JPH11183803A - Confocal microscope device - Google Patents

Abstract

PROBLEM TO BE SOLVED: To process images with a dynamic range above the dynamic range of an AD converter by varying the quantity of measurement light or the signal level of a slice image according to the quantity of reflected light from a sample, weighting the variation quantity, and searching for the slice image. SOLUTION: The photographic signal of a camera 6 has its level controlled by an AGC circuit 21 and is inputted as an NTSC signal to the AD converter 22. When the sample 4 has a steep slope or a small peak, the gain is increased by passing the signal through an AGC circuit 21 to send the NTSC signal which is raised in signal level to the AD converter 22. Gain values of the AGC circuit 21 by frames are sent to the AD converter 23. A CPU 24 while weighting a slice image of the NTSC signal by a gain makes a picture search and displays the image on a CRT 12 through a frame memory 11. The use of the AGC circuit 21 expands the dynamic range and even if the quantity of the reflected light from the sample 4 is small, the picture search can securely be made.

Description

DETAILED DESCRIPTION OF THE INVENTION

[0001]

BACKGROUND OF THE INVENTION 1. Field of the Invention The present invention relates to a confocal microscope apparatus, and more particularly to a confocal microscope apparatus for reconstructing a plurality of confocal slice images to obtain a three-dimensional stereoscopic image for expanding a dynamic range when acquiring a slice image. It is about improvement.

[0002]

2. Description of the Related Art In recent years, there has been an increasing demand for measurement of a micro three-dimensional shape with the improvement of processing techniques. In the field of biotechnology, attention has been paid to the three-dimensional structure of molecules. There are interference microscopes, confocal microscopes, and the like that respond to these.

Here, a confocal microscope will be described. FIG.
1 is a configuration diagram illustrating an example of a conventional confocal microscope device.
The laser beam emitted from the light source 1 through the confocal scanner 2 is focused by the objective lens 3 of the microscope and is irradiated on the sample 4. The light reflected by the sample 4 returns to the confocal scanner 2 via the objective lens 3 again. An image of the sample surface formed by the scanner 2 is picked up by a camera 6, and an output signal thereof is converted into a digital signal by an analog / digital converter 7 and read by a central processing unit (hereinafter referred to as a CPU) 8.

The confocal scanner 2 can two-dimensionally scan the light beam irradiating the sample 4 in a direction perpendicular to the optical axis (XY axis direction). Therefore, the camera 6 can capture an image of the scanning surface of the sample, that is, a slice image.

On the other hand, the stage 5 on which the sample 4 is placed can be moved in the optical axis direction (also called the height direction or the Z axis direction) by the stage moving mechanism 9. The stage moving mechanism 9 is controlled by the CPU 8, and the moving amount is controlled by the CPU 8.
8 can be confirmed.

A slice image taken while moving the stage can be stored in the memory 10. CPU
Reference numeral 8 reconstructs, for example, a three-dimensional display image of the sample from the Z-axis movement amount and each of the slice images, and stores the image data in the frame memory 11. The image in the frame memory 11 is CR
It is displayed on T12.

[0007] Further, since such a confocal microscope apparatus employs a pinpoint illumination method, a combination of laser light or non-laser light and a pinhole is used. According to such a combination, an effect is obtained that scattered light from other than the measurement point can be easily prevented.

In addition, a pinhole is installed as a spatial filter in front of the light receiver, and light from other than the measurement point is cut off. That is, noise light existing in the same plane of the measurement point forms an image beside the pinhole, and is cut so as not to enter the light receiver.

Further, the light at the point shifted in the optical axis direction is spread by the objective lens in front of the pinhole, so that the received light is drastically reduced. In short, the light amount increases only when there is a focus on the optical axis, and the light amount becomes almost zero at a point out of focus. From this, it is known that the confocal microscope has a resolution also in the optical axis direction.

With the confocal microscope having the above-described configuration, only one point in a three-dimensional space can be measured. If the light irradiating the sample is two-dimensionally scanned in a direction perpendicular to the optical axis, a slice image of the sample in a three-dimensional space can be obtained.

Since the amount of light peaks at the focal position, when a sample having a surface such as a semiconductor is measured, the optical axis position where the amount of light becomes maximum is considered to be the surface of the sample. Therefore, a position where the maximum amount of light is provided in the optical axis direction among a plurality of slice images is peak-searched for each pixel, a height at which the peak value of each pixel is obtained is obtained, and an image is reconstructed based on those heights. Thereby, a three-dimensional surface shape image of the sample can be obtained.

