JP2000275530A - Confocal microscope - Google Patents

Abstract

PROBLEM TO BE SOLVED: To obtain an optimum confocal image while nearly equalizing the lightness of a light field image to the lightness of a confocal image of a composite image. SOLUTION: This microscope is equipped with a random pinhole pattern part 201 which has pinhole parts 206 arranged at random and also has transmissivity (k), a rotary disk 104 which is k2 as large as the area of the random pinhole pattern part 201 and has an opening part 202 transmitting light, a means which guides the light from a light source to the disk 104, a means which guides the light transmitted through the disk 104 to a sample, a means which irradiates the rotary disk 104 with the light from the sample again, a means which selectively picks up the composite image including a confocal component and a nonconfocal component of the sample obtained by being transmitted through the pinhole pattern part 201 at random and the light field image of the sample obtained by being transmitted through the opening part 202, and a means which obtains the confocal image of the sample by subtracting the light field image from the composite image, on a light-shielding mask 205.

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. 5, 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 50 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. 6, a rotating disk 501 is provided with a random pinhole pattern portion 601 in which pinholes are randomly arranged, and an opening 602 through which light can freely pass. Further, shielding portions 603 and 604 for blocking light are provided between the random pinhole pattern portion 601 and the opening portion 602. The rotating disk 501 is connected to a motor shaft (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 501, the half mirror 103, and the condenser lens 108, and then enter the CCD camera 109. The imaging timing of the CCD camera 109 is controlled in synchronization with the rotation speed of the rotating disk 501, and each of the CCD cameras 109 captures two images that have passed through the random pinhole pattern unit 601 and the opening 602.

[0007] The output image of the CCD camera 109 is stored in the computer 502. The image obtained by passing through the random pinhole pattern portion 601 is an image obtained by adding a non-confocal image to a confocal image because the density of pinholes is several tens times that of a normal Nipkow disc.

[0008] Only the confocal image is obtained by subtracting the non-confocal image containing the confocal component from the bright-field image obtained from the opening 602. This calculated confocal image is
It 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%. Wilson et al. Report that the available reflected light for incident light is 25-50%, resulting in a brighter image.

Also, NAANeil, T. Wilson and R. Juska
itis, "A Light Efficient Optically Sectioning Micr
oscope "Journal of Microscopy, Vlo.189, pt.2, (199
8), PP.114-117 has a line pattern section 701 in which a number of light-shielding portions and transmissive portions are arranged in a straight line as shown in FIG. There is an opening 702 that can pass through the line pattern portion 7
01 and an opening 702, a light shielding unit 70 for blocking light
It is described that a confocal image can be obtained by performing the same processing on a disk having 3,704.

[0011]

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. It is necessary to subtract the bright-field image obtained in the transmission portion of the disc from the image in which the confocal image and the non-confocal image obtained through the pinhole portion overlap (hereinafter, referred to as a composite image).

However, the ratio of the brightness of the confocal image portion to the brightness of the non-confocal image portion of the composite image changes depending on the pinhole density and the NA of the objective lens. However, the relationship between the brightness of the non-confocal image portion of the composite image in the random pinhole pattern portion and the brightness of the bright field image obtained in the opening has not been known. Therefore, it has been difficult to obtain an optimal confocal image.

Further, in a disc having a line pattern portion whose transmission portion is linear, a confocal component is not included in a direction parallel to the straight line, but a confocal component is included in a direction perpendicular to the straight line. There is a disadvantage that the confocal effect varies depending on the direction of the aperture line in the image.

It is an object of the present invention to make the brightness of the non-confocal image portion of the composite image substantially equal to the brightness of the bright-field image,
An object of the present invention is to provide a confocal microscope capable of obtaining an optimal confocal image.

Another object of the present invention is to obtain a relatively uniform confocal image in a rotating plate for a confocal microscope having a transmission portion in which linear light-shielding portions and transmission portions are alternately arranged. It is to provide a confocal microscope with a possible rotating plate.

[0016]

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

(1) In the confocal microscope according to the present invention (claim 1), the illumination means for irradiating the sample with light, and a portion through which light emitted from the illumination means is transmitted and a portion which is shielded are provided. A semi-transmissive region that is mixed and has a light transmittance of k, and a switching unit that can alternately switch between an opening region that transmits light emitted from the illumination unit and a translucent region that transmits the semi-transmissive region. An image pickup unit for selectively picking up a composite image obtained by superimposing a confocal image and a non-confocal image of the sample obtained, and a bright field image of the sample obtained through the opening region, A confocal microscope comprising means for obtaining a confocal image of the sample from a composite image and a bright field image taken by an imaging means, wherein the area of the aperture region is
It is characterized by being approximately k ² times the semi-transmissive region.

