JP2003005078A - Confocal microscope - Google Patents

Abstract

PROBLEM TO BE SOLVED: To provide a confocal microscope which is simple and inexpensive in constitution and has a high-velocity focus detecting function. SOLUTION: This microscope has a light source 1, a rotary disk 4 which is alternately formed with transparent sections to transmit rays and light shielding sections to shield the light to a parallel line form, an objective lens 8 which irradiates the surface of a sample 9 with the light transmitted through the rotary disk 4, an image forming optical system 200 which forms the image by condensing the reflected light from the sample 9 by means of the objective lens 8 and the rotary disk 4 and a CCD 13 which is arranged in the position of the image formed by the image forming optical system 200. The image forming optical system 200 is provided with a beam splitter 30 in its optical path and the beam splitter 30 is provided with a pinhole 301 and a photodetector 302 in its reflection side optical path.

Description

Detailed Description of the Invention

[0001]

TECHNICAL FIELD The present invention relates to an optical microscope, and more particularly to a confocal microscope.

[0002]

2. Description of the Related Art In recent years, a disk rotating confocal microscope capable of achieving high resolution in real time has been commercialized. In this microscope, a disc with a transmission part such as a line pattern is inserted in the optical path of a normal microscope, and the disc is rotated to aim for the confocal effect.The image of the out-of-focus surface is removed. , Only the image of the in-focus surface is extracted.

FIG. 9 shows the basic structure of a conventional confocal microscope. In this figure, the light emitted from a light source 1 passes through a first lens 2 and is incident on a beam splitter (light splitting member) 3. It is partially reflected and projected onto the rotating disk 4.
The disc 4 has a so-called line pattern in which linear light-transmitting portions and light-shielding portions are alternately formed as shown in FIG. 10, and can be rotated by a motor 5 at a substantially constant speed. ing. Only the light that has passed through the transparent portion of the disk 4 passes through the second lens 6, the quarter-wave plate 7 and the objective lens 8 and is focused on the sample 9. The reflected light from the sample 9 follows the above path in reverse, and only the light that has passed through the transparent portion of the rotating disk 4 again passes through the beam splitter 3, the third lens 11 and the image forming lens 12, and the CCD 1
Form a confocal image on 3.

In this case, the second lens 6, the quarter-wave plate 7 and the objective lens 8 constitute a confocal optical system 100, and the third lens 11 and the image forming lens 12 form the image forming optical system 2.
00 is configured. The light source 1 and the rotating disk 4 are the first
Of the sample 2, and the rotating disk 4 and the sample 9 are arranged at conjugate positions with the confocal optical system 100 interposed therebetween, whereby an image in which only the in-focus portion of the sample 9 is extracted. It is obtained on the CCD 13.

Usually, when observing a sample or measuring its height, it is necessary to move the sample in the direction of the optical axis so that the sample surface is substantially in focus. Therefore, the operator must move the sample stage or the objective lens 8 in the optical axis direction for focusing while viewing the confocal image from the CCD 13 on the monitor as a preparation for the observation, which requires a lot of man-hours. Was there. In order to reduce such man-hours,
A confocal microscope having a focus detection function has been proposed.

This is because, for example, the image obtained by the CCD 13 is image-processed by the image processing device 14, and the contrast of the sample image when the sample is moved in the optical axis direction is calculated and detected as a defocus amount. , The position with the highest contrast is clearly indicated as the in-focus position, and the drive unit 1 automatically
A method of adjusting the distance between the sample 9 and the objective lens 8 via 5, or a separate focusing optical system that does not include a rotating disk is provided so that the shield does not enter the optical path. In this way, the optical system is used to obtain a defocus amount by a conventional microscope focus detection method or to perform automatic focusing.

[0007]

As described above, the conventional confocal microscope requires an image processing device and a dedicated optical system for focusing, which is expensive. there were. Further, in the case of focus detection using an image processing apparatus, it takes time to process because it is a calculation for a two-dimensional image, and there is a problem that the apparatus becomes large in size when a dedicated optical system is used.

