JPH03185335A - Apparatus and method for measuring sample - Google Patents

Abstract

PURPOSE:To obtain varaible constants whose number is more than the number of photodetectors by selectively guiding the light having the first optical characteristic when a sample passes the first position, and selectively guiding the light having the second optical characteristic to a photodetector when the sample passes the second position. CONSTITUTION:When a particle to be detected passes through the position of a region to be detected 1a, four kinds of light beams are generated. At this time, shutters 23a and 33a are opened and 23b and 33b are closed. Therefore, the intensity of the fluorsence of the FITC which is selected with the BPT 22a is detected with a detector 25. The intensity of the fluorescence of the PE which is selected with the BPF 32a is detected with a detector 35. When the same particle to be detected passes through the position of 1b, the shutters are controlled so that 23a and 33a are closed and 23b and 33b are opened. The intensity of the SS which is selected with the BPF 22b is detected with the detector 25. The intensity of the fluorescence of the Duochrome which is selected with the BPF 32b is detected with the detector 35. Therefore, four kinds of measured values of the light beams are obtained with two detectors. When the intensity of the forward scattering light is obtained with a photodetector 8 and added, five kinds of the measured variable constants are obtained.

Description

[Detailed Description of the Invention] [Industrial Application Field] The present invention is a specimen testing device that performs specimen analysis, antigen-antibody reaction measurement, etc. by irradiating light onto individual specimens in a sample and performing optical measurements. Regarding.

[Prior Art] FIG. 6 shows the configuration of a flow cytometer as an example of a conventional specimen testing device.

The sample liquid, which has been adjusted to an appropriate reaction time and concentration and further stained with a fluorescent reagent or the like as necessary, is placed in the sample liquid container 115 shown in FIG. Further, a sheath liquid such as distilled water or physiological saline is placed in the sheath liquid container 114. The sample liquid container 115 and the sheath liquid container 114 are each pressurized by a pressurizing mechanism (not shown). Then, according to the sheath flow principle, the sample liquid is wrapped in the sheath liquid in the flow cell 104 and converged into a thin flow, and the flow cell 1
It passes through approximately the center of the flow section in 04. At this time, individual test particles, which are specimens contained in the sample liquid, are separated and sequentially flow one by one or one lump at a time. A laser beam emitted from a laser light source 101 is converged into an arbitrary shape by a pair of cylindrical lenses 102 and 103 whose generatrix directions are in the direction of the flow section and perpendicular to the direction of the flow section, respectively. be done. 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. Of the scattered light, the forward scattered light emitted in the forward direction of the optical path is photometered by a condenser lens 105 and a photodetector 106. In order to prevent the irradiated light beam from directly entering the photodetector 106, a small light-absorbing stopper 100 is provided in front of the optical lens 105 in the middle of the optical path, so that the direct light from the irradiation light source and It is designed to remove the transmitted light that has passed through the test particles. This makes it possible to photometer only the scattered light from the test particles.

Of the scattered light, light emitted in the lateral direction perpendicular to the laser optical axis and the flow of the particles to be detected is transmitted through the condenser lens 10.
The light is focused at 7. The focused light is reflected by the dichroic mirror 108, and the wavelength of the scattered light, that is, the wavelength of the laser light (
The side scattered light is measured by the photodetector 111 through a band pass filter 121 that selectively transmits the light (488 nm in the case of an Ar'' laser). In order to photometer multiple colors of fluorescence generated together with light, among the fluorescence that is collected by a condenser lens 107 and passed through a dichroic mirror 108, a dichroic mirror 109, a bandpass filter 122 for green fluorescence wavelength (near 530 nm), Green fluorescence is detected by a set of photodetectors 112, and total internal reflection is detected by a set of photodetectors 112.
, a bandpass filter 123 for red fluorescence wavelength (near 57 Onm), and a photodetector 113 detect red fluorescence. Photodetector 106, 111.112% 113
are input to the arithmetic circuit 116, and the arithmetic circuit 1
In step 16, calculations such as analysis of particle types and properties, measurement of antigen-antibody reactions, etc. are performed.

[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 drugs 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 problem, and aims to provide an analyte measuring device that can obtain numerical measurement parameters of a photodetector.

