JPH07199073A - Confocal laser scanning microscope and its optical tomographic image display method - Google Patents

Abstract

PURPOSE:To provide a confocal laser scanning microscope which can correct the distortion of an optical tomographic image of a sample due to the difference in refractive index between the dipping medium of an objective and a sealing medium wherein the sample is charged. CONSTITUTION:A computer 24 which controls a Z stage 28 where the sample 18 is mounted is stored with the refractive index n1 of the dipping medium (air or water) for the objective 16 and the refractive index n3 of the sealing medium in which the sample 18 is charged. The computer 24 controls the Z stage 28 according to the combination of the refractive indexes n1 and ns so as to correct the distortion of the optical tomographic image of the sample 18 due to the difference between the refractive indexes n1 and n3. Consequently, the optical tomographic image of the sample 18 whose distortion is corrected is displayed on an image monitor 26.

Description

Detailed Description of the Invention

[0001]

BACKGROUND OF THE INVENTION 1. Field of the Invention The present invention relates generally to a confocal laser scanning microscope, and more particularly to a confocal laser scanning microscope capable of correcting the distortion of an optical tomographic image, and an optical distortion-corrected optical microscope for the confocal laser scanning microscope. And a method for displaying a tomographic image.

[0002]

2. Description of the Related Art Confocal laser scanning microscope (CLSM)
The light from the light source is narrowed to a minute spot by the objective lens, the sample is scanned with this spot, and the transmitted light or the reflected light from the sample is detected by the photoelectric converter. As a result, the sample image is displayed on the CRT.

With such a CLSM, an image with less flare and good contrast can be obtained, and an optical tomographic image of the sample is obtained by scanning the sample while moving the focal position in the Z direction according to the confocal detection method. be able to.

[0004]

However, the tomographic image is distorted due to the difference between the refractive index of the medium in which the objective lens is immersed and the refractive index of the medium in which the sample is enclosed. For example, when spherical fluorescent beads or the like are used as a sample for obtaining a tomographic image, the observation result of the tomographic image is as shown in Table 1.

Table 1 Type of immersion medium Refractive index n ₁ Type of enclosed medium Refractive index n ₃ Observation results 1. Air 1.00 Glycerin 1.475 Horizontal oblong 2. Oil 1.52 Glycerin 1.475 Substantially circular 3. Oil 1.52 Water 1.333 Vertical elliptical shape It is therefore an object of the present invention to provide a confocal laser scanning microscope in which distortion-free tomographic images are obtained.

[0006]

According to one aspect of the present invention, there is provided a confocal laser scanning microscope for obtaining an optical tomographic image of a sample encapsulated in an encapsulation medium. This microscope includes a laser light source that emits laser light, an objective lens that is immersed in an immersion medium, and a focusing optical system that converges the laser light from the light source to a sample in an encapsulating medium,
Scanning means for scanning the focal position of the objective lens with respect to the sample along the optical axis direction of the objective lens by the confocal detection method, and photoelectric conversion means for detecting reflected light or transmitted light from the sample and performing photoelectric conversion. , Means for displaying an optical tomographic image of the sample based on the output signal of the photoelectric conversion means, means for storing the refractive index of the immersion medium and the refractive index of the encapsulating medium, and the stored refractive index of the immersion medium, Means for controlling the scanning means according to the refractive index of the encapsulating medium.

According to another aspect of the present invention, there is provided a converging optical system which includes a laser light source for emitting a laser beam and an objective lens immersed in an immersion medium, and converges the laser beam from the light source to a sample in an encapsulating medium. By the system and confocal detection method,
Scanning means for scanning the focal position of the objective lens with respect to the sample stepwise along the optical axis direction of the objective lens;
Confocal laser provided with photoelectric conversion means for detecting reflected light or transmitted light from the sample and performing photoelectric conversion, and electronic display means for displaying an optical tomographic image of the sample based on the output signal of the photoelectric conversion means. A method of displaying an optical tomographic image of a sample with a scanning microscope is provided. In this method, the length per pixel of the electronic display means is calculated from the actual size of the sample in the lateral direction, and the first minimum scanning unit Z of the stepwise scanning by the scanning means is calculated based on the calculated length. determining _s and based on the refractive index n ₁ of the immersion medium of the objective lens and the refractive index n ₃ of the encapsulating medium, a first minimum unit Z _s
Of the second minimum scanning unit Z _s ′ corrected by the above, and the optical tomographic image of the sample is electronically displayed while controlling the stepwise scanning of the scanning unit according to the second minimum unit Z _s ′. And displaying it on the means.