[0012]

However, if the sample has a steep slope or the surface reflectance is low and the peak is very small during scanning, the peak search as it is will result in noise. There is a problem that image quality is deteriorated due to picking up. That is, for example, in the case of an 8-bit AD converter 7, the dynamic range is limited to 8 bits.

The present invention solves the above problems,
An object of the present invention is to provide a confocal microscope device capable of easily expanding a dynamic range.

[0014]

In order to achieve the above object, according to the first aspect of the present invention, a substrate having a large number of minute openings is rotated and irradiation light passing through the minute openings of the substrate is rotated. Is configured to scan the sample, receive reflected light from the sample to obtain a slice image of the sample, and relatively move the sample in the optical axis direction to obtain the slice image. In the confocal microscope apparatus, a control unit that changes a measured light amount or a signal level of a slice image in accordance with a light amount of reflected light from the sample, and weights a change amount of the measured light amount or a signal level of the slice image to obtain the sample. Image reconstruction means for peak-searching a slice image at each relative movement position in the optical axis direction for each pixel to obtain a three-dimensional image of the sample or an image with a deep focal depth. It is characterized in.

According to the first aspect of the present invention, the measured light amount or the signal level of the slice image is changed by the control means in accordance with the light amount of the reflected light from the sample. That is, control is performed so that the measured light amount or signal level always falls within a certain predetermined range. This improves the SN ratio. Then,
The peak search is performed on the slice image at each position in the optical axis direction of the sample while weighting the amount of the change when the image is reconstructed by the image reconstructing means. In this way, a three-dimensional image of the sample or an image with a large depth of focus can be easily obtained.

[0016]

DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS The present invention will be described below in detail with reference to the drawings. FIG. 1 is a configuration diagram showing one embodiment of a confocal microscope device according to the present invention. FIG. 1 shows an embodiment of a confocal microscope apparatus for calculating a height image of a sample. 1, the same reference numerals are given to the same parts as those in FIG. 4, and the description of those parts will be omitted.

According to the present invention, there is provided a control means for changing an image signal or a measured light amount according to the amount of reflected light from a sample,
It is characterized by having an image reconstructing means for peak-searching each slice image while weighting the changed amount to the slice image to obtain a three-dimensional image of the sample or an image having a deep focal depth.

In this embodiment, an automatic gain control circuit (AGC circuit) and an AD converter are used as the control means, and a CPU is used as the image reconstruction means. Hereinafter, each component and operation will be described in detail.

The AGC circuit 21 controls the signal level of a slice image output from the camera 6, and the output (slice image) of the controlled signal level is sent to the AD converter 22 as an NTSC signal. The gain value of the AGC circuit 21 is sent to the AD converter 23.

The CPU 24 performs a peak search while weighting the slice image of the AD converter 22 with the gain value of the AD converter 23. The CPU 24 drives the stage moving mechanism 9 and can read the Z-direction moving amount via the AD converter 25.

The operation of such a configuration will be described below. The operation up to the point where the camera 6 takes a slice image of the sample 4 is the same as in the conventional example. The photographing signal of the camera 6 is guided to the AGC circuit 21, the level of which is controlled, and input to the AD converter 22 as an NTSC signal.

At this time, if the sample 4 has a steep slope or the peak is very small while scanning, the confocal slice image from the camera 6 is data with large noise and small contrast, but AGC. N through which the gain is increased and the signal level is raised by passing through the circuit 21
The signal is sent to the AD converter 22 as a TSC signal.

AGC for each frame at this time
The gain value of the circuit 21 is sent to the AD converter 23. CP
U24 performs peak search while weighting the slice image of the NTSC signal read from the AD converter 22 with the gain value read from the AD converter 23.

In a conventional apparatus, when an 8-bit AD converter is used, the dynamic range is limited by the 8-bit AD converter. However, in the present invention, AG
By using the C circuit 21, the dynamic range can be expanded to (gain value of the AGC circuit) × (8-bit AD converter), and even if the amount of reflected light from the sample 4 is small, noise is not mixed in. The peak search can be performed reliably.

The slice image subjected to the peak search in this manner is written into the frame memory 11 and displayed on the CRT 12.

The foregoing description has been directed to specific preferred embodiments for the purpose of describing and illustrating the invention. Therefore, the present invention is not limited to the above-described embodiments, and includes many more modifications without departing from the spirit thereof.
This includes deformation.