[Kth Means for Solving Problem n] The present invention that solves the above-mentioned problems includes a means for sequentially flowing the individual analytes in a sample, and a means for sequentially flowing the first and second irradiation light beams at intervals in the flow direction of the analyte. a common light detection means for receiving each light emitted from the specimen passing through the first and second test positions; When the specimen passes through the first position, the light having the first optical characteristic is selectively guided to the photodetector, and when the specimen passes through the second position, the second optical characteristic emitted from the specimen is guided to the photodetector. and a selection means for selectively guiding characteristic light to the photodetector.

[Embodiment] Hereinafter, a basic configuration diagram of an embodiment of the present invention will be explained using FIG. 1 and FIG. 2. In addition, after explaining the basic form, some more detailed examples will be explained later. FIG. 1 is a basic configuration diagram of an embodiment of the present invention, and FIG. 2 is a view of FIG. 1 viewed from above, showing the side optical system in detail.

In the figure, reference numeral 1 denotes a flow cell for sequentially flowing test particles, such as living cells and latex particles, one by one using a sheath flow system, and the test particles flow from the top to the bottom of the page. 2.3 is a laser light source, and various light sources such as a general Ar+ laser, He-Ne laser, dye laser, and semiconductor laser can be used in this field. Reference numeral 4 denotes a condenser lens that focuses the irradiation light beam on the test region of the flow cell section, and focuses the laser beams from the laser light sources 2 and 3 on positions 1a and 1b in the flow cell, respectively. The shape of the imaging beam is preferably an ellipse with the major axis in the direction perpendicular to the flow. Here, the distance between both irradiation positions 1a and lb is approximately 100 μm. A member 5 disposed in the straight beam direction is a light stopper, 6 is a condenser lens for condensing forward scattered light, 7 is a field stop, and 8 is a photodetector for detecting forward scattered light. The members 11 arranged in orthogonal directions include a condensing lens 2 that condenses side scattered light;
1 a, 2 l b, 31 a, 3 l b
25.35.45 is a dichroic mirror for color-separating the side scattered light and fluorescence generated by the test particles; 25.35.45 is a photodetector for detecting the side scattered light and fluorescence; A photomultiplier with high detection sensitivity is suitable.

Laser light emitted from the laser light source 2 is focused by a condenser lens 4 and irradiated onto the test area 1a. When test particles pass through the test region 1a, light scattering occurs due to the test particles, and at this time, if the test particles are fluorescently dyed, fluorescence is also excited and generated together with the scattered light. A part of the generated scattered light travels in the forward direction of the optical path and becomes forward scattered light. This forward scattered light passes through a stopper 5 that blocks the zero-order light of the laser beam, a condensing lens 6, and a field diaphragm 7, and then its intensity is detected by a photodetector 8 to obtain a forward scattered signal.

On the other hand, the fluorescence excited by the test particles is condensed by the condenser lens 11 together with the side scattered light that is part of the scattered light. The focused light passes through a condensing lens 12, a field diaphragm 13,
Dichroic mirrors 21a, 2 through the condensing lens 14
1b. The field stop 13 has an aperture large enough to allow light from circumferential positions la and lb to pass through. Here, dichroic mirrors 21 having mutually different characteristics
The side scattered light and fluorescence color-separated and reflected by a and 21b are filtered through a bandpass filter 2 according to the characteristics of the reflected light.
2a and 22b, and a shutter 23a that can block the light beam, such as a mechanical shutter.

23b. The shutters 23a and 23b may be high-speed shutters that can be driven independently of each other, and various shutters such as a liquid crystal shutter, AO, or Bockel cell can be used in addition to mechanical shutters.

Next, the electrical processing procedure of the device of the embodiment will be explained.

The signal obtained by the photodetector 8 is connected to an analog processing circuit 51 and a comparison circuit 52. The output of the analog processing circuit 51 is sequentially connected to an A/D converter 53,
Similarly, the outputs of the photodetectors 25, 35, and 45 are output to analog processing circuits 61, 62, 63, and A/D converters 64.6.
The output of each A/D converter is led to one CPU circuit 67, and a memory 68 is connected to the CPU circuit 67. Further, the reference voltage v0 is also input to the above-mentioned comparison circuit 52, and its output is connected to the shutter control circuit 54, which is further connected to the shutter control circuit 54.
The outputs of 4 are 23a, 23b, 33a, 33b, 43a,
43b. When driving the shutter, when the voltage applied to the shutter is 0, the shutter opens and transmits light, and when a constant voltage v1 is applied, the shutter closes and blocks the light.