According to still another aspect of the present invention, a laser light source for emitting a laser light and an objective lens immersed in an immersion medium are included, and the laser light from the light source is converged to a sample in an encapsulation medium. An optical system, a confocal detection method, a scanning means for scanning the focal position of the objective lens with respect to the sample stepwise along the optical axis direction of the objective lens, and detecting reflected light or transmitted light from the sample. A digital computer for an optical tomographic image of a sample by a confocal laser scanning microscope including photoelectric conversion means for photoelectrically converting and an electronic display means for displaying an optical tomographic image of the sample based on an output signal of the photoelectric conversion means. A method for displaying using is provided. This method provides the computer with a database containing at least the refractive index n ₁ of the immersion medium of the objective lens and the refractive index n _{3 of the} encapsulating medium, and in the computer, from the actual lateral dimension of the sample. Calculating the length per pixel of the electronic display means, and determining the first minimum scanning unit Z _s of the stepwise scanning by the scanning means based on the calculated length;
In the computer, a second minimum scanning unit Z _s ′ is calculated by correcting the first minimum unit Z _s based on the refractive index n ₁ of the immersion medium of the objective lens and the refractive index n ₃ of the enclosing medium. And a second minimum unit Z by the computer.
_While controlling the stepwise scanning of the scanning means according to _s ′,
According to an embodiment of the method of the invention, comprising displaying an optical tomographic image of the sample on an electronic display means, a second minimum unit Z _s ′.
Is calculated according to Z _s ′ = Z _s · n ₃ / n ₁ .

[0009]

In the present invention, the scanning means is controlled according to the combination of the refractive index of the immersion medium of the objective lens and the refractive index of the sealing medium in which the sample is sealed. According to such control, the distortion of the optical tomographic image of the sample due to the difference between the refractive index of the immersion medium and the refractive index of the inclusion medium is suppressed.

[0010]

DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS Referring to FIG. 1, in a confocal laser scanning microscope (CLSM) generally designated by reference numeral 10, a laser beam emitted from a laser light source 12 passes through a dichroic mirror 14 and an objective lens 16 causes a sample to pass through a sample. Converges to 18.

On the other hand, the objective lens 1 is reflected by the sample 18.
Only light in a predetermined wavelength region of the light transmitted through 6 is reflected by the dichroic mirror 14, passes through the resonance point pinhole 20, and is detected by the photoelectric converter 22. The output of the photoelectric converter 22 is given to the image monitor 26 as an image signal via the digital computer 24 connected in the vicinity of the CLSM 10. Sample 1 is displayed on the image monitor 26.
Eight tomographic images are displayed.

The sample 18 is encapsulated in an encapsulating medium (not shown) such as glycerin or water, and the sample 18 is CLSM1.
0 is mounted on the stage 28. This stage 2
8 has a piezo element (not shown) as a drive source, and C
It is automatically driven in the vertical direction (Z-axis direction) under the control of a computer 24 connected near the LSM 10. The stage 28 is preferably also automatically drivable in the horizontal direction (X and Y axis directions) under the control of the computer 24.

The computer 24 is provided with a man-machine interface 30 such as a keyboard. Immersion medium the objective lens 16 is immersed in CLSM10 (e.g., air or oil) the refractive index n ₁ and encapsulation medium (e.g., glycerine or water) database containing the refractive index n ₃ of the in the computer 24 through the interface 30 It can be stored in a memory (not shown).

Next, the operation of the CLSM 10 will be described with reference to FIG. Here, the above-mentioned refractive indexes n ₁ and n ₃ are stored in a memory (not shown) in the computer 24.
Stored in.

First, the observer selects and sets the refractive index n _{1 of the} immersion medium and the refractive index n ₃ of the enclosed medium that are being used (S1).