In the present invention, the switching means includes the semi-transmissive region in which a plurality of linear openings through which light is transmitted are arranged and the transmittance of the light is k. Preferably, the disk is a rotatable disk, and the semi-transmissive region of the disk has a fan shape having a central angle of 90 ° or more with the rotation axis as a vertex.

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

By making the area of the aperture region k ² times the area of the translucent region having a transmittance of k, the confocal and non-confocal composite image taken by the imaging means and the aperture can be obtained. The brightness of the non-confocal image becomes almost the same as that of the non-confocal image, and an optimal confocal image can be obtained.

A linear portion through which light is transmitted and a linear portion through which light is shielded are alternately arranged so that the angle of the semi-transmissive region where the light transmittance is k is 90 ° or more. Variations in confocal components due to the direction of a linear portion through which light passes in the image are averaged, and a relatively uniform confocal image 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. 2A, the rotating disk 104 has a random pinhole pattern portion 201 which is randomly arranged so that the area of the pinhole is 25 to 50% of the whole, and freely rotates light. An opening 202 that can pass through is provided. Further, between the random pinhole pattern portion 201 and the opening portion 202, a shielding portion 2 for blocking light is provided.
03 and 204 are provided.

The rotating disk 104 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. Half mirror 1
A CCD camera 109 including a condensing lens 108 and an imaging element in which a plurality of pixels are formed and arrayed on a transmission optical path 03.
Is arranged.

An A / D board 110 is connected to an image output terminal of the CCD camera 109.
0 is a CPU 112 via a bus 111, an image memory 113 for storing images, and a difference program 1 for subtracting images.
14 ₁ monitor 115 as an output means for outputting an image processing result and the like are connected by the memory 114, and a differential program 114 ₁ stored.

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. Light passing through the random pinhole pattern portion 201 and the opening 202 of the rotating disk 104 forms an image by the objective lens 105 and is incident on the sample 106.

Then, the light from the sample 106 passes through the random pinhole pattern portion 201 and the opening 202 of the rotating disk 104 again via the objective lens 105. Further, the light passes through the half mirror 103 and enters the CCD camera 109 via the condenser 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 random pinhole pattern portion 201 and the opening 2
02 are captured.

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 random pinhole pattern unit 201 is a composite image in which a non-confocal image is added to a confocal image. Also, the opening 202
Is a bright-field image which is a non-confocal image.

Here, when the portion of the random pinhole pattern portion 201 in FIG. 2A is enlarged, as shown in FIG. 2B, light is transmitted through a portion having the pinhole 206 and light is shielded without the pinhole. The mask 205 is deposited with Cr or the like so as not to transmit light. The radius r of the pinhole 206 is set such that r = bMλ / NA (1) using a constant b, where M is the magnification of the objective lens, NA is the aperture ratio, and λ is the wavelength of light. Usually, the constant b is 0.35
The wavelength λ is 550 nm and the magnification M is 10
0, NA = 0.9, the radius r of the pinhole is 21.
4 μm.

The sector area of the random pinhole pattern portion 201 is S ₀ , and the total area of a large number of transparent pinholes is S ₁ . The transmittance k in the fan-shaped portion of the random pinhole pattern portion 201 is k = S ₁ / S ₀ (2).

At a certain point (x, y) of a general microscope image,
Brightness data of the bright-field image_{(x, y )}And like this
The brightness data of the focused bright-field image
M _{fo (x, y)}Of out-of-focus brightfield image out of focus
Data m_{defo (x, y)}Is included, so a bright-field image
Luminance data m_{(x, y)}Is m_{(x, y)}= M_{fo (x, y)}+ M_{defo (x, y)} (3)

As compared with the conventional Nipkow disk, an image obtained by a rotating disk having a very high density of pinholes is a composite image in which not only in-focus but also out-of-focus is mixed. Let the luminance data of the composite image at a certain point (x, y) on the image be cm _{co (x, y)} . Consider a case where there is an image passing through a pinhole at this point (x, y). The luminance data cm _{co (x, y)} of the composite image is the luminance data c _{fo (x, y ) of the} focused confocal component and the data cm of the unfocused component.
_Since it is the sum with _{defo (x, y)} , cm _{co (x, y)} = c _{fo (x, y)} + cm _{defo (x, y)} (4) Here, the focused confocal component is the light that comes out of a certain pinhole, is reflected by the sample, and passes through the pinhole again.