When the test particle passes the point la and crosses the laser beam, the output signal of the forward scattered light intensity obtained by the photodetector 8 becomes a signal as shown in FIG. 3(a).

Also, Fig. 3(b) shows the output of the photodetector 25, 35, 45,
That is, it is an output signal of side scattered light intensity or fluorescence detection intensity. Here, by comparing the output signal of the photodetector 8 shown in FIG. 3(a) with the reference signal v0, which is a threshold value, by the comparator 52, a timing pulse as shown in FIG. 3(C) is obtained.

Here, when the test particles pass through 18, the scattered light and fluorescence caused by the laser beam L1 are transmitted to the shutter 23a.

33a and 43a, and when the particles pass through 1b, the scattered light and fluorescence caused by the laser beam L2 are transmitted to the shutter 2.
In order to pass only 3b, 33b, and 43b,
The driving number f3 of each shutter 23a, 33a, and 43a is as follows:
As shown in FIG. 3(d), the shutter control circuit 54 closes the shutter again at the falling edge of the timing pulse and opens the shutter again at the rising edge of drive No. 13 of each shutter 23b, 33b, and 43b. Further, drive signals for the shutters 23b, 33b, and 43b are also generated by the shutter control circuit 54. This is shown in Figure 3 (e
), after 11 seconds from the falling edge of the main pulse signal (C), which is slightly shorter than the time required for the test particle to pass between the two lasers, the test particle is detected! b
The opening and closing of each shutter is controlled by a control signal of 0 or more, which is a timing signal such that the shutter is opened at the point just before , and the shutter is closed again 17 seconds after the test particle finishes passing through 1b. When the test particles pass through the position la, the shutters 22a, 32A') *
l', I k 9 9 k
1 9 h j')
hA(n is the same, and when the same test particle subsequently passes the position 1b, the opening and closing of each shutter is reversed.

On the other hand, the outputs of the photodetectors 25, 35, and 45 are sent to the analog processing circuits 6ri and 62.63, respectively, and the peak value and area integral value of the pulse signal generated when the test particle passes through the laser beam irradiation area. etc. are measured. Furthermore, the analog signals that are their output are A/D converters 64, 65, 6.
6, each of the converted digital signals is input to a CPU circuit 67 and stored in a memory 68. Based on the measurement data stored in the memory 68 in this way, a particle analysis circuit 69 performs calculations such as analysis of particle types and properties, detection of antigen-antibody reactions, etc., and the results are sent to an output device such as a CRT or printer. 70.

Next, the measurement principle according to the present invention will be explained.

Normally, the shutters 23a, 33a, and 43a are open, and the shutters 23b and 43a are open.
33b and 43b are in the closed state and when the test particles pass through the test region 1a, the optical path of the shutter 23a is selected, so that the scattered light and the scattered light generated by the test particles in the test region 1a are selected. Dichroic mirror 2 of fluorescence
Only light having a specific wavelength (the wavelength of the bandpass filter 22a) that is reflected by the light beam 1a and passes through the bandpass filter 22a selectively reaches the photodetector 25 and is detected. Similarly, the scattered light and fluorescence transmitted through the dichroic mirrors 21a and 21b are color-separated by the dichroic mirrors 31a and 31b, which have different wavelength characteristics.
The reflected light similarly reaches the shutters 33a and 33b, but
Here, only the light that has reached the shutter 33a is selected.

Therefore, the photodetector 35 detects only light of a specific wavelength (the wavelength of the bandpass filter 32a) that has passed through the dichroic mirror 21a, been reflected by the dichroic mirror 31a, and passed through the bandpass filter 32a.

Furthermore, the light that has passed through the dichroic mirrors 31a and 31b is reflected by mirrors 41a and 41b, and passes through bandpass filters 42a and 42b having mutually different wavelength characteristics and reaches shutters 44a and 44b, but only the light that has reached shutter 44a is is selected. Therefore, the photodetector 45 is a dichroic mirror 21a.

Only light of a specific wavelength (the wavelength of the bandpass filter 42a) that has passed through the bandpass filter 42a and the bandpass filter 42a is detected.

On the other hand, the test particles that have passed through the test region 1a move,
When the test area 1b is reached after a predetermined time, the control circuit drives the shutter to open and close, and the shutter 2 is opened and closed in the opposite direction.
3a, 33a, 43a are closed, 23b, 33b, 43b are open, and the optical path from the test area to the photodetector is switched. Therefore, each detector detects light of a specific wavelength, which is selected by the bandpass filters 22b, 32b, and 42b arranged in the switched optical path.

