JP2000097857A - Scanning sight meter - Google Patents

Abstract

PROBLEM TO BE SOLVED: To impart a CLSM(confocal laser scanning microscope) function while suppressing an economical burden for a user and increase of an occupied area. SOLUTION: This scanning sight meter (LSC) 10 has an excited light introducing part 100 for emitting excited light, a dichroic mirror 12 for separating the excited light and fluorescence, a light deflecting system 20 for scanning the excited light two-dimensionally, a pupil projection lens 32, an objective lens 34, and a fluorescence detecting part 200. The introducing part 100 and the detecting part 200 are provided with two systems of an optical system for LSC measurement and an optical system for CSLM measurement respectively. The introducing part 100 emits a laser beam incident to a pupil position of the objective lens 34 with a light flux diameter sufficiently smaller than an opening diameter of a pupil of the lens 34 in the LSC measurement, and emits the laser beam incident to the pupil position of the lens 34 with the light flux larger than the opening diameter of the lens 34 in the CSLM measurement.

Description

DETAILED DESCRIPTION OF THE INVENTION

[0001]

BACKGROUND OF THE INVENTION 1. Field of the Invention The present invention relates to a scanning cytometer for two-dimensionally scanning a specimen with laser light to measure cells.

[0002]

2. Description of the Related Art A cell population on a slide glass is scanned with a spot in which a laser (laser beam) is converged, fluorescence emitted from individual cells of the cell population is detected, and scanned image data is image-processed. "Method and apparatus for measuring multiple optical properties of biological specimen" for extracting and measuring cell data ("Laser scanning cytometer (LSC: Laser)
Scanning Cytometer) ”) is disclosed in JP-A-3-255365.
Is disclosed.

[0003] A laser scanning cytometer (hereinafter, referred to as a "scanning cytometer") (LSC) is a method in which an isolated cell suspension sample is fixed to a fixed laser and a spot where the laser beam is focused. Unlike a flow cytometer that measures a cell by dividing a cell population into tens of thousands of water droplets per second by ultrasonic vibration and passing it over a laser spot as a jet stream, it is placed on a slide glass. There is a difference in the basic configuration in that each cell is measured by optically and mechanically scanning a laser spot with respect to the collected cell population. However, a scanning cytometer (LSC)
Irradiates and excites a laser spot on a cell population that is biochemically labeled with a fluorescent dye, measures the fluorescence emitted by individual cells at high speed, and measures the immunological properties of the cell population,
It is the same as the flow cytometer in that the purpose is to present the measurement results as statistical data representing genetic characteristics, cell proliferation, and the like.

Further, a scanning cytometer (LSC) is
Record the coordinate position of each cell on the slide glass, and
Reproduction of the coordinate position by the scanning stage is possible. As a result, individual cells exhibiting a photometric value corresponding to a specific subset of the statistical data can be moved into the visual field of the microscope and observed under a microscope. That is, a cytometer (LSC) is superior to a flow cytometer in that a microscope observation image can be associated with cell data. A function of sequentially reproducing the position of each cell under a microscope and observing the microscopic shape after measuring the cell population specimen is hereinafter referred to as a “recall observation function” of a scanning cytometer (LSC).

[0005] A typical medical and biological research application of a scanning cytometer (LSC) is in the cell cycle analysis of cancer tumors. The property of "proliferative" is particularly important in cancer tumors, but this cannot be achieved by observing individual cells under a microscope, and hundreds to thousands of Among cells, a statistical numerical index can be objectively expressed by a ratio of the number of cells belonging to a stable phase, a DNA synthesis phase, and a mitotic phase of the cell division cycle. Scanning cytometers (LSCs) provide such statistical numerical indices while specifying individual cell data belonging to a particular phase within the cell division cycle (eg, metaphase) to isolate individual cells within a microscopic field. The morphological examination can be performed under a microscope by the recall observation function that is called up sequentially.