For example, the AD converter 2 of the AGC circuit 21
Frame memory 11 can be used for both 2 and 23. In this case, since the frame memory has an AD conversion function, not only the AD converters 22 and 23 are unnecessary, but also the advantage that the slice image and the gain value can be simultaneously processed by skillfully using the frame memory as follows. Also occurs.

Successful use of a frame memory is as follows. The NTSC signal has 525 scanning lines per frame, but about 490 lines are actually displayed on the CRT screen. As shown in FIG. 2, about 15 upper and lower lines of the frame are blank areas. Therefore, the gain value of the AGC circuit 21 is recorded in this blank area.

FIG. 3 shows an NTSC signal in this case, in which a gain value is superimposed in a blank period between the vertical synchronizing signal and the actual image signal. Therefore, CPU2
On the side 4, the slice image of the frame and the gain value thereof can be simultaneously read and processed. The image signal input from the AGC circuit 21 to the frame memory is NTS
Not only the C system but also the PAL system may be used.

The gain value of the AGC circuit 21 may be determined based on the light amount of a specific part of the slice image. Further, instead of changing the signal level of the slice image as in the above embodiment, the light amount of the light source 1 may be changed. For the light quantity control, for example, a method of controlling the light quantity with a diaphragm can be applied.

[0031]

As described above, according to the present invention, the following effects can be obtained. According to the first aspect of the present invention, the control unit changes the measured light amount or the signal level of the slice image in accordance with the light amount of the reflected light from the sample, and the image reconstruction unit weights the change amount to obtain the slice image. Is performed, so that an image can be processed with a dynamic range equal to or greater than the dynamic range of the AD converter. Further, in the peak search, the peak search is performed based on the slice image whose level has been changed. Therefore, even when the amount of reflected light is small, there is an effect that the peak can be reliably searched without being mixed with noise.

According to the second aspect of the invention, the same effect as in the first aspect can be obtained by changing the light amount of the light source of the irradiation light for irradiating the sample.

According to the third aspect of the present invention, the signal level of the slice image can be easily changed by the automatic gain control circuit, and the amount of change in the signal level can be easily read by the gain value. effective.

According to the fourth aspect of the present invention, there is an advantage that the gain value of the automatic gain control circuit can be determined based on the light receiving amount of a part of the slice image.

According to the present invention, the NTSC signal output from the automatic gain control circuit is stored in the frame memory, and the gain value of the automatic gain control circuit is recorded in a blank area of the NTSC signal. As a result, effective use of the frame memory and effective use of the blank area can be achieved.

[Brief description of the drawings]

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

FIG. 2 is an explanatory diagram of a blank area.

FIG. 3 is an explanatory diagram of an NTSC signal.

FIG. 4 is a configuration diagram showing an example of a conventional confocal microscope device.

[Explanation of symbols]

 Reference Signs List 1 light source 2 confocal scanner 3 objective lens 4 sample 5 stage 6 camera 9 stage moving mechanism 11 frame memory 12 CRT 21 automatic gain control circuit 22, 23, 25 AD converter 24 CPU

Claims (5)

[Claims] A substrate having a large number of minute openings is rotated;
The sample is scanned with irradiation light that has passed through the minute opening of the substrate, reflected light from the sample is received, and a slice image of the sample is obtained. In a confocal microscope device configured to be able to obtain an image, a control unit that changes a measured light amount or a signal level of a slice image according to a light amount of reflected light from the sample; and Image reconstruction means for weighting the amount of change in the signal level and performing a peak search for a slice image at each relative movement position of the sample in the optical axis direction for each pixel to obtain a three-dimensional image of the sample or an image with a deep depth of focus. A confocal microscope device comprising: 2. A control means for changing a measured light amount or a signal level of a slice image according to a light amount of reflected light from the sample, the control means having a function of changing a light amount of a light source of irradiation light for irradiating the sample. 2. The confocal microscope device according to claim 1, wherein: 3. The confocal microscope device according to claim 1, wherein an automatic gain control circuit is used as said control means. 4. The apparatus according to claim 1, wherein said control means includes means for receiving a part of the slice image, and determines a gain value of said automatic gain control circuit in accordance with an amount of light received by said means. Item 6. A confocal microscope device according to Item 3. 5. The control means includes a frame memory.
4. The confocal device according to claim 3, wherein the image signal output from the automatic gain control circuit is stored in a frame memory, and a gain value of the automatic gain control circuit is recorded in a blank area of the image signal. Microscope equipment.