The present invention has been made in view of the above problems of the prior art, and an object of the present invention is to have a simple and inexpensive structure and to provide a high-speed focus detection function. To provide a focusing microscope.

[0009]

In order to achieve the above object, a confocal microscope according to the present invention has a light source, a light-transmitting portion and a light-shielding portion which are alternately formed in a linear shape. Rotating member, an objective lens that irradiates the sample with light that has passed through the rotating member, and imaging optics that collects reflected light from the sample through the objective lens and the rotating member to form an image. A confocal microscope including a system and an imaging member arranged at a position of an image formed by the imaging optical system, the image position formed by the imaging optical system, or before and after the image position. It is characterized in that a light detecting means having a minute opening is provided. According to the present invention, a light splitting member is provided in the optical path of the imaging optical system, and the light detecting means is provided in the reflection-side optical path of the light splitting member. Further, according to the present invention, the light detecting means is provided around the image pickup member.

[0010]

BEST MODE FOR CARRYING OUT THE INVENTION Hereinafter, an embodiment of the present invention will be described based on illustrated examples. FIG. 1 shows a first embodiment of the present invention. In the figure, substantially the same members and portions as those shown in the conventional example are designated by the same reference numerals, and detailed description thereof is omitted. In this first embodiment, after the reflected light from the sample 9 passes through the rotating disk 4,
A confocal image of the sample 9 is formed on the CCD 13 by the imaging optical system 200, and is partially reflected by the beam splitter 300 as a light splitting member to reach the pinhole 301. Behind the pinhole 301 is the photo detector 3
02 is arranged, and the output end of the photo detector 302 is connected to the focus detection unit 303.

When the sample 9 is at the focal position of the objective lens 8, the reflected light reflected by the sample 9 is condensed by the light transmitting portion of the rotating disk 4. In this case, a spot having a diameter substantially equal to the line width L is formed on the light transmitting portion. On the other hand, the sample 9 is the objective lens 8
In the focal position of 1, the reflected light reflected by the sample 9 is not collected by the light transmitting portion of the rotating disk 4, but is collected before or after the light transmitting portion. In this case, a spot having a diameter larger than the line width L (see FIG. 10) is formed on the transparent portion. Therefore, in the direction of the line width L (direction A in FIG. 10), light is transmitted only in the range of the line width L, which is the same as the state in which almost no light is transmitted. On the other hand, since the transmission range is not limited in the direction orthogonal to the direction of the line width L (direction B in FIG. 10), the reflected light is transmitted through the translucent portion even though it is out of the focus position. Will pass through. Therefore, the amount of light that has passed through the translucent portion does not change significantly before and after the focused state.
As a result, even if an attempt is made to detect the in-focus state due to a change in the amount of light passing through the transparent portion, it becomes difficult to detect it.

Therefore, although the pinhole 301 is arranged in this embodiment, for example, the magnification of the imaging optical system 200 is set to 1
If it is doubled, the opening diameter of the pinhole 301 is made substantially equal to or smaller than the line width L of the transmission part of the line pattern of the rotary disk 4. By doing so, it is possible to remove light from other than the focus position even in the direction orthogonal to the direction of the line width L. Thereby, also with respect to the light transmitted in the direction orthogonal to the direction of the line width L, when the sample deviates from the focal position of the objective lens 8, the amount of light passing through the opening of the pinhole 301 can be reduced. Conversely,
Only when the sample is at the focal position of the objective lens 8, the sample passes through the opening of the pinhole 301. As a result, the amount of light that has passed through the opening of the pinhole 301 largely changes before and after the focused state, and accurate focus detection can be performed. When the magnification of the imaging optical system 200 is other than 1, the size of the opening of the pinhole 301 is determined as follows. That is, assuming that the opening of the pinhole 301 is projected onto the position of the rotating disk 4 by the imaging optical system 200, the size of the image of the opening at this time is substantially equal to or smaller than the line width L of the light transmitting portion. The size of the opening may be set as follows.

