JP2000275540A - Confocal microscope - Google Patents

Abstract

PROBLEM TO BE SOLVED: To vary the lightness rate of a confocal image part and the rate of a nonconfocal image part. SOLUTION: An optical lens 102 and a half-mirror 103 are arranged on the optical path of the light emitted by a light source 101. On the optical path of the reflected light from the half-mirror 103, a rotary disk 104 which rotates at a constant speed and extracts a composite image formed of a confocal image and a nonconfocal image overlapping with each other and a light field image by turns, an objective 105, and a sample 106 are arranged. On the optical path of the transmitted light from the half-mirror 103, an image forming lens 108 and a CCD camera 109 which alternately picks up the composite image and light field image are arranged. A memory 114 store d with a coefficient program 1142 which multiplies the light field image by a coefficient and an operation program 1142 which subtracts data of the light field image multiplied by the coefficient from the data of the composite image and a monitor 115 which displays the image process result obtained by the programs are connected.

Description

DETAILED DESCRIPTION OF THE INVENTION

[0001]

BACKGROUND OF THE INVENTION 1. Field of the Invention The present invention relates to a confocal microscope for observing and measuring a microstructure or a three-dimensional shape of a sample at high speed using light.

[0002]

2. Description of the Related Art As a conventional typical high-speed confocal microscope, there is a confocal microscope using a Nipkow disk in which a large number of pinholes are spirally arranged at intervals of about 10 times the diameter. In a confocal microscope using a Nipkow disc, it is necessary to eliminate crosstalk from an adjacent pinhole, and therefore the arrangement interval of the pinholes must be increased. Therefore, the utilization rate of light from the light source was as low as about 1%, and only a dark image was obtained.

[0003] As an improvement of a confocal microscope using a disk, R.S. Juskaitis, T .; Wilson et al.
“Efficient real-time confocal microscopy with whi
te light soreces ”Nature Vol1., 383 Oct. 1996 p804-8
There is a technology announced in 06. FIG. Wilso
1 shows the configuration of a confocal microscope of N et al.

As shown in FIG. 7, an optical lens 102 and a half mirror 103 are arranged on an optical path of light emitted from a light source 101 such as a halogen light source or a mercury light source. The rotating disk 70 is placed on the reflected light path of the half mirror 103.
1. The objective lens 105 and the sample 106 are arranged.

As shown in FIG. 8, a rotating disk 701 is provided with a random pinhole pattern section 801 in which pinholes 802 are randomly arranged, and an opening 803 through which light can freely pass. Further, shielding portions 804 and 805 are provided between the random pinhole pattern portion 801 and the opening 803 to block light. Also,
The rotating disk 701 is connected to a shaft of a motor (not shown) via a rotating shaft 107, and rotates at a constant rotation speed.

[0006] The reflected light from the sample 106 is
05, the rotating disk 701, the half mirror 103, and the condenser lens 108, and is incident on the CCD camera 109. The imaging timing of the CCD camera 109 is controlled in synchronization with the rotation speed of the rotating disk 701, and the CCD camera 109 captures two images passing through the random pinhole pattern portion 801 and the opening 803, respectively.

The output image of the CCD camera 109 is stored in the computer 702. The image obtained by passing through the random pinhole pattern portion 801 is an image obtained by adding a non-confocal image to a confocal image because the density of pinholes is several tens of times that of a normal Nipkow disc.

A non-confocal image containing the confocal component and a non-confocal image obtained from the opening 803 are subtracted to obtain only a confocal image. The calculated confocal image is displayed on the monitor 115.

In the Nipkow disk type confocal microscope, the reflected light from the sample which can be used for the incident light from the light source is 0.5 to 1%. In a confocal microscope of Wilson et al., It is reported that reflected light that can be used for incident light is 25 to 50%, and a brighter image can be obtained.

