JPH03221838A - Method and device for measuring body to be tested - Google Patents

Abstract

(57) [Summary] This bulletin contains application data before electronic filing, so abstract data is not recorded.

Description

[Detailed Description of the Invention] [Industrial Application Field] The present invention is capable of analyzing specimens and detecting antigen-antibody reactions by irradiating light onto individual specimens in a sample and performing optical measurements such as scattered light and fluorescence. The present invention relates to a sample testing method and device for performing measurements, etc.

[Prior Art] A flow cytometer is known as an example of a conventional specimen testing device. This involves fluorescently labeling specimens such as blood cells in a sample solution, and passing these specimens one by one onto a test area that is irradiated with a light beam. By selecting these parameters, these parameters are photometrically measured for each specimen, and the specimens are analyzed based on statistical trends in the measured parameters for a large number of specimens.

This makes it possible to analyze cell DNA and search for surface antigens.

[Problems to be Solved by the Invention] However, conventionally, a dedicated photodetector is used for each fluorescence to measure fluorescence of a plurality of colors.

Until now, it was common to have a configuration that simultaneously measured two colors, red fluorescence and green fluorescence, or three colors with yellow fluorescence added, but in recent years there has been an increasing demand for even more colors, and new fluorescence Development of materials is also progressing.

As a result, if the number of fluorescence channels used simultaneously increases, the number of photodetectors will also increase accordingly. That is, there is a problem in that the optical arrangement becomes complicated and a large number of expensive photodetectors such as photomultipliers are required.

The present invention has been made to solve the above-mentioned problems, and an object of the present invention is to provide an analyte measurement method and apparatus that can obtain different fluorescence parameters similar to the number of photodetectors.

[Means for Solving the Problems] The present invention for solving the above-mentioned problems includes a step of fluorescently labeling an analyte in a sample, a step of irradiating each analyte in the sample with light, and a step of irradiating the individual analytes in the sample with light. A method for measuring a specimen, comprising the step of photometrically measuring light emitted from the specimen and fluorescence emitted from the specimen during non-irradiation after a predetermined period of time from the time of light irradiation using the same photodetector.

Also, means for irradiating light onto an irradiation position, and means for sequentially passing individual fluorescently labeled specimens to the irradiation position;
Specimen measurement characterized by having means for measuring light emitted from the specimen at the irradiation position and fluorescence emitted from the specimen at a predetermined non-irradiation position downstream of the irradiation position in time series using the same detector. It is a device.

[Example] Embodiments of the present invention will be described in detail below with reference to the drawings.

1 and 2 show the optical arrangement of an embodiment of the invention.

A sample solution such as blood or latex suspension adjusted to an appropriate reaction time and concentration and simultaneously labeled with multiple types of fluorescent reagents, and a sheath solution such as physiological saline or distilled water are prepared. These liquids are pressurized by the pressurizing mechanism and guided to the flow cell 3, and according to the sheath flow principle, the sample liquid is wrapped in the sheath liquid within the flow cell 3 and is converged into a thin flow, which passes through the flow section within the flow cell 3. At this time, the test particles such as individual cells and latex contained in the sample liquid are separated and blown away one by one from the top to the bottom of the page. A laser beam emitted from a laser light source 1 (in this example, an ultraviolet laser with a wavelength of 300 nm) is directed against this flow of particles to be detected.
The light is converged into an arbitrary elliptical shape by an imaging optical system 2 consisting of two cylindrical lenses whose generatrix directions are in the direction of the flow section and perpendicular to the direction of the flow section, respectively. Generally, it is preferable that the shape of the light beam irradiated onto the test particles is an ellipse having a major axis in a direction perpendicular to the flow.

This is to ensure that the light beam is irradiated with uniform intensity onto the test particles even if the flow position of each test particle varies slightly in the fluid.

When a light beam is irradiated onto a particle to be examined, scattered light is generated. Among the generated scattered lights, the forward scattered lights emitted in the forward direction of the optical path are photometered by the condenser lens 6, the aperture, and the photodetector 8. The opening of the aperture is arranged conjugately with the light irradiation position 4, so that only the light from the irradiation position 4 is guided to the photodetector 8. In order to prevent the irradiated strong light beam from directly entering the photodetector 8, a small light-absorbing stopper 5 is provided in the optical path in front of the condensing lens 6, so that the direct light from the irradiation light source, And the transmitted light that has passed through the test particles is removed. Thereby, only the light scattered from the light irradiation position 4 can be photometered. Note that in this configuration, the combined light intensity of forward scattered light and fluorescence is measured, but this poses no problem since the fluorescence intensity is generally extremely weak compared to the forward scattered light intensity. Of course, a bandpass filter that selectively transmits the wavelength of the scattered light may be placed in front of the photodetector 8.