On the other hand, as an apparatus for observing a fluorescently labeled cell structure, a confocal laser scanning microscope (hereinafter, “CLS”) is used.
M: Conforcal Laser Scanning Microscope ”). The CLSM enables high-resolution optical section observation with respect to a conventional fluorescence microscope, and acquires moving means in the optical axis (Z) direction and an optical cut image data sequence in the optical axis (Z) direction. In combination with image processing means for constructing and displaying a three-dimensional image, it has the advantage that it can observe the cell structure three-dimensionally and analyze its morphology.

In the above-mentioned analysis of the cell cycle by the scanning cytometer (LSC), it is an important application to analyze the behavior of the chromosome three-dimensionally in the above-mentioned, middle and late stages during the cell division, in particular. Done CLS
This is expected to be shared with M's recall observation function. Further, from the viewpoint of apparatus configuration technology, the function items such as laser irradiation, two-dimensional scanning, fluorescence detection, and scanning image data acquisition are performed by a scanning cytometer (LSL).
It can be said that C) is common to CLSM.

On the other hand, the specifications of common function items between the scanning cytometer (LSC) and the CLSM are different due to the difference between the purpose of the apparatus and the performance to be achieved. No device having the function of CLSM is provided.

[0009] If the user needs to share, the same stage as the scanning stage provided in the scanning cytometer (LSC) is mounted on the CLSM, and a different one from the scanning cytometer (LSC). By using the CLSM for recall observation as a system, it is feasible to use two system products together.

However, the user has to use and use two system component units, such as a microscope, a scanning stage, a laser light source, a scanning device, a fluorescence detector, and a computer for image capturing. There is a disadvantage that the cost burden increases and the space occupied by the apparatus in a laboratory or the like increases. Also, the user
The two devices must be operated separately, which is inconvenient to use.

For this reason, although the use of the scanning cytometer (LSC) and the CLSM in medical and biological applications is advantageous, the combined use of the scanning cytometer (LSC) and the CLSM is almost impossible. The fact is that it has not been done.

[0012]

Although there is no prior art which claims to share a scanning cytometer (LSC) and a CLSM, the scanning cytometer (LSC) and the CLS do not.
The technical problems will be described based on M's prior application.

JP-A-3-255365 shows the idea of a scanning cytometer (LSC) as described at the beginning. Here, the spot size of the laser that irradiates the sample and scans it two-dimensionally is assumed to be “equivalent to the cell size (within the same order of magnitude).” Regarding the fluorescence detection system, both confocal and non-confocal systems are described. Not. Regarding the spot size, it is described that the cell population sample should be scanned faster.

On the other hand, according to Japanese Patent Application Laid-Open No. 7-209187,
In order to acquire a “high-resolution image” for morphological observation at the same time as scanning for photometry, it is assumed that two or more aperture diameters having different sizes are prepared as confocal detection apertures (“openings arranged at image forming positions”). I have. According to the present invention, since the “high-resolution image” for morphological observation is acquired in advance at the same time as the photometry, the sample is not relocated during the recall observation of the scanning cytometer (LSC) and the captured “high-resolution image” is acquired. Just call

However, storing “high-resolution images” of thousands to tens of thousands of cell populations as data for each cell measurement of each cell population specimen requires a huge data storage capacity. And For this reason, the system scale increases and the cost rises, and at the same time, the time for storing and retrieving data increases, and the throughput as the system decreases.

When acquiring a "high-resolution image", since the optical depth of focus becomes shallow, when acquiring a high-resolution image, focus adjustment is repeated in order to correct defocus during sample scanning. You have to do it.

Further, in combination with moving means in the direction of the optical axis (Z) and image processing means for acquiring a sequence of optically cut image data in the direction of the optical axis (Z) to construct and display a three-dimensional image, individual cells In order to take advantage of the CLSM's feature of being able to observe the structure three-dimensionally and analyze its morphology, acquisition of a “high-resolution image” sequence requires more time and data storage capacity.