As described above, the opening and closing of the two-part shutter placed in front of each photodetector is controlled in synchronization with the passage of the test particles. In other words, the optical path from the test region to the photodetector is divided into two, and the optical path is switched in synchronization with the passage of the test particles. Therefore, it is possible to distinguish and sample the optical signal generated by the first laser beam and the optical signal generated by the second laser beam in time series, and by using the same photodetector and analog processing system, two Various types of optical signals can be measured. That is, in the apparatus of this embodiment, which includes three photodetectors in the side system, these detectors can obtain fluorescence and side scattered light with six different parameters.

In addition, although the above explanation has shown an example in which the number of side photodetectors is three, the number of photodetectors is not limited to this, and if it is one, it is a general conventional example. Similarly, two colors of fluorescence can be obtained with a single photodetector. At this time, since a dichroic mirror is not required, the device configuration becomes extremely simple. Further, if the number of photodetectors is four or more, even more parameters can be obtained. Note that the parameters to be measured do not necessarily have different optical characteristics, and the reliability of the measured values may be further improved by detecting a plurality of lights having the same optical characteristics.

In addition, as a further development, by increasing the irradiation position of the irradiation light, the dichroic mirror bandpass filter, and the number of shutter divisions to 3 or more, more types of parameters can be obtained without increasing the number of photodetectors. Obtainable. For example, if the flow is to be divided into three, the light irradiation position may be set at three locations in the flow direction, and the shape of the shutter etc. may be equally divided into three as shown in FIG.

Now, the basic configuration of the apparatus of the embodiment of the present invention has been explained above, and some more specific embodiments will be explained below. It goes without saying that the types and combinations of fluorescence that can be used are not limited to these.

[Example 1] In Example 1, test particles were stained with three types of fluorescence, and 2
This system uses four photodetectors to detect four lateral channels.

A total of five types of measurement parameters can be obtained by adding the forward scattered light parameter to these parameters. Note that since the detection optical system including the photodetector 45 is not used in this embodiment, it may be omitted.

In Figures 1 and 2, two Ar" laser light sources with a wavelength of 488 nm are used as laser light sources 2 and 3. In addition, instead of preparing two laser light sources, a single laser light source is used as shown in Figure 4. The laser beam from the light source may be optically split into two beams using a half mirror and a total reflection mirror.At this time, the excitation efficiency of each fluorescent agent can be adjusted by changing the intensity ratio of the two laser beams. It is more preferable to have a strength suitable for.

The type of fluorescence that stains the test particles is selected from fluorescence excited by excitation light with a wavelength of 488 nm. For example, FITC (5
30nm), PE (570nm).

If staining is performed using three types of fluorescence using Duochrome (6 Onm), the wavelength from the test particle is 488 nm.

Light of four different wavelengths, 530 nm, 570 nm, and 610 nm, is generated simultaneously. In addition, duochro
Me is not directly excited at a wavelength of 488 nm, but due to the fluorescence (570 nm) generated when PE is excited, duo
It has a stepwise excitation process in which chrome is excited.

Dichroic mirrors 21a, 21b.

The optical separation wavelengths of 31a and 31b are 510 nm and 310 nm, respectively.
Bandpass filters 22a and 22b are set to about 590nm, 450nm, and 650nm.

32a and 32b are each 530 nm.

FITC, 33 (side scattered light), P E ,
Performs duochrome intensity detection.

When the particle to be detected passes through the position 18, the above four types of light r are generated. At this time, the shutter 238° 33a is open and the shutters 23b and 33b are closed, so the detector 25 uses a bandpass filter. The fluorescence intensity of FITC selected by 22a is detected, and the fluorescence intensity of PE selected by bandpass filter 32a is detected by the detector 35. When the same test particle passes the position 1b in the 0th order,
Shutters 23a, 33a close, 23b.

33b is controlled to open, the detector 25 detects the SS intensity selected by the bandpass filter 22b, and the detector 35 detects the SS intensity selected by the bandpass filter 32b.
The fluorescence intensity of the duochrome selected in 2b is detected.

In this way, measurements of light with four different optical characteristics can be obtained using two detectors. By adding the forward scattered light intensity obtained by the photodetector 8 to this, a total of five types of measurement parameters are obtained.