When the site to be observed of the sample 18 placed on the stage 28 is set on the optical axis of the objective lens 16,
On the image monitor 26, one cross-sectional image of the sample 18 corresponding to the position of the stage 28 in the Z-axis direction (focal position of the objective lens 16 with respect to the sample 18) is displayed (S2).

Next, the observer designates the X-section to be observed in the cross-sectional image through the interface 30 while referring to the image monitor 26 (S3).

Further, after the scanning start position on the Z axis is designated (S4), the actual scanning is started (S5).

At this time, in order for the tomographic image of the sample 18 displayed on the monitor 26 to have the same vertical and horizontal scales, an arithmetic unit (not shown) in the computer 24 firstly measures the horizontal direction of the sample 18. The length per pixel of the monitor 26 is calculated from the dimensions (S6). This length determines the Z-axis step Z _s to be scanned.

The conventional CLSM executes scanning while moving the stage in the Z-axis direction according to this step Z _s . In contrast, in the present invention, the stage 28 is moved in the Z-axis direction according to step Z _s ′ in which Z _s is corrected by the refractive index n ₁ of the immersion medium of the objective lens 16 and the refractive index n ₃ of the enclosing medium. Here, Z _s ′ is Z _s ′ = Z _s · n ₃ / n ₁ (I), and the calculation unit of the computer 24 according to the refractive indices n ₁ and n ₃ stored in the memory of the computer 24. Is calculated (S7). The correction formula (I) will be described later.

A control unit (not shown) in the computer 24
Displays an image captured in one line scanning (S8) of the image monitor 26 (S9), while shifting the stage 28 by _Z's in the Z-axis direction (S11), the data for one screen is obtained The sample 18 is scanned and displayed until (S10), and the images obtained thereby are arranged in the vertical direction on the monitor 26. As a result, a tomographic image of the sample 18 is displayed on the monitor 26.

The introduction of the correction formula (I) will be described with reference to FIG.

According to Snell's law, n ₁ · sin i = n ₃ · sin i ′ (i) When the boundary surface rises Z ₁ as shown by the dotted line in FIG. 3, the observation point moves from 0 to 0 ″. , the moving distance Z ₁ from the interface 0'after rising 'is, Z _1' = d / tan i 'Meanwhile, Z ₁ = d / tan i _{Thus, Z 1' = Z 1 ·} tan i / tan i ' (Ii) In (i) and (ii), in the region where i and i ′ are sufficiently small (paraxial region), sin i = i, sin
Since i '= i', tan i = i, tan i '= i' holds, (i) and (ii) can be expressed as follows.

N ₁ · i = n ₃ · i ′ Z ₁ ′ = Z ₁ · i / i ′ When i and i ′ are deleted from this, Z ₁ ′ = Z ₁ · n ₃ / n ₁ As a result, the above formula (I) is obtained.

The present invention is not limited to the above embodiment, but various changes and modifications can be made. For example, in the above embodiment, the refractive indices n ₁ and n ₃ are calculated in advance by the computer 24.
However, it may be input each time one tomographic image is observed.

In the above-described embodiment, the system in which the computer 28 controls the stage 28 using a piezo element as a driving source for scanning the sample 18 in the Z-axis direction is shown. However, instead of this, the objective lens 16 is used. A piezo element may be incorporated and the objective lens 16 may be moved in the Z-axis direction by computer control. Alternatively, the objective lens 16 and / or the stage 28 may be moved in the Z-axis direction by a computer-controlled step motor. Although the microscope of the epi-illumination system is shown in the above embodiment, a microscope of the trans-illumination system may be constructed.

In the above embodiment, the scanning of one line has been described, but the invention can be applied to the one for obtaining a continuous cross section by XY scanning.

[0028]

As described above, according to the confocal laser scanning microscope of the present invention and the optical tomographic image display method thereof, the refractive index of the immersion medium of the objective lens and the refractive index of the enclosed medium in which the sample is enclosed. The focus position of the objective lens along the optical axis direction of the objective lens is corrected in accordance with the combination with. As a result, a distortion-corrected tomographic image can be obtained in real time.

[Brief description of drawings]

FIG. 1 is a block diagram showing a confocal laser scanning microscope of the present invention.

2 is a flow chart showing the operation of the confocal laser scanning microscope of FIG.