Of the scattered light, the light emitted in the side direction perpendicular to the laser beam and the flow of the particles to be detected is emitted by the condenser lens 9.
The light is focused. The focused light is passed through dichroic mirror 1
0 and the wavelength of the scattered light, that is, the wavelength of the laser light (30
The side scattered light is measured by a photodetector 17 through a bandpass filter 11 that selectively transmits light (near 0 nm), a condensing lens 13, and an aperture 15. As shown in the figure, the aperture 15 is provided with an opening 15a at the center of the optical axis, and the opening 15a is in a conjugate relationship with the light irradiation position 4, so that only the scattered light emitted from the light beam position 4 is detected by the photodetector 17. It is now detected in .

In addition, in order to measure the fluorescence of multiple colors generated together with the scattered light from the test particles, the fluorescence that has been condensed by the condenser lens 9 and passed through the dichroic tube 10 is collected by the condenser lens 14.
, aperture 3o, and photodetector 18, fluorescence of a specific wavelength is detected. The detailed structure of the aperture 30 is shown in FIG. In FIG. 6, there are two openings 3 in the center of the light-shielding aperture 30 and in the vicinity thereof.
0a and 3Qb are provided. Each opening 30a, 30
Bandpass filters 31 and 32 are attached to each of the filters b, and each bandpass filter has the property of selectively transmitting only fluorescence of a specific wavelength. Opening 3
0a is provided at the center of the aperture 30, and an opening 30b is provided at the center of the aperture 30. Here, the opening 30a is conjugate with the light irradiation position 4, and the opening 30b is conjugate with a position slightly downstream of the light irradiation position 4. That is, the opening 30a selectively guides fluorescence of a specific wavelength emitted from the light irradiation position 4 to the photodetector 18, and the opening 30b allows fluorescence of a specific wavelength emitted from the light irradiation position 4 to be guided from a non-irradiation position downstream of the light irradiation position 4. Only fluorescence of a specific wavelength is guided to the photodetector 18. In other words, the photodetector 18 is configured to obtain two types of fluorescence photometric output pulses in time series with time intervals, and two types of fluorescence parameters can be obtained in time series with the same photodetector. It will be done.

Note that the characteristics of the pan 1 to the bus filter 31 are different from those that selectively transmit scattered light wavelengths, so that the same photodetector 18 can obtain two different parameters: side scattered light intensity and fluorescence intensity. In this case, since there is a large difference in intensity between the scattered light and the fluorescence, it is preferable to use a photodetector 18 with a wide dynamic range or a bandpass filter 31 with a high degree of optical attenuation.

The signals from the photodetectors 8.17.18 are processed in a signal processing section as shown in FIG. The forward scattered light output of the photodetector 8 is input to a trigger circuit 21 and an analog processing circuit 22 as a pulsed electrical signal. Further, the side scattered light output of the photodetector 17 and the fluorescence output of the photodetector 18 are input and summed into analog processing circuits 23 and 24, respectively.

In each analog processing circuit, the peak value and integral value of the signal pulse are detected. When the forward scattered light signal exceeds a certain level, i.e., when a particle to be inspected approaches the light irradiation position, a trigger signal is generated for a certain period of time, and this signal is sent to the analog processing circuit 22. and is manually input to the analog processing circuit 23. Each analog processing circuit has a sample and hold function, so that the human input signal is processed only during the period when the l-rigger signal is generated. Further, the trigger signal from the trigger circuit 21 is manually inputted to the fluorescence trigger generation circuit 25, and the fluorescence trigger generation circuit 25 operates only while the scattered light detection trigger signal is being generated and from time t to time t2. Trigger signal 2
The signal is manually input to the analog processing circuit 24. This allows the signal of the fluorescence photodetector 18 to be sampled twice, when the particles pass through the light irradiation position 4 and thereafter from t1 to t2. The output of each analog processing circuit 22, 23, 24 is the A/D conversion circuit 26.
The signals are converted into digital values and stored in the memory 27 as forward scatter signals, side scatter signals, and two-color fluorescence signals, respectively.

The memory 27 obtained with respect to a large number of samples as described above.
Based on the measurement parameters stored in the arithmetic circuit 28
Sample analysis calculations are performed at , and the results are output to an output section 29 such as a CRT or printer.

Next, the measurement principle of the present invention will be explained using FIG. In the present invention, the fluorescent dyes used are a combination of commonly used fluorescent dyes with short fluorescence lifetimes and fluorescent dyes with longer fluorescence lifetimes than ordinary autofluorescence. Alternatively, select multiple types of fluorescent materials with long fluorescence lifetimes. FIG. 4 is a graph showing the lifetime curve of fluorescence excited by light irradiation. The horizontal axis represents elapsed time, and time O is the moment when the test particle is irradiated with light in the test section. Moreover, the vertical axis is the amount of fluorescence generated. In the figure, curve 19 is due to commonly used fluorescent dyes or autofluorescence possessed by cells and dirt, and the fluorescence lifetime from excitation until the fluorescence intensity reaches O is about 100 nsec. . On the other hand, curve 20 is a lifetime curve of a fluorescent dye having a longer lifetime than usual used in the present invention, and for example, Eu3+ chelate is used as the fluorescent dye.

The fluorescence lifetime in this case is 1000 nsec or more. Note that this fluorescence lifetime curve has approximately the same shape as long as the intensity of the laser light that excites the fluorescence is constant.