[0010]

However, the conventional disk scanning confocal microscope has the following disadvantages.
T. The disc-scanning confocal microscope of Wilson et al. (International Publication No. WO 97/31282) is several tens times brighter than the disc-scanning confocal microscope using a conventional Nipkow disc. From a superimposed image of a confocal image and a non-confocal image obtained through a pinhole portion (hereinafter referred to as a composite image), a non-confocal image obtained through a transmitting portion of a disc (hereinafter referred to as a bright-field image) ) Must be subtracted.

However, the brightness ratio of the confocal image portion and the brightness ratio of the non-confocal image portion of the composite image vary depending on the pinhole density and the NA of the objective lens. Also,
Depending on the sample, there are times when it is desired to reduce the confocal effect so as to observe not only the confocal image but also a little non-confocal image and observe the upper and lower relationship of the sample.

In a conventional microscope that obtains a confocal image using subtraction, the ratio of subtracting a bright-field image from a composite image is as follows:
Since the area ratio between the pinhole portion and the transmissive portion on the disk is determined, it was necessary to replace the disk itself in order to change the ratio of the subtraction.

It is an object of the present invention to provide a confocal microscope capable of changing the ratio of the brightness of the confocal image portion and the ratio of the non-confocal image portion without changing the rotating disk. is there.

[0014]

Means for Solving the Problems [Configuration] The present invention is configured as follows to achieve the above object.

(1) A confocal microscope according to the present invention (claim 1) is an illumination means for irradiating a sample with light, and a confocal image and a non-confocal image of the sample from light from the sample. Means for extracting a superimposed composite image, means for extracting a bright-field image from light from the sample, and imaging for extracting the extracted composite image and bright-field image, respectively, to obtain a composite image and a bright-field image Means, arithmetic means for subtracting the composite image data obtained by the imaging means from the bright field image at a desired ratio, output means for outputting an image obtained by the arithmetic means, and variable ratio And means for performing the following.

(2) A confocal microscope according to the present invention (claim 2) is an illumination means for irradiating a sample with light, and a confocal image and a non-confocal image of the sample from light from the sample. Means for extracting a superimposed composite image, means for extracting a bright field image from light from the sample, and imaging means for capturing the extracted composite image and bright field image to obtain a composite image and a bright field image A brightness varying means for changing the brightness of at least one of the composite image and the bright-field image; and calculating the confocal image data by calculating data of the composite image and the bright-field image obtained by the imaging means. And an output unit for outputting an image obtained by the operation of the operation unit.

Preferred embodiments of the present invention are described below.

[0018] The means for extracting the composite image includes a mask portion provided with a mixture of a portion for transmitting incident light to the sample and a portion for blocking light, and a device for reflecting or transmitting light from a plurality of regions of the sample. Means for making the light incident on the mask, and means for forming an image of the light transmitted through the mask portion out of the reflected light or the transmitted light, wherein the means for extracting the bright-field image includes the incident light and the sample An opening for transmitting reflected light or transmitted light from the sample; and an imaging unit for forming an image of reflected light or transmitted light from the sample.

The mask and the opening are provided on the same disk. It is more preferable that the area ratio between the mask portion and the opening is approximately 4: 1. In the mask section, a plurality of pinholes are randomly arranged. In the mask portion, linear light shields and opening regions are alternately arranged and formed. It comprises means for rotating the disk and means for rotating the disk.

[Operation] The present invention has the following operation and effects by the above configuration.

The ratio between the non-confocal image component and the confocal image component of the obtained subtracted image can be changed, and an image having a confocal effect according to the pinhole density, the objective lens, and the state of the sample can be obtained.

[0022]

Embodiments of the present invention will be described below with reference to the drawings.

[First Embodiment] FIG. 1 is a block diagram showing a schematic configuration of a confocal microscope according to a first embodiment of the present invention. FIG. 2 is a plan view showing the configuration of a rotating disk used in the confocal microscope of FIG.

As shown in FIG. 1, an optical lens 102 and a half mirror 103 are arranged on an optical path of light emitted from a light source 101 such as a halogen light source or a mercury light source. The rotating disk 10 is placed on the reflected light path of the half mirror 103.
4, the objective lens 105 and the sample 106 are arranged.