FIG. 3 is an optical path diagram for explaining correction of a focal position of the confocal laser scanning microscope of FIG.

[Explanation of symbols]

10 ... Confocal laser scanning microscope, 12 ... Laser light source, 16 ... Objective lens, 18 ... Sample, 22 ... Photoelectric converter (photoelectric conversion means), 24 ... Digital computer (storage means, control means), 26 ... Image monitor ( Electronic display means), 28 ... Stage (scanning means).

Claims (5)

[Claims] 1. A confocal laser scanning microscope for obtaining an optical tomographic image of a sample encapsulated in an encapsulation medium, comprising: a laser light source for emitting a laser beam; and an objective lens immersed in an immersion medium, A converging optical system for converging the laser light from the light source to the sample in the encapsulation medium; and a scanning means for scanning the focal position of the objective lens with respect to the sample along the optical axis direction of the objective lens by a confocal detection method. A photoelectric conversion unit that detects reflected light or transmitted light from the sample and performs photoelectric conversion, a unit that displays an optical tomographic image of the sample based on an output signal of the photoelectric conversion unit, and a refractive index of the immersion medium and A confocal laser scanning microscope comprising means for storing the index of refraction of the encapsulation medium and means for controlling the scanning means according to the stored index of refraction of the immersion medium and the index of refraction of the encapsulation medium. 2. A laser light source for emitting laser light, an objective lens immersed in an immersion medium, a focusing optical system for converging the laser light from the light source to a sample in an encapsulating medium, and a confocal detection method. Scanning means for scanning the focal position of the objective lens with respect to the sample stepwise along the optical axis direction of the objective lens; photoelectric conversion means for photoelectrically detecting reflected light or transmitted light from the sample; A method of displaying an optical tomographic image of a sample by a confocal laser scanning microscope, comprising: an electronic display unit that displays an optical tomographic image of the sample based on an output signal of a photoelectric conversion unit, wherein The length per pixel of the electronic display means is calculated from the actual size of the scanning means, and the first minimum scanning unit Z _s of the stepwise scanning by the scanning means is calculated based on the calculated length.
And determining a second minimum scanning unit Z _s ′ obtained by correcting the first minimum unit Z _s based on the refractive index n ₁ of the immersion medium of the objective lens and the refractive index n ₃ of the encapsulating medium. And displaying an optical tomographic image of the sample on the electronic display means while controlling the stepwise scanning of the scanning means according to the second minimum unit Z _s ′. Optical tomographic image display method. 3. The second minimum unit Z _s ′ is Z _s ′ = Z _s
The optical tomographic image display method according to claim 2, which is calculated according to n ₃ / n ₁ . 4. A laser light source for emitting a laser beam, an objective lens immersed in an immersion medium, a converging optical system for converging the laser beam from the light source to a sample in an enclosure medium, and a confocal detection method. Scanning means for scanning the focal position of the objective lens with respect to the sample stepwise along the optical axis direction of the objective lens; photoelectric conversion means for photoelectrically detecting reflected light or transmitted light from the sample; A method for displaying an optical tomographic image of a sample by a confocal laser scanning microscope using a digital computer based on an output signal of the photoelectric conversion device and an electronic display unit for displaying an optical tomographic image of the sample. Te, and providing at least comprises a database and a refractive index n ₃ of the refractive index n ₁ and the encapsulating medium of the immersion medium of the objective lens to said computer, before In the computer, the length per pixel of the electronic display means is calculated from the actual lateral size of the sample, and the calculated minimum length is the first minimum scanning unit of the stepwise scanning by the scanning means. Determining Z _s , and, in the computer, a second minimum corrected first minimum unit Z _s based on the refractive index n ₁ of the immersion medium of the objective lens and the refractive index n ₃ of the encapsulating medium. Calculating a scanning unit Z _s ′ and displaying an optical tomographic image of the sample on an electronic display unit while controlling the stepwise scanning of the scanning unit by the computer according to the second minimum unit Z _s ′. And a method for displaying an optical tomographic image of a confocal laser scanning microscope. 5. The second minimum unit Z _s ′ is Z _s ′ = Z _s
The optical tomographic image display method according to claim 4, which is calculated according to n ₃ / n ₁ .