In such a structure, since the rotating disk 4 is rotating at a substantially constant speed, when the distance between the sample 9 and the objective lens 8 is constant, the amount P of light entering the photodetector 302 is as shown in FIG. Rotating disc 4 as shown
The waveform is repetitive at each time t required for one rotation. Here, assuming that the sample 9 or the objective lens 8 moves at a constant speed in the optical axis direction, the received light amount P becomes maximum when the sample 9 is in focus and becomes small before and after that, and thus is shown in FIG. The amount of received light changes as described above.

FIG. 4 shows an example of the circuit configuration of the focus detection section 303. As is clear from this figure, the received light current from the photodetector 302 is converted into a current voltage by the amplifier 304, input to the comparator 306 via the low-pass filter (LPF) 305, and compared with the threshold value 307. In this case, the threshold value 307 is set to about a minute amount of light as shown by a broken line in FIG. When the amount of received light P exceeds the threshold value 307, focus detection is permitted, and when it falls below the threshold value 307, focus detection is prohibited (see FIG. 5). The reason for doing this is as follows.

In the focus detection method according to this embodiment, the position where the amount of received light is maximum is determined as the in-focus position. For example,
Focusing on the signal indicated by X in FIG. 3, since the amount of received light is zero before and after that, the signal of X becomes maximum. Therefore, if focus detection is always permitted, the X position will be erroneously determined to be the in-focus position. On the other hand, in the present embodiment, the focus detection is performed only when the reflected light from the sample 9 reaches the photodetector 302, so the above-mentioned erroneous determination does not occur.

Next, referring to FIG. 4 again, the output of the comparator 306 is used as a hold trigger of the sample hold amplifier 308, and the voltage corresponding to the amount of received light at that time is held. In this case, the output of the sample hold amplifier 308 is as shown in FIG. 6, and the position where the largest output is obtained is the in-focus position. Therefore, if this is displayed by a level meter or the like, it becomes possible to clearly notify the defocus. As is clear from this description, if the distance between the sample 9 and the objective lens 8 is adjusted so that the output signal of the sample hold amplifier 308 has a peak, focusing can be automatically performed.

As described above, according to the present embodiment, the number of additional parts on the optical path is small, and since the focusing is performed from the information of one point on the sample by the electrical processing, the high-speed focusing is possible. It is possible to provide a confocal microscope capable of taking out.

FIG. 7 is a diagram showing the essential parts of a second embodiment of the present invention. In the figure, the same members as those in the first embodiment are designated by the same reference numerals, but the configuration of other parts not shown is the same as that in the first embodiment. In this embodiment, another beam splitter 400 is arranged in the optical path reflected by the beam splitter 300 to form two optical paths, and the pinhole 301a and the photodetector 3 are provided in the transmitted optical path.
02a also has a pinhole 301b in its reflected light path.
And a photo detector 302b are arranged respectively. In this case, the image of the transmission part of the rotating disk 4 is formed at a position separated from the beam splitter 400 by a distance d ₁ . Therefore, the pinhole 301a is arranged at a distance d ₁ at a position away from the distance d ₂ by the beam splitter 400 as a reference, whereas, pinhole 301b is a distance d ₂ on the basis of the position of the distance d ₁ beam They are arranged at positions close to the splitter 400, respectively.

According to the second embodiment, when the transmitting portion of the rotary disk 4 has a linear shape, the light beam diameter in the direction orthogonal to the direction of the line width L of the transmitting portion depends on the focused state. Since the pinholes 301a and 301b are arranged in this manner, each photodetector 302 can be changed.
It is possible to detect the shift amount and the shift direction of the focus from the outputs of a and 302b.

In any of the above embodiments, instead of using a pinhole and a photodetector corresponding thereto,
A photodetector having a light-receiving surface having the same size as the pinhole can be used, but this can further simplify the configuration.