As shown in FIG. 2, the rotating disk 104 has a line pattern portion 201 in which a plurality of linear light shielding members 202 for shielding light are arranged in parallel at regular intervals, and an opening portion 203 for transmitting light. And a shielding unit 204 for shielding light.
205 is provided.

Light shielding body 202 and shielding portions 204 and 205
Is a light-shielding film formed by a vapor deposition method or the like. In the line pattern portion 201, the width of the light-shielding body 202 is preferably set to be about 1 to 3 times the arrangement interval. The rotating disk 104 is connected to a motor shaft (not shown) via a rotating shaft 107, and rotates at a constant rotation speed. An imaging lens 108 and a CCD camera 109 including an imaging element in which a plurality of pixels are arrayed are arranged on the transmission optical path of the half mirror 103.

An A / D board 110 is connected to an image output terminal of the CCD camera 109.
0 is connected to a CPU 112 via a bus 111. The bus 111 has an image memory 113 for storing an image, a subtraction operation program 114 ₁ for performing image subtraction, and a coefficient program 114 for changing the contrast of a digital image.
₂ is connected to a memory 114 in which a ₂ is stored, and a monitor 115 for displaying an image processing result by a program.

Next, the operation of the present apparatus will be described. Light emitted from the light source 101 passes through an optical lens 102, is reflected by a half mirror 103, and enters a rotating disk 104 that rotates at a constant speed. The light passing through the line pattern portion 201 and the opening 203 of the rotating disk 104 forms an image by the objective lens 105, and the sample 1
06.

Then, the light reflected on the surface of the sample 106 passes through the objective lens 105 and is again turned on the rotating disk 104.
Pass through the line pattern portion 201 and the opening 203. Then, the light passes through the half mirror 103 and enters the CCD camera 109 via the imaging lens 108 to capture an optical image. The imaging timing of the CCD camera 109 is controlled in synchronization with the rotation speed of the rotating disk 104, and the CCD camera 109 captures two images passing through the line pattern unit 201 and the opening 203.

The output image of the CCD camera 109 is A / D
The data is converted into digital data by the board 110, and
Through the image memory 113. The image passing through the line pattern unit 201 is a composite image in which a non-confocal image is added to a confocal image. The image obtained through the opening 203 is a bright-field image that is a non-confocal image.

The image data of the composite image corresponding to the pixel position (x, y) of the image sensor of the CCD camera 109 is represented by cm
_{(x, y)} , if the image data corresponding to the bright-field image is m _{(x, y)} , the image data c _{(x, y)} of the confocal image corresponding to the position _{(x, y)} is c _{( x, y)} = cm _{(x, y)} -m _{(x, y)} (1). Then, if this calculation is performed for all the pixels, a confocal image can be obtained.

Incidentally, the ratio of the brightness of the composite image to the brightness of the bright-field image depends on the line pattern portion 201 and the aperture 20.
3 is determined by the area ratio. In order to eliminate the non-confocal portion in the composite image, the brightness of the bright-field image and the brightness of the non-confocal image in the composite image must be the same. If the brightnesses of the two images are different, there is a possibility that a non-confocal image component remains in the subtraction result, or a part of the confocal image is removed by subtraction.

[0033] Therefore, in this apparatus, in order to adjust the brightness of the non-confocal image and the bright field image in the composite image, after the coefficient program 114 ₂ multiplied by the coefficient data of the bright-field image, subtract operation program by _1141, the calculation of confocal images by subtracting the data of the corresponding position of the coefficients multiplied by the bright field image from the data of the composite image.

The result of the subtraction is displayed on the monitor 115. An image of the subtraction result confirmed by the monitor 115, the confocal effect by shifting the focal point (portion other than the focus state condition to be removed from the image) while watching the changes the coefficients stored in the coefficient program 114 ₂ The resulting image.

