where the width is 10
The case of 00 μm is a case where the opening 36 is inserted into the optical path.
According to the present invention, it is understood that extremely high-performance image formation can be performed by using the slit 33 having a width of 100 μm or less. As described above, according to the confocal microscope of the present invention, it is understood that an image with extremely high resolution and contrast can be obtained even when the low-magnification objective lens is used.

The present invention is not limited only to the above-described embodiment, and many modifications and variations are possible. For example, in the above-described embodiment, the reflection type confocal microscope receives the reflected light from the sample, but the transmission type confocal microscope may receive the transmitted light of the sample. Further, in the above-described embodiment, the polarizing beam splitter is used to separate the light incident on the sample and the reflected light from the sample, but other optical path splitting means such as a half mirror can be used. In that case, the 1/4 λ plate is unnecessary. Furthermore, in the above-described embodiment, a scanning mirror configured to vibrate the reflecting mirror is used to deflect the light beam in the sub-scanning direction. However, a scanning mirror that rotates the reflecting mirror may be used.

[0025]

As described above, in the confocal microscope according to the present invention, an acousto-optical device having a small deflection angle is not used for deflecting the light beam in the main scanning direction. Light can be incident, so that the effective NA does not decrease,
It is possible to form a high-resolution image by effectively utilizing the NA inherent in the objective lens, and to observe a wide-field of a sample having a rough surface. Such advantages become remarkable especially when a low NA and low magnification objective lens is used.
Furthermore, as compared with a conventional confocal microscope using a slit, the present invention uses a fiber bundle having a linear output end, so that the amount of light transmitted through the slit is uniform at both the center and the end, so-called A high-quality image without shading can be obtained. in this case,
If the distance between the line end of the fiber bundle and the slit is adjusted according to the objective lens used, a higher-quality image free from stray light and flare can be displayed.

[Brief description of the drawings]

FIG. 1 is a diagram showing a configuration of a confocal microscope using a conventional acousto-optical device.

FIG. 2 is a diagram for explaining a relationship between NA of an objective lens and resolution.

FIG. 3 is a diagram for explaining a relationship between a magnification of an objective lens and an effective NA;

FIG. 4 is a graph showing the correlation between the magnification of an objective lens used in a microscope and NA.

FIG. 5 is a diagram for explaining a relationship between an NA of an objective lens and a sample inclined surface.

FIG. 6 is a diagram showing a configuration of an embodiment of a confocal microscope according to the present invention.

FIG. 7 is a perspective view showing the configuration of the fiber bundle.

FIG. 8 is a diagram showing a configuration of another embodiment of the confocal microscope according to the present invention.

FIG. 9 is a graph showing performance when an objective lens with a magnification of 10 is used.

FIG. 10 is a graph showing performance when an objective lens with a magnification of 20 is used.

[Explanation of symbols]

 16 Relay lens 17 Polarizing beam splitter 18 Scanning mirror 19 Relay lens 20 1/4 λ plate 21 Objective lens 22 Sample surface 23 Imaging lens 24 One-dimensional image sensor array 31 Light source 32 Fiber bundle 32a Incident end 32b Exit end 33 Slit 34 Polarized Plate 35 Opaque plate 36 Aperture 41 Objective lens drive 42 Laser horoscale 43 Encoder 44 System controller 45 Computer 46 Console 47 Amplifier 48 Image processing circuit 49 Monitor

Claims (3)

[Claims] 1. A light source device, comprising a number of fibers, one end of which is bundled to receive light emitted from the light source to form an incident end, and the other end is formed so as to emit this light as linear light. A fiber bundle arranged as an emission end arranged in a line, a slit for limiting light emitted from the emission end of the fiber bundle, and a line-shaped bright line formed on a sample placed at a position conjugate with the slit. A confocal optical system including an objective lens that forms an image and reflects reflected light or transmitted light from the sample on a predetermined imaging surface; and light inserted into the optical path of the confocal optical system and restricted by the slit. A deflecting device for deflecting the sample in a direction perpendicular to the longitudinal direction thereof, a one-dimensional image sensor array arranged on the predetermined imaging plane to receive the sample image formed by the confocal optical system, and An image processing circuit that processes an output signal from the one-dimensional image sensor array to output an image signal; and converts the image signal output from the image processing circuit into a main scanning direction with the slit extending direction as a main scanning direction. A confocal microscope, comprising: display means for displaying a direction of deflection by the device as a sub-scanning direction. 2. The confocal microscope according to claim 1, wherein the light source device is a light source device including a high-pressure mercury vapor discharge lamp. 3. The slit is formed in an opaque plate member, an opening larger than the slit is formed in the plate member, and the slit and the opening formed in the plate member are selectively placed in the optical path. 2. The confocal microscope according to claim 1, wherein the confocal microscope is configured to switch between confocal and non-confocal so as to allow insertion.