Also, the "high-resolution image" achieved here
Irradiates a laser spot for photometry with a relatively large spot size ("equivalent to cell size") and reduces only the aperture on the fluorescence detection side, so the CLSM theory in both the in-plane direction and the optical axis direction is used. It is impossible to achieve a typical resolution limit. In other words, the “high-resolution image” obtained in JP-A-7-209187 has both resolution and optical cutting performance.
The CLSM is unsatisfactory, and has a drawback that it cannot fulfill the purpose of the above-mentioned common use of the scanning cytometer (LSC) and the CLSM. The present invention provides a CL system that suppresses the burden on the user and the increase in occupied area.
It is an object of the present invention to provide a scanning cytometer having the function of the SM.

[0019]

A scanning cytometer according to the present invention comprises: a laser light source for emitting laser light; an objective lens for focusing the laser light from the laser light source on a sample;
A light beam diameter switching means for switching a light beam diameter of laser light incident on the pupil of the objective lens from the laser light source; a scanning means for scanning a sample with the laser light from the laser light source; An optical path switching means capable of switching between an optical path of a focus detector and an optical path for non-confocal detection, and a detector for detecting light from the sample are provided.

[0020]

FIG. 1 shows the configuration of a scanning cytometer according to an embodiment of the present invention. The scanning cytometer (LSC) 10 has the function of CLSM,
Either C measurement or CLSM measurement can be selectively performed.

The apparatus 10 includes an excitation light introducing section 100 for emitting excitation light, a dichroic mirror 12 for separating excitation light and fluorescence, and an optical deflector 2 for two-dimensionally scanning the excitation light.
0, a pupil projection lens 32, an objective lens 34, and a fluorescence detection unit 200.

Excitation light introduction unit 100 and fluorescence detection unit 200
Are each provided with two systems, an optical system for LSC measurement and an optical system for CLSM measurement. Excitation light introduction unit 100
Is a laser light source 1 for emitting a laser beam as excitation light.
02 and a spot projection lens 104 for LSC measurement
And

Further, the pumping light introducing unit 100 includes a CLSM
For this purpose, two convex lenses 118 and 120 constituting a beam expander and four optical path bending mirrors 106, 114, 116 and 110 for passing light through the beam expander are provided.

Two optical path bending mirrors 106 and 110
Are selectively removably disposed on the optical path between the laser light source 102 and the spot projection lens 104, and between the spot projection lens 104 and the dichroic mirror 12, respectively. That is, the two optical path bending mirrors 10
6 and 110 are slide mechanisms 108 and 112, respectively.
Depending on the type of measurement, it is configured to be inserted into the optical path as shown by a solid line or removed from the optical path as shown by an imaginary line, depending on the type of measurement.

The optical deflector 20 has two galvanometer mirrors whose driving directions are different from each other by 90 °. By changing the direction of each reflecting surface, the light deflector 20 can deflect a light beam in XY directions orthogonal to each other. . In the figure, only one galvanometer mirror is shown for convenience of explanation.

The fluorescence detector 200 has a collector lens 204 for LSC measurement and a photodetector for detecting fluorescence, for example, a photomultiplier tube 202. Fluorescence detector 20
0 is a convex lens 2 for condensing fluorescence for CLSM
18 and the confocal pinhole 2 located at the focal position
22, a convex lens 220 for converting the fluorescent light passing through the confocal pinhole 222 into a parallel light beam, and four optical paths for guiding the fluorescent light to the confocal pinhole 222 and guiding the passing light to the photomultiplier tube 202 The folding mirrors 206, 214, 216, and 210 are provided.

Two optical path bending mirrors 206 and 210
Are selectively removably disposed on the optical path between the dichroic mirror 12 and the collector lens 204 and between the collector lens 204 and the photomultiplier tube 202, respectively. That is, the two optical path bending mirrors 206 and 2
10 is inserted into the optical path as shown by a solid line or removed from the optical path as shown by an imaginary line, depending on the type of measurement, by slide mechanisms 208 and 212 (optical path switching means), respectively. It is composed of

The apparatus 10 further includes a stage 50 on which the specimen 40 is placed, and a controller 60 for controlling each unit.
An external input device 62 for inputting necessary information;
It has a monitor 64 for displaying measurement results and the like.