This operation will be described in more detail with reference to the flowchart of FIG. First, from the data of the composite image which is the image that has passed through the line
The coefficient α used when subtracting the data of the bright-field image (non-confocal image), which is the image passing through No. 3, is input (step S1). The coefficient α differs depending on the transmittance of the line pattern unit 201, the area ratio between the line pattern unit 201 and the opening 203, the magnification of the objective lens, and the NA, but approximately 0.
Enter a value between 5 and 1.5.

Subsequently, the fetching of the data of the composite image (scan)
Step S2) and capture of bright-field image data
Step S3) is performed, and the data of each image is stored in the image
It is stored in the memory 113. And the coefficient program 11
4_TwoBrightfield image data m_{(x, y)}Above
After multiplying the coefficient α input in step S1, the coefficient calculation is performed.
Program 114₁By c_{(x, y)}= Cm_{(x, y)}-Αm_{(x, y)} (2) is executed and calculated by the CPU, and corresponds to the pixel position (x, y).
Confocal image data c _{(x, y)}(Step S
4). Then, the image data corresponding to the positions of all the pixels
The above calculation is performed for.

Next, the image obtained by the calculation is displayed on the monitor 115 (step S5), and the displayed image is observed to determine whether the coefficient α is appropriate (step S6). If the coefficient α is not appropriate, the coefficient α is re-input (step S7), and steps S2 to S6 are executed again. When the coefficient α is re-input, if the resolution in the Z direction is too good, the coefficient α is reduced and re-input, and if the resolution is too bad, the coefficient α is increased and re-input.

If it is determined in step 6 that the coefficient α is appropriate, the composite image and the bright field image are fetched again, and the respective images are stored in the image memory 113 (steps S8 and S9).

Then, using the coefficient α again, the data cm (x, y) and m (x, y) of the composite image and the bright-field image corresponding to the pixel position (x, y) are expressed by the following equation (2). Assign and operate,
Data c of the confocal image corresponding to the pixel position (x, y)
(x, y) is obtained (step S10). This calculation is performed on the image data corresponding to the positions of all the pixels to obtain a confocal image.

Then, the confocal image obtained by the calculation is displayed on the monitor 115 (step S11). Then, the user determines whether or not to end (Step S1)
2). If it is determined that the processing is not to be ended, the above steps S8 to
Execute S12 again. If it is determined that the processing is to be ended, the above processing is ended.

When obtaining a stereoscopic image near the surface of the sample 106, the sample 106 is moved up and down by a vertical movement stage or a piezo element to obtain a confocal image while changing the image in the height direction. Is obtained by synthesizing a plurality of obtained images.

As described above, the confocal image is obtained by adjusting the brightness ratio between the composite image and the bright-field image by software. , The optimum confocal image can be easily obtained without changing the rotating disk.

Depending on the sample, it may be desired to reduce the confocal effect so as to observe not only the confocal image but also a little non-confocal image and observe the upper and lower relation of the sample. It can be easily adjusted by changing α without changing the rotating disk.

[Second Embodiment] FIG. 4 is a block diagram showing a schematic configuration of a confocal microscope according to a second embodiment of the present invention. FIG. 5 is a plan view showing the configuration of a rotating disk used in the confocal microscope of FIG. 4 and 5, the same parts as those in FIGS. 1 and 2 are denoted by the same reference numerals, and detailed description thereof will be omitted.

This apparatus differs from the microscope of the first embodiment in that the light amount ratio between the composite image and the bright-field image is not changed by software, but the area of the shielding portion is changed.

As shown in FIG. 5A, the rotating disk 401 has a random pinhole pattern in which pinholes 502 are randomly arranged and the number thereof is such that the area of the pinholes 502 is 25 to 50% of the whole. A portion 501 and an opening 503 through which light can freely pass are provided. Further, the random pinhole pattern portion 501 and the opening 50
3 are provided with shielding portions 504 and 505 for blocking light. The shielding portions 504 and 505 are as shown in FIG.
As shown in FIG.
The light shielding plates 506 and 507 can rotate around the rotation axis in the directions A and B.