FIG. 8 is a diagram showing the essential parts of a third embodiment of the present invention. In each of the above embodiments, the beam splitter 300 splits the light and guides it to the photodetector. Instead, in the present embodiment, as shown in FIG. Is used. The effective area of the CCD 13 is usually rectangular, but the image (field of view) area formed by the optical system is often circular. Therefore, the peripheral portion of the image formed by the optical system is not formed on the CCD 13. Therefore, by disposing the pinhole 301 and the photodetector 302 in this portion, it is possible to cope with the loss of light amount that may occur when the beam splitter is used.

If photodetectors are arranged at a plurality of positions on the image plane so that one of them can be selected,
It is possible to make a judgment in a place where the aberration of the objective lens used is small. Further, if signals at a plurality of locations are added and used, it is convenient because focus detection can be performed while reducing the influence of edge portions where the reflected light from the sample is reduced.

Further, in the above embodiment, the amplifier 304
Subsequent electric circuits may be replaced with a CPU and software. In this case as well, the determination can be made based on the information of one point, so that the speed can be increased as compared with the case of performing image processing of a two-dimensional image. Further, it becomes possible to easily cope with automation.

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 2 or 3, wherein the light detecting means is a photodetector, and the opening is formed on a light receiving surface of the photodetector.

(2) The confocal microscope according to claim 2 or 3, wherein the light detecting means comprises a pinhole and a photodetector, and the opening is an opening of the pinhole.

(3) A light splitting member is provided in the optical path of the imaging optical system, and the light detecting means is provided at the image position in one optical path formed by the light splitting member. The confocal microscope according to claim 2.

(4) The first light splitting member is provided in the optical path of the imaging optical system, and the light detecting means is provided before and after the image position in one optical path formed by the light splitting member. The confocal microscope according to claim 2.

[0028]

As described above, according to the present invention, it is possible to provide a confocal microscope which has a simple and compact structure and can perform focus detection at high speed.

[Brief description of drawings]

FIG. 1 is an overall configuration diagram of a first embodiment of the present invention.

FIG. 2 is a diagram showing changes in the amount of light received by a photodetector due to the rotation of a rotating disk.

FIG. 3 is a diagram showing changes in the amount of received light when the photodetector is in focus with the rotation of a rotating disk.

FIG. 4 is a block diagram showing an example of a circuit configuration of a focus detection unit used in the first embodiment.

FIG. 5 is a diagram showing changes in focus detection inhibition and permission signals.

FIG. 6 is a diagram showing a change in output of a sample hold amplifier used in the first embodiment.

FIG. 7 is a diagram showing a main part of a second embodiment of the present invention.

FIG. 8 is a diagram showing a main part of a third embodiment of the present invention.

FIG. 9 is a diagram showing an overall configuration of a conventional confocal microscope.

FIG. 10 is a plan view of a rotating disk.

[Explanation of symbols]

1 light source 2,6,11 lens 3,300,400 beam splitter 4 rotating discs 5 motor 7 1/4 wave plate 8 Objective lens 9 samples 10 sample table 12 Imaging lens 13 CCD 14 Image processing device 15 Drive 100 confocal optical system 200 Imaging optical system 301, 301a, 301b Pinhole 302, 302a, 302b Photodetector 303 Focus detection unit 304 amplifier 305 Low-pass filter 306 comparator 307 threshold 308 sample hold amplifier L line width

Claims (3)

[Claims] 1. A rotating member in which a light source, a transmitting portion that transmits light, and a light shielding portion that shields light are alternately formed in a linear shape, and an objective lens that irradiates the sample with the light transmitted through the rotating member. When,
An imaging optical system that collects reflected light from the sample through the objective lens and the rotating member to form an image, and an imaging member arranged at the position of the image formed by the imaging optical system. A confocal microscope comprising: a confocal microscope, which is provided with a photodetection means having a minute aperture portion at the image position formed by the imaging optical system or before and after the image position. 2. The confocal lens according to claim 1, wherein a light splitting member is provided in the optical path of the image forming optical system, and the light detecting means is provided in the optical path on the reflection side of the light splitting member. microscope. 3. The confocal microscope according to claim 1, wherein the light detection means is provided around the image pickup member.