The stage 50 includes two tables movable in directions orthogonal to each other, and can move the placed sample 40 in XY directions orthogonal to each other. The controller 60 controls the optical deflector 20, the stage 50, and the four slide mechanisms 108, 112, 208, and 212, processes the measurement data obtained from the photomultiplier tube 202, and monitors the processing results and necessary information. 64 is displayed.

In the LSC measurement, the four optical path bending mirrors 106, 110, 206, and 210 are moved to the positions shown by imaginary lines by the four slide mechanisms 108, 112, 208, and 212, respectively, and both of them are out of the optical path. Is done.

The laser emitted from the laser light source 102 is reflected by the dichroic mirror 12 through the spot projection lens 104, and is reflected by the optical deflector 20 and the pupil projection lens 3
Then, the light enters the objective lens 34 via the second lens 2 and is focused on the surface of the sample 40.

The laser beam emitted from the laser light source 102 is generally a parallel beam, but strictly speaking, the laser beam travels while gradually expanding from the beam inside the laser tube. The spot projection lens 104 is a weak convex lens that suppresses the spread, and correctly forms an image of the light beam in the laser tube on the surface of the sample 40.

Accordingly, the laser beam enters the pupil position of the objective lens 34 with a light beam diameter sufficiently smaller than the aperture diameter of the pupil of the objective lens 34. In LSC measurement, in order to scan a wide range, the optical deflector 20 includes one galvanomirror 22,
For example, only the galvanomirror 22 that scans the light beam in the X direction is driven, and in response to this, the sample 50 is moved by the stage 50 in a direction (Y direction) orthogonal to the scanning direction of the light beam by the galvanomirror 22.

As described above, by combining the movement of the spot by the optical deflector 20 at a high speed but limited to a narrow range and the movement of the sample 40 by the stage 50 at a low speed but over a wide range, the spot formed on the sample surface becomes Scanned over a large area on the specimen.

The fluorescent light emitted from the specimen 40 is collected by the objective lens 34, passes through the pupil projection lens 32 and the optical deflector 20, passes through the dichroic mirror 12, passes through the collector lens 204, and passes through the head-on type photoelectron. Multiplier tube 202
And is photoelectrically converted.

The fluorescent light traveling from the light deflector 20 to the photomultiplier tube 202 is substantially a parallel light flux, but strictly diverges when the specimen defocuses in a direction approaching the objective lens. The collector lens 204 is a convex lens for causing the light to converge, and connects the light receiving surface of the photomultiplier tube 202 to the optical deflector 20 in an almost image-forming relationship. Since the pupil plane of the objective lens 34 and the light deflector 20 are connected in an image forming relationship by the pupil projection lens 32, the pupil plane of the objective lens 34 and the light receiving surface of the photomultiplier tube 202 are connected in an image forming relation. I have. As a result, the light intensity distribution on the light receiving surface is hardly changed by the pupil surface photometry even when scanning is performed by the optical deflector 20 or defocusing occurs due to variations in the thickness or position of the sample.

Since the fluorescence from the specimen 40 is emitted from the entire pupil opening of the objective lens 34, the aperture diameter of the collector lens 204 is determined by the maximum pupil diameter of the objective lens 34 to be used and the diameter when the specimen 40 approaches the objective lens. It is determined in consideration of the worst defocus condition.

The output of the photomultiplier tube 202 is input to the controller 60. The controller 60 processes the scanned image data based on the photoelectric signal from the photomultiplier 202 to quantitatively measure the amount of intracellular components of each cell in the specimen. The controller 60 further records coordinate position data of each cell in the specimen, statistically analyzes the photometric value of each cell,
The position of the specimen 40 is controlled by the stage 50 based on the coordinate position data of the individual cells belonging to the specific subset, and the desired cells are rearranged in the microscope field of view, whereby the device 10 realizes the recall observation function. I can do it.

