JPS62274241A - Cell analyzing instrument - Google Patents

Abstract

PURPOSE:To obtain a two-dimensional sample and to obtain detailed information by obtaining the fluorescent image from a preliminarily fluorescent-dyed sample by another magnifying lens on a photodetector. CONSTITUTION:A laser beam diameter of a laser luminous flux 2 from a laser 1 of a cell analyzing instrument 1 is magnified by lenses 3, 4 and is made into parallel luminous fluxes which are condensed by a light lens 5 of a short focal length. The lens 5 is attached to a bimorph oscillator consisting of two sheets of piezo-electric elements and the oscillator 6 is fixed by a fixing jig 7. The condensed laser beam is controlled by an oscillator driving circuit 12 in such a manner that a beam west comes near to the center of a transparent glass cell 8 in which an observation sample such as cells is allowed to flow. The sample to be passed in the cell 8 is preliminarily subjected to fluorescent dyeing to emit fluorescence by absorbing laser light. The fluorescent image is projected by an objective lens 9 onto a one-dimensional image sensor 10. The two-dimensional sample is obtd. by moving the sample by which the detailed information on the cells, etc., is obtd.

Description

Detailed Description of the Invention 3. Detailed Description of the Invention [Field of Industrial Application] The present invention relates to a flow cytometer, and particularly to a cell analysis device suitable for obtaining two-dimensional information about cells.

[Conventional technology]

A conventional cell analysis device known as a flow cytometer is a device that irradiates the entire cell with a laser beam and detects the intensity of fluorescence emitted from the cell using a photodetector, as described in Japanese Patent Application Laid-Open No. 75473/1983. Information about the cells was obtained from the fluorescence intensity. but.

It was not possible to obtain two-dimensional information such as the spatial distribution of fluorescence intensity within each cell.

[Problem that the invention seeks to solve]

In the above-mentioned conventional technology, no consideration is given to scanning the laser beam to obtain an intracellular two-dimensional fluorescence image, and up to now there has been little need to obtain two-dimensional information.

However, to classify cells in more detail, cell morphological information is required. Conventionally, cell morphological information has been obtained using an optical microscope, but in order to perform image measurement of a large number of cells, a series of processing steps such as sample movement, focus adjustment, and input of one image are required, which reduces processing time. It takes a very long time.

For example, an automatic leukocyte classification device has a limit of image processing of 100 to 500 cells per minute.

An object of the present invention is to provide high-speed image input means that enables high-speed processing of two-dimensional fluorescent images of cells.

[Means for solving interlayer points]

The above object is achieved by means for focusing the laser beam at a sufficiently narrow speed and by scanning the beam waist portion where the beam diameter is the narrowest in the optical axis direction.

The method of scanning the beam waist in the optical axis direction is as follows:
Attach the condenser lens to the bimorph type pressure 11:J74,
A sawtooth wave voltage is applied to mechanically vibrate the condenser lens in the optical axis direction. As a result, the beam waist position of the laser beam moves back and forth in the optical axis direction. From this, the beam waist position, that is, the position where the light intensity per unit volume is maximum, is the position that is optically scanned. . One-dimensional information on cells, etc. can be obtained by detecting the fluorescence intensity of the fluorescence emitted from the beam waist position using a one-dimensional optical sensor described below, which can be switched in synchronization with optical scanning.

Actual cells are in solution, and if the solution is allowed to flow through a glass container at a constant speed, two-dimensional image information can be obtained by repeating optical scanning.

[Effect]

As is well known, a bimorph piezoelectric pendulum is a device in which two piezoelectric vibrators are stacked one on top of the other and a voltage of opposite polarity is applied to each piezoelectric vibrator to obtain mechanical vibration with a large vibration amplitude. . The vibration state is determined by the piezoelectric element constant dimension, applied voltage, etc. When an optical lens is attached to this bimorph type piezoelectric vibrator and a parallel beam of laser light is made incident on the lens, the beam waist position where the laser beam converges will be shifted along the optical axis due to the vibration of the lens that accompanies the mechanical vibration of the vibrator. Move back and forth. Since the light intensity is high at the beam waist position, the fluorescence intensity emitted from fluorescently stained cells is also high at this position. Before and after the beam waist, the laser beam rapidly spreads and the light intensity is low, and as a result, the fluorescence intensity is also low. When the voltage applied to the vibrator is made into a sawtooth wave, the moving speed of the beam waist becomes constant, resulting in linear one-dimensional scanning. When a fluorescence image obtained by the above-mentioned -dimensional scanning is received by a one-dimensionally arranged photodetector using a separately prepared microscope objective lens, a fluorescence image converted into a voltage signal is obtained. When a target object such as a cell is moved in a direction perpendicular to the optical scanning direction, in combination with a series of one-dimensional optical scanning,
A two-dimensional image signal is obtained.

Examples will be explained below.

〔Example〕

An embodiment of the present invention will be described below with reference to FIG. A laser beam 2 from a laser 1 is adjusted to have a diameter expanded by two lenses 3 and 4 and to become a parallel beam. The obtained light beam is focused by an optical lens 5 having a short focal length. The lens 5 is attached to a bimorph vibrator 6 made up of two piezoelectric elements, and the vibrator is fixed with a fixing jig 7.

The laser beam 11 focused by the condensing lens 5 is arranged so that its beam waist is near the center of the transparent glass cell 8 through which the m81 sample such as cells is passed. As is done in a flow cytometer, a sample such as cells is localized at the center of the transparent glass cell 8 and allowed to flow stably. This is to prevent the sample from straying from the laser beam and from staining the surface of the transparent glass cell 8. In FIG. 1, the sample flows in a direction perpendicular to the plane of the paper.