On the other hand, an unfocused bright-field image at a certain point is a reflection from a point other than the corresponding point on the sample, and is the same except for the out-of-focus component and the brightness. Assuming that the transmittance of light in the pinhole portion is k, at point (x, y), the luminance data cm _{defo (x, y)} of the out-of-focus component in the pinhole portion is
_K of luminance data m _{defo (x, y)} of the out-of-focus image of the bright field part
Since the luminance is doubled, the following expression holds: cm _{defo (x, y) ∝km defo (x, y)} (5)

Since the brightness of a composite image obtained from an actual pinhole is k times that of an image obtained from a transparent portion having the same area, the luminance data of the obtained composite image can be obtained from equations (4) and (5). cm _{(x, y)} becomes cm _{(x, y)} = kcm _{(x, y)} = kc _{fo (x, y)} + k ² m _{defo (x, y)} (6)

Comparing Equations (3) and (6), it is necessary to eliminate the luminance data m _{defo (x, y)} of the out-of-focus image component in order to obtain only a focused image. I understand. Therefore, by multiplying equation (3) by k ² and subtracting from equation (6), cm _{(x, y)} −k ² m _{(x, y)} = kc _{fo (x, y)} −k ² m _{fo ( x, y)} (7). And if this calculation is performed on the whole image,
A confocal image is obtained.

The image obtained by the equation (7) is different from the data of the confocal image exactly, but only the focused image component remains and the component of the unfocused image is removed. In other words, a sectioning image in the z direction can be obtained similarly to the confocal image. From the above discussion, it is sufficient that the area of the bright field image portion is k ² times the area of the pinhole portion.

For example, the random pinhole pattern section 2
The transmittance of the portion 01 is 1/2, and the central angle of this sector is 1
If it is 20 °, the central angle of the sector of the opening 202 is 30 °. CPU112 performs the calculation from the composite image and the bright field image of the image memory 113 by the differential program 114 _1, and displays the result on the monitor 115. At this time, since the brightness of the bright-field image is the same as that of the non-confocal image in the composite image, a confocal image can be obtained only by simple subtraction.

As described above, by setting the area of the opening to be k ² times the area of the pinhole pattern, an optimal confocal image can be obtained.

[Second Embodiment] A confocal microscope according to a second embodiment of the present invention will now be described with reference to FIG.
The description will be made by using the rotating disk. Here, the same portions as those in FIG. 1 are denoted by the same reference numerals, and description thereof will be omitted. In the present embodiment, since the configuration other than the rotary disk is the same as that of the first embodiment, only the rotary disk is shown.

The rotating disk 300 of the present embodiment is similar to that of FIG.
As shown in FIG. 2, a line pattern portion 301 and an opening portion 302 in which line-shaped shields and opening regions are alternately arranged, and a light shielding portion 30 between the line pattern portion 301 and the opening portion 302.
3,304.

The width of the opening region of the line pattern portion 301 is substantially the same as the diameter of the pinhole of the first embodiment, and is determined by equation (1). Further, the width of the shield is about 1 to 3 times the width of the opening area, but in the present embodiment, the width of the shield is equal to the width of the opening area. At this time, the center angles of the fan-shaped line pattern portion 301 and the opening are 90 ° and 22.5 °, respectively.

The reason why the center angle of the fan-shaped line pattern portion 301 is 90 ° will be described below. Consider a case where an image is captured by the CCD camera 109 through the line pattern unit 301. When the shutter of the CCD camera 109 is open, the rotating disc 300 is rotating.
The image of the line pattern portion 301 captured by the camera changes from the image illustrated in FIG. 4A to the image illustrated in FIG.

In the image shown in FIG. 4A, the aperture line is parallel to the direction A in the figure, so that the image obtained in this state contains only the bright field component and the confocal component is 0. On the other hand, the direction B is perpendicular to the longitudinal direction of the linear shield, so that the confocal component is maximized and the bright field component is minimized.

In the image shown in FIG. 4B, since the longitudinal direction of the aperture line is parallel to the direction B, in this direction only the bright field component and the confocal component are zero. On the other hand, since the direction of A is perpendicular to the longitudinal direction of the aperture line, the confocal component is maximized and the bright field component is minimized.