As shown in FIG. 6A, the light shielding plates 506 and 507 have a structure in which a hole through which a rotating shaft is passed is formed at a vertex of a sector. FIG. 6B shows a state in which the light shielding plates 506 and 507 are attached to the rotating disk 401.

As shown in FIG. 6B, the light shielding plate 506,
507 is attached to each of the shielding portions 504 and 505, but only one is shown because it is symmetrical.
The light shielding plates 506 and 507 are fixed to the rotating disk 401 by using holes provided at the vertices.
b. The light blocking plate fixing member 508 is
There is a hole in the center that passes through the axis of rotation. Further, the outer periphery of the light shielding plate fixing member 508a and the light shielding plate fixing member 508b
Are threaded on the inner periphery of the light shielding plates 506 and 507 by tightening the screws.

To change the area of the shielding portions 504 and 505, loosen the screws of the light shielding plate fixing members 508a and 508b and move the light shielding plates 506 and 507 around the rotation axis.
After adjusting the areas of the opening 503 and the random pinhole pattern 501, the light-shielding plate fixing members 508a and 508b are adjusted.
And fix again so that the light shielding plates 506 and 507 do not move.

The rotating disk 401 is connected to the rotating shaft 10.
7, and is connected to a shaft of a motor (not shown) so as to rotate at a constant rotation speed.

Next, the operation of the real device will be described. As in the device of the first embodiment, the CCD camera 109 controls the imaging timing in synchronization with the rotation speed of the rotating disk 401, and captures two images that have passed through the random pinhole pattern portion 501 and the opening 503. I do.

The output image of the CCD camera 109 is A / D
The data is converted into digital data by the board 110, and
Through the image memory 113. As described above, the image obtained by the random pinhole pattern unit 501 is a composite image obtained by adding a non-confocal image to a confocal image. The data of the composite image and the bright-field image corresponding to the pixel position (x, y) are expressed as cm _{(x, y)} and m _, respectively.
_{(x, y)} , the corresponding confocal image data c _{(x, y)}
Can be calculated as c _{(x, y)} = cm _{(x, y)} -m _{(x, y)} (1). Then, if this calculation is performed for all pixels, a confocal image can be obtained.

By executing the subtraction program 114 ₁ stored in the memory 114, the CPU 112 performs the above calculation from the data of the composite image and the data of the bright-field image stored in the image memory 113, and monitors the result on the monitor 11.
5 is displayed.

At this time, the brightness of the composite image and the brightness of the bright-field image depend on the pinhole pattern portion 501 and the opening portion 50.
3 is determined by the area ratio. In order to eliminate the non-confocal part of the composite image, the brightness of the bright-field image must be the same as the non-confocal image in the composite image. If the brightness of the two images is different, there is a possibility that a non-confocal image component remains in the subtraction result, or a portion of the confocal image is subtracted by the subtraction and disappears.

Then, the image of the subtraction result is confirmed on the monitor 115, and the light-shielding plate 506 of the rotating disk 401 is observed while shifting the focus and observing the confocal effect (a state in which portions other than the focused state are removed from the image). , 507 in the direction of A or B so that the pinhole pattern portion 501 and the opening
The area ratio of both is adjusted by changing the area of at least one of the three.

As described above, the brightness of the composite image and the bright field image can be adjusted by changing the area ratio between the random pinhole pattern portion and the opening portion, so that the rotating disk can be easily changed. A confocal image can be obtained.

Depending on the sample, it may be desirable to reduce the confocal effect and observe a vertical relation of the sample while leaving a small amount of a non-confocal image as well as a confocal image. By changing the area ratio between the pinhole pattern portion and the opening, it is possible to easily observe without changing the rotating disk.