In the CLSM measurement, four optical path bending mirrors 106, 110, 206, and 210 are moved to four slide mechanisms 108, 112, 208, and 212, respectively.
To the position shown by the solid line, and both are arranged on the optical path.

The laser beam emitted from the laser light source 102 is reflected by two optical path bending mirrors 106 and 114, passes through a beam expander composed of two convex lenses 118 and 120 having different focal lengths, and is bent into two optical paths. After being reflected by mirrors 116 and 110, it is reflected by dichroic mirror 12, and is deflected by optical deflector 20 and pupil projection lens 3.
Then, the light enters the objective lens 34 via the light source 2 and is focused on the sample surface 40.

The laser beam has a luminous flux diameter equal to the focal length of the convex lens 120 / convex lens 11 by a beam expander.
The image is enlarged at an enlargement magnification determined by the focal length of 8. As a result, the laser enters the pupil position of the objective lens 34 with a light beam diameter larger than the aperture diameter of the pupil of the objective lens 34.

In the CLSM measurement, since the spot is moved with high positional accuracy, scanning by the spot on the specimen 40 is performed by the optical deflector 20 having high speed and high position controllability.
Using two galvanometer mirrors.

The fluorescence emitted from the specimen 40 is collected by the objective lens 34, passes through the pupil projection lens 32 and the optical deflector 20, passes through the dichroic mirror 12, and is reflected by the two optical path bending mirrors 206 and 214. After that, the light is condensed by the convex lens 218 to form a confocal spot. The light that has passed through the confocal pinhole 222 is
And two optical path bending mirrors 21
After being reflected by 6 and 210, the light enters a head-on type photomultiplier tube 202 and is photoelectrically converted.

The convex lens 218 is designed to form a confocal spot having a diffraction limit diameter. The confocal pinhole 222 is configured so that the controller 60 can adjust the position and select the aperture size by using a turret (not shown) so that the confocal pinhole 222 is accurately arranged in conformity with the confocal spot size. Is preferred.

Generally, the CLSM measurement is performed following the LSC measurement. For example, after grasping the overall tendency of the sample by LSC measurement, CLS is performed on the part that attracts the user's attention.
Generally, an M measurement is performed.

As described above, since the coordinate position data of each cell in the specimen has already been acquired at the time of LSC measurement, CL
In the SM measurement, the controller 60 controls the external input device 62
The apparatus 10 moves the stage 50 in accordance with the information input from the user, for example, a location desired by the user to observe, and arranges specific cells in the specimen 40 on the optical axis.

The stage 50 preferably has a function of moving the specimen 40 along the optical axis direction, that is, the Z direction, so that the apparatus 10 can generate an optical cross-sectional image utilizing the features of the confocal optical system. Can get. Further, the device 10
By controlling the Z-direction position of the specimen 40 by the controller 60 and acquiring the optical cut image data sequence,
A three-dimensional image can be constructed.

As can be seen from the above description, the device 1
0 controls the positions of the optical path bending mirrors 106, 110, 206, and 210 inside the excitation light introducing unit 100 and the fluorescence detecting unit 200, so that the scanning cytometer (LS
C) and the function of CLSM can be switched.

For this switching, preferably, a dedicated GUI (Graphical User Interface) is prepared in the external input device 62, and the slide mechanisms 108 and 112 are
, 208 and 212 and the optical deflector 20 and the stage 50. Thus, the user can switch between the scanning cytometer (LSC) and the CLSM without being particularly conscious.

Therefore, at the time of recall observation with a scanning cytometer (LSC), high-resolution three-dimensional morphological observation by CLSM can be applied. In the present embodiment, an apparatus having one laser light source 102 and one photomultiplier tube 202 has been described as an example. Of course, the present invention provides, for example, The present invention can also be applied to an apparatus having one or more laser light sources and a plurality of photomultipliers for simultaneously measuring a plurality of different fluorescences.