Samples such as cells are fluorescently stained in advance and emit fluorescence when they absorb laser light.

This fluorescent image is projected onto a one-dimensional photodetector 10 by an objective lens 9. A -dimensional photodetector is made up of a one-dimensional array of multiple independent photodetectors. As the bimorph oscillator vibrates, the position of the beam waist moves, and the beam waist image moves linearly on the -dimensional image sensor.

The bimorph oscillator 6 and the -dimensional image sensor 10 are controlled by signals from a scan control circuit 11. The photodetector elements of the bimorph vibrator are switched by a vibrator drive circuit 12, and the photodetector elements of the -dimensional image sensor 10 are switched by a sensor switching circuit 13.

The photodetector output is amplified to a required signal level by an amplifier 14 and subjected to necessary signal processing in a signal processing bag [15]. 1
Reference numeral 6 denotes an optical filter, which is inserted to block incident laser light and allow only fluorescence light to pass through. The signal processing device 15 is C
A signal processing device 15, which is an RT display device, writes data into a digital memory after AD conversion, and performs various image processing, is controlled by signals from the scan control section 11.

FIG. 2 is an enlarged view of the transparent glass cell 8 from the lateral direction. It can be seen that the beam waist of the laser beam moves due to the vibration of the bimorph oscillator. 1
7 is an example of a cell sample.

As described above, primary image information is obtained by vibrating the bimorph oscillator in a sawtooth pattern and receiving a fluorescent image from the sunburi with a one-dimensional image sensor, and
By flowing the sample at a constant flow rate, a two-dimensional image can be formed by multiple optical scans. As a result, more detailed observation data can be obtained based on two-dimensional information within a cell, rather than information on the entire cell as in the conventional method.

It is assumed that the one-dimensional image sensor 10 is a non-accumulating photodetector, and the switching position of each photodetector is switched in synchronization with the beam waist. As a result, the fluorescence signal at the beam waist position can be made to correspond one-to-one with the photodetector output. By making the dimensions of each photodetector of the -dimensional image sensor 10 correspond to the size of the beam waist, it is possible to obtain a two-dimensional image signal with high resolution.

In the first embodiment, a non-accumulative photodetector was used as the one-dimensional image sensor 10, but? # CCD with multi-layer characteristics
Two-dimensional image information can be obtained by a type-dimensional image sensor. In this case, due to accumulation, a single photodetecting element detects not only the beam waist but also the integrated light of multiple optical scans, which causes image blurring, but the optical scanning speed is faster than the moving speed of cells, etc. This can be dealt with by making it sufficiently fast. In addition, the resolution decreases because the fluorescence from the part where the beam diameter is widened before and after the beam waist is also detected, but if the convergence angle of the laser beam is large, the fluorescence from the part where the beam spread is large is detected. It is weak and there is little decrease in resolution.

In addition, in the present invention, as can be seen from Fig. 2, if there is something with a high light absorption rate and a large area before the laser beam is focused, the light amount at the beam waist will be small and the fluorescence intensity will also be small. .

In this case, the effects of the present invention may not be fully exhibited.

The laser that is the light source of the present invention is capable of concentrating light into a minute spot size compared to a normal lamp.
As a result, the fluorescence intensity also increases, making light detection easier.

In the previous embodiments, a convex lens was used as the condenser lens to condense light to one point, but if a cylindrical convex lens is used as the condenser lens 5, the following first-order advantages arise. In Example 1, information can only be obtained from one point where the laser is focused, but by using a cylindrical lens, the laser is focused in a straight line due to the nature of the lens, and as a result, -dimensional image sensor 10
Integral fluorescence data for the entire range of light focused in a straight line is obtained. Therefore, a two-dimensional image with the same effect as a fluorescence microscope image can be obtained.

Furthermore, the above object can be achieved by using one linear photodetector instead of the -dimensional image sensor. In this case, light from areas other than the beam waist is detected in the same way, which may cause a reduction in resolution depending on the target sample.

〔Effect of the invention〕

According to the present invention, it is possible to realize secondary optical scanning by taking into account the effect of the flow of a sample such as a cell, compared to a conventional two-dimensional image input device using a fluorescence microscope or an "I" V camera.

(]) Two-dimensional fluorescence intensity distribution of cells, especially white blood cells, etc.
It is possible to obtain it at high speed.

(2) Since the laser beam can be focused on a minute spot, the fluorescence intensity is stronger than that of a lamp or the like.

Light detection is easy.

(3) By knowing the two-dimensional fluorescence intensity distribution, it is possible to obtain more detailed information about the cells.

[Brief explanation of the drawing]

FIG. 1 is an overall configuration diagram showing an embodiment of the present invention. FIG. 2 is a conceptual diagram explaining the present invention in detail. DESCRIPTION OF SYMBOLS 1... Laser, 5... Condensing lens, 6... Bimorph resonator, 8... Transparent glass cell, 9... Objective lens, 10... -dimensional image sensor, 11... Scanning control circuit, 12... Vibrator drive circuit, 13... Sensor switching circuit, °.

Claims (1)

[Claims] 1. A means for parallelizing the spread of the laser beam, a means for vibrating the light converging lens attached to the bimorph piezoelectric vibrator back and forth in the optical axis direction by applying a voltage, and as a result, the condensed beam waist section is moved. A sample sample previously stained with fluorescence is moved at a constant speed by a means capable of moving the sample sample, and a fluorescent image from the sample sample is generated on a photodetector using another magnifying lens. A cell analysis device characterized by obtaining a two-dimensional sample image.