The present invention is not limited to the above embodiment. For example, although a pinhole is used in the first embodiment and a line is used in the second embodiment, a line may be used in the first embodiment and a pinhole may be used in the second embodiment. Further, the present invention is not limited to a rotating disk, and for example, rotates a liquid crystal for displaying a cylindrical rotating body having a random pinhole portion and an opening or a random pinhole portion and an opening, respectively. The present invention can also be applied to a confocal microscope provided instead of a disk.

In addition, the present invention can be variously modified and implemented without departing from the gist thereof.

(Features of Embodiment) In the measuring method of the confocal microscope described in the embodiment, a sample is irradiated with light, and a confocal image of the sample is obtained from reflected light or transmitted light from the sample. A composite image in which the composite image and the non-confocal image overlap each other, and a bright field image of the sample is extracted from reflected light or transmitted light from the sample. Obtaining a field-of-view image, and data cm _{(x, y) of the} composite image corresponding to the position (x, y) of the image
Calculating α ₁ cm _{(x, y)} −α ₂ m _{(x, y)} for the entire image with respect to the bright-field image data m _{(x, y)} . Displaying an image to be obtained.

[0061]

As described above, according to the present invention, a multiplication of a composite image including a confocal image and a non-confocal image and a bright-field image are multiplied by a coefficient and the area ratio between the two is made variable. Thus, the brightness ratio between the two can be easily adjusted, and an appropriate confocal image can be obtained.

[Brief description of the drawings]

FIG. 1 is a block diagram showing a configuration of a confocal microscope according to a first embodiment.

FIG. 2 is a diagram showing a configuration of a rotating disk in the confocal microscope of FIG. 1;

FIG. 3 is a flowchart for explaining a measuring method of the confocal microscope of FIG. 1;

FIG. 4 is a block diagram showing a configuration of a confocal microscope according to a second embodiment.

FIG. 5 is a diagram showing a configuration of a rotating disk in the confocal microscope of FIG. 4;

FIG. 6 is a diagram showing a configuration of a rotating disk in the confocal microscope of FIG. 4;

FIG. 7 is a block diagram showing a configuration of a conventional confocal microscope.

FIG. 8 is a diagram showing a configuration of a rotating disk in the confocal microscope of FIG. 7;

[Explanation of symbols]

DESCRIPTION OF SYMBOLS 101 ... Light source 102 ... Optical lens 103 ... Half mirror 104 ... Rotating disk 105 ... Objective lens 106 ... Sample 107 ... Rotation axis 108 ... Imaging lens 109 ... CCD camera 110 ... D board 111 ... Bus 112 ... CPU 113 ... Image memory 114 … Memory 114 ₁ … Subtraction program 114 ₂ … Coefficient program 115… Monitor 201… Line pattern part 202… Light shield 203, 503… Opening 204, 205 504, 505… Shield part 401… Rotating disk 501… Random pinhole pattern Part 502: Pinholes 506, 507: Light shielding plate 508: Light shielding plate fixing member

Claims (2)

[Claims] An illumination unit configured to irradiate a sample with light; a unit configured to extract a composite image in which a confocal image and a non-confocal image of the sample are superimposed from light from the sample; Means for extracting a bright field image from the light of the above, and taking the extracted composite image and bright field image, respectively,
Imaging means for obtaining a composite image and a bright-field image; computing means for subtracting the data of the composite image obtained by the imaging means and the bright-field image at a desired ratio; outputting an image obtained by the computing means A confocal microscope comprising: an output unit that performs the operation; and a unit that changes the ratio. 2. Illumination means for irradiating a sample with light; means for extracting a composite image in which a confocal image and a non-confocal image of the sample are superimposed from light from the sample; Means for extracting a bright-field image from the light of the image, imaging means for capturing the extracted composite image and bright-field image to obtain a composite image and a bright-field image, and at least one of the composite image and the bright-field image Brightness varying means for changing brightness; computing means for calculating data of the composite image and bright-field image obtained by the imaging means to obtain confocal image data; and an image obtained by the calculation of the computing means A confocal microscope, comprising: output means for outputting an image.