The present invention is not limited to the above-described embodiment, but includes all implementations that do not depart from the gist of the invention. For example, even when one laser light source is used as in the present embodiment, in addition to switching the optical path and switching the light beam diameter in each optical path, as a configuration for switching the optical path system, for example, on the optical path of the laser light source, The lens used in the present embodiment can switch the beam diameter by moving the lens on the optical path through a lens capable of performing an afocal zoom, or by providing a plurality of lenses having different focal lengths, for example, in a slide mechanism, By switching a lens having a desired focal length in the diameter-enlarged optical path, the light beam diameter can be switched.

The present invention includes the embodiments exemplified in the following sections. 1. A laser is condensed by a microscope objective lens on a cell population specimen, two-dimensional scanning is performed, fluorescence from cells excited by the laser is detected by a detector, and a scan formed by a photoelectric signal detected by the detector is performed. Image data is taken into a computer, the amount of intracellular components of each cell in the cell population sample is quantitatively measured by image processing on the scanned image data, and coordinate position data of each cell in the cell population sample is recorded. A scanning type that statistically analyzes the photometric value of the cells and controls the stage based on the coordinate position data of the individual cells belonging to a specific subset to move each cell within the field of view of the microscope and observe with a microscope. The cytometer has a means for irradiating the sample with a light beam equivalent to the diffraction limit size determined by the numerical aperture of the objective lens to perform two-dimensional scanning and a means for detecting confocal fluorescence. And it features. 2. 2. The apparatus according to claim 1, wherein at least one of the means for irradiating the sample with a light beam equivalent to the diffraction limit size determined by the numerical aperture of the objective and performing two-dimensional scanning and the means for detecting confocal fluorescence is used. At least in part,
It is characterized by being shared with the scanning type cytometer. 3. 3. The apparatus according to claim 2, further comprising moving means in the optical axis (Z) direction and image processing means for acquiring an optical cut image data sequence in the optical axis (Z) direction to construct and display a three-dimensional image. It is characterized by the following. 4. 3. The apparatus according to claim 2, wherein at least the laser and the optical deflector of the means for irradiating the sample with a light beam equivalent to the diffraction limit size determined by the numerical aperture of the objective lens and performing two-dimensional scanning include the scanning cell measuring device and It is shared, and is characterized by comprising a laser beam diameter converting means. 5. 3. The apparatus according to claim 2, wherein at least the fluorescence detector of the confocal fluorescence detection means is shared with the scanning cytometer, and at least one of the fluorescence focusing lens switching means and the confocal pinhole insertion / removal switching means. It is characterized by having one. 6. 3. The apparatus according to claim 2, wherein the means for irradiating the sample with a light beam equivalent to the diffraction limit size determined by the numerical aperture of the objective lens to perform two-dimensional scanning and the means for detecting confocal fluorescence are a multi-pinhole disk scanner and an image sensor. Is characterized by the following.

[0053]

According to the present invention, there is provided a scanning cytometer having a CLSM function while suppressing an economic burden on a user and an increase in occupied area. This allows the CLSM to be used during recall observation of the scanning cytometer (LSC).
Can be applied, and more preferable morphological observation can be performed.

[Brief description of the drawings]

FIG. 1 shows a configuration of a scanning cytometer according to an embodiment of the present invention.

[Explanation of symbols]

 DESCRIPTION OF SYMBOLS 20 Optical deflector 34 Objective lens 100 Excitation light introduction part 118 Convex lens 120 Convex lens 200 Fluorescence detector 202 Photoelectron image multiplier 218 Convex lens 222 Confocal pinhole

Claims (1)

[Claims] 1. A laser light source for emitting laser light, an objective lens for focusing laser light from the laser light source on a sample, and a light beam diameter of the laser light incident on a pupil of the objective lens from the laser light source. Possible beam diameter switching means; scanning means for scanning a sample with laser light from the laser light source; and an optical path capable of switching light from the sample to an optical path of a confocal detector and an optical path for non-confocal detection. A scanning cytometer comprising: a switching unit; and a detector that detects light from the sample.