As described above, when a rotating disk having a linear opening area is used, the ratio between the confocal component and the bright field component differs depending on the longitudinal direction of the shield of a captured image. If the longitudinal direction of the shield rotates 90 °, the confocal and non-confocal components are averaged in various directions. Therefore, the image obtained by the fan-shaped line pattern portion 301 having a central angle of 90 ° is a composite image in which the non-confocal image is added to the confocal image, as in the case of the random pinhole pattern portion in the first embodiment. Become an image. A confocal image can be calculated by subtracting a bright-field image, which is a non-confocal image obtained at the opening 302, from this composite image.

Further, since the transmittance of the line pattern portion 301 is １ ／ and the area of the opening 302 is ４ of the area of the line pattern portion 301, similar to the first embodiment, the transmittance is simple without multiplication by a coefficient. A confocal image can be obtained by subtraction.

As described above, in the present embodiment, the portion for obtaining the composite image of the rotating disk is formed by alternately arranging the linear openings and the light-shielding members. By setting the center angle of the line pattern portion to 90 °, the confocal component does not differ depending on the direction.

The present invention is not limited to the above embodiment. For example, in the second embodiment, the central angle of the line pattern portion is 90 °, but may be 90 ° or more. In this case, the confocal component slightly differs depending on the direction. However, if the central angle is 90 ° or more, the confocal component is equal in each direction in a region where the central angle is 90 °. It does not result in unnatural images with different confocal effects. Further, the present invention is not limited to a rotating disk.For example, a cylindrical rotating body having a random pinhole portion and an opening or a liquid crystal displaying a random pinhole portion and an opening, respectively, is a rotating disk. Can be applied to a confocal microscope provided instead of. In addition, the present invention can be variously modified and implemented without departing from the gist thereof.

[0051]

As described above, according to the present invention, the area of the aperture region where a bright-field image is obtained is reduced by the area of the semi-transmission region where a composite image including a confocal image and a non-confocal image is obtained. by ^doubling the k, the brightness of the non-confocal obtained composite image and the aperture of the confocal and non-confocal captured by the imaging means to obtain substantially the same, and the optimal confocal images be able to.

In a rotating disk having a semi-transmissive area in which linear aperture areas and light-shielding bodies are alternately arranged, a relatively uniform confocal point is obtained by setting the central angle of the semi-transmissive area to 90 ° or more. Images can be obtained.

[Brief description of the drawings]

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

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

FIG. 3 is a plan view showing a configuration of a rotating disk used in a confocal microscope according to a second embodiment.

FIG. 4 is a diagram showing an image of a line pattern portion captured by a CCD camera.

FIG. 5 is a block diagram showing a configuration of a conventional confocal microscope.

FIG. 6 is a plan view showing a configuration of a rotating disk having a random pinhole pattern used in the confocal microscope of FIG. 5;

FIG. 7 is a plan view showing a configuration of a rotating disk having a line turn portion used in the confocal microscope of FIG. 5;

[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 ... Condensing lens 109 ... CCD camera 110 ... A / D board 111 ... Bus 112 ... CPU 113 ... Image Memory 114 ... Memory 114 ₁ ... Difference program 115 ... Monitor 201 ... Random pinhole pattern part 202 ... Opening 203.204 ... Shielding part 205 ... Light-shielding mask 206 ... Pinhole 300 ... Rotating disk 301 ... Line pattern part 302 ... Opening 303.304 Shielding section 501 Rotating disk 502 Computer 601 Random pinhole pattern section 602 Opening section 603.604 Shielding section

Claims (2)

[Claims] An illumination means for irradiating a sample with light, a part for transmitting the light emitted from the illumination means and a part for shielding are mixed, and a half of which has a light transmittance of k. A transmissive region, a switching unit that can alternately switch an opening region that transmits light emitted from the illumination unit, and a confocal image and a non-confocal image of the sample obtained by transmitting the semi-transmissive region. Imaging means for selectively capturing a composite image obtained by superimposing the images and a bright-field image of the sample obtained through the opening area, and a composite image and a bright-field image captured by the imaging means. A confocal microscope comprising: means for obtaining a confocal image of a sample, wherein the area of the opening region is approximately k ² times the semi-transmissive region. 2. The switching means includes a semi-transmissive region in which a plurality of linear openings through which light is transmitted are arranged and the light transmittance is k, and the switching means is a disk which is rotatable about a rotation axis. The confocal microscope according to claim 1, wherein the semi-transmissive region of the disk has a fan shape having a central angle of 90 ° or more with the rotation axis at the apex.