Assuming that a particle is irradiated with light at the instant of time O, the generation of scattered light ends instantaneously, and even for ordinary fluorescent dyes with short fluorescence lifetimes, the amount of fluorescence generated reaches O by time t1. The opening 30a of the aperture 30 shown in FIG. 5 is arranged conjugately with the light irradiation position 4 where the light spot hits, and selectively guides only the fluorescence 19 of a specific wavelength from the specimen flowing through this position to the photodetector. It looks like this. Further, the opening 30b is arranged conjugately with a non-irradiation position slightly downstream of the light irradiation position 4, so that only the fluorescence 20 of a specific wavelength 1 from this part is guided to the photodetector. In other words, assuming that the particle flow velocity is constant in the opening 30b, only the light in the shaded area between time t1 and time t2 in FIG. 4 is photometered. As is clear from Figure 4, 1. From t2 to t2, only the fluorescence of the Eu3+ chelate substance, which has a long fluorescence lifetime, is generated, so it is not affected by the contamination of autofluorescence, ordinary fluorescent dyes, or scattered light, which are other than the target fluorescence, and is pure. The objective fluorescence intensity can be measured.

Assuming that the test particles are double-stained with two types of fluorescence: a red fluorescent dye with a long fluorescent life, such as Eu3+ chelate, and a green fluorescent dye with a normal fluorescent lifetime, each bandpass filter 31 of the aperture 30 The characteristics of .32 are:
Select materials that have the property of selectively transmitting green fluorescence and red fluorescence, respectively. In such a configuration, green fluorescence and red fluorescence are selectively incident on the photodetector 18 in chronological order each time the test particle passes the light irradiation position, and one photodetector can detect Two different 2 fluorescences are measured. At this time, since the red fluorescence due to the Eu'' chelate, which has a long luminescence lifetime, is measured in the time range t1 to t2 in which normal fluorescence does not occur, extremely accurate measurement values can be obtained without being contaminated by any stray light.

Although the fluorescence intensity measured between tl and t2 is not the peak value, the fluorescence lifetime curve has almost the same shape as long as the intensity of the laser light that excites the fluorescence is constant, and a value proportional to the peak output can be obtained. This is not a particular problem as it can be done. If necessary, the shape of this curve and the time 1. ．． The peak output can also be predicted by giving a correction coefficient to the photometric output based on 12. This correction may be performed by providing a detection level changer when photometering with the photodetector 18, or
- The correction may be performed by performing arithmetic processing based on the measured values that have been previously stored in the memory 27.

In addition, in the above embodiment, the number of openings of the aperture is 2.
The number of parameters is not limited to 1, and the number may be further increased to obtain more parameters with the same detector. in this case,
By preparing multiple types of fluorescent dyes with long fluorescence lifetimes, labeling the target particles with stiff particles at the same time, and detecting these fluorescence individually in time series after the generation of stray light, we can eliminate the influence of scattered light. Fluorescence intensities of multiple parameters that cannot be detected can be obtained by measuring degrees with a small number of photodetectors.

[Effects of the Invention] According to the present invention, a plurality of different fluorescence parameters can be measured in time series using a dual-purpose detector by utilizing the time difference in fluorescence lifetime.

This results in a low-cost, compact device configuration.

[Brief explanation of drawings]

1 and 2 are block diagrams of a device according to an embodiment of the present invention, FIG. 3 is a block diagram of a signal processing section of a device according to an embodiment, and FIG. 4 is a graph and diagram showing a fluorescence lifetime curve. Figure 5 is a diagram showing the relationship between aperture shape and measurement time, and the main symbols in the diagram are: 1...laser light source, 2...imaging optical system, 3...・Flow cell, 4...Light irradiation position, 8.17.18...Photodetector, 10...Dichroic mirror 11.12...Band pass filter, 7.15.3
0...Aperture, 15a, 30a, 30b...Aperture opening 31
．． 32...Band pass filter, O 1゜2

Claims (4)

[Claims] (1) A step of fluorescently labeling the analyte in the sample, a step of irradiating each analyte in the sample with light, light emitted from the analyte at the time of the light irradiation, and non-irradiation after a predetermined time from the time of the light irradiation. A method for measuring a specimen, comprising: measuring fluorescence emitted from the specimen in time series using the same photodetector. (2) means for irradiating light to an irradiation position; means for sequentially passing individual fluorescently labeled specimens to the irradiation position; A specimen measuring device comprising: means for measuring fluorescence emitted from a specimen at a non-irradiated position in time series using the same detector. (3) The fluorescent labels of the specimen are first, fluorescent labels with different fluorescence lifetimes,
labeling with a second fluorescent dye, measuring the first fluorescence, and measuring the first fluorescence;
Claim (1), wherein the second fluorescence is measured after the generation of the fluorescence.
Or the specimen measuring method or specimen measuring device described in (2). (4) Specify the analyte in the sample in the first and second tubes with different fluorescence lifetimes.
irradiating each analyte in the sample with a single light beam to excite the first and second fluorescent dyes; 2. A method for measuring a specimen, comprising: a step of photometrically measuring the fluorescence of step 2 in time series;