[Embodiment 2] Next, a second embodiment in which six lateral channels can be detected will be described.

In Fig. 1 and Fig. 2, the wavelength 4 is used as the laser light source 2.
An 88 nm Ar'' laser light source is used, and a dye laser light source with a wavelength of 600 nm is used as the laser light source 3.A multi-oscillation laser light source that simultaneously outputs light of multiple wavelengths is used as the laser light source, and the half mirror shown in FIG. - may be replaced by a dichroic mirror to obtain a plurality of beams with different wavelengths from a single light source.

As the type of fluorescence that stains the test particles, select fluorescence excited by excitation light with a wavelength of 488 nm and fluorescence excited with a wavelength of 600 nm. For example, FITC (530 nm) is suitable for 488 nm.

Staining is performed using PE (570 nm), TR (610 nm) and APC (660 nm) suitable for 600 nm, a total of four types of fluorescence.

Dichroic Yoler 21a, 21b.

The optical separation wavelengths of 31a and 31b are 570 nm,
Bandpass filters 22a and 22b are set to about 605nm, 550nm, and 630nm.

488 as 32a, 32b, 42a, 42b respectively
nm, 600nm, 530nm, 610nm, 570n
A material having a characteristic of selectively transmitting light with a wavelength around 660 nm is selected.

When the test particle passes the position 1a where the Ar'' laser beam is irradiated, FITC and PE are excited by the 488 nm irradiation light, and the 488 nm and 530 nm.

Three types of light of 570 nm are generated. At this time, Shirt 2
2a, 32a, 43a open, 22b.

Since 32b and 43b are closed, SS is detected at the detector 25.
(488 nm), FITC is detected by the detector 35, and PE is detected by the detector 45. Next, after a predetermined time, when the same test particle passes through the lb position where the dye laser light is irradiated, , 600 nm irradiation light excites TR and APC, and three types of light of 600 nm, 610 nm, and 660 nm are generated. At this point, the shutters 22a, 32a, 42a are closed, and the shutters 22b, 32b are closed, contrary to the previous case.

42b is controlled to open, the detector 25 detects SS (600 nm), the detector 35 detects TR, and the detector 45 detects APC.

Now, in this embodiment, each shutter is not necessarily necessary.
Only light of 8 nm, 530 nm, and 570 nm is generated, and each photodetector selectively detects only light of these wavelengths even without a shutter. Further, since the dye laser beam irradiated to the lb position generates only light of 600 nm, 610 nm, and 660 nm, each photodetector selectively detects only light of these wavelengths even without a shutter. That is, depending on the combination of the irradiation light wavelength, the type of fluorescence, and the wavelength selection characteristics, it is possible to distinguish and detect each parameter even without a shutter.

Generalizing this, the wavelength of each irradiation light is different, and the characteristics of the first wavelength selection member and the second wavelength selection member, such as a bandpass filter placed in front of the photodetector, are changed depending on the wavelength of the first wavelength. The selection member has a characteristic of selecting light of a wavelength that is generated by the first irradiation light but not generated by the second irradiation light, and
The wavelength selection member has a characteristic of selecting light of a wavelength that is generated by the second irradiation light but not generated by the first irradiation light, thereby eliminating the need for a shutter.

As described above, in this embodiment, the three side detectors can detect six channels, and in addition to this, forward scattered light can be added, making it possible to detect a total of seven types of light with different optical characteristics.

[Effects of the Invention] As described above, according to the present invention, it is possible to obtain several kinds of parameters of a photodetector. This makes it possible to achieve the same performance as conventional models at a more compact size and lower cost. Also, conventionally,
It is now possible to easily detect four or more types of light having different optical characteristics, which has been difficult due to the optical arrangement.

[Brief explanation of drawings]

Figure 1 is a configuration diagram of a conventional sample testing device, and the main symbols in the diagram are: 1. Flow cell, 2. 3. Laser light source, 8. Photodetector, 7. 13. Field of view. Aperture, 21a, 21b, 31a
, 31b, 41a. 41b...Dichroic mirrors 22a, 22b, 32a, 32b, 42a. 42b...Band pass filter, 23a, 23b, 33a, 33b, 43a. b...Shutter, 25. 35, 5... Photodetector, Mouse Z diagram tt tz Anvil 11 Miu-

Claims (8)

[Claims] (1) means for sequentially flowing the individual specimens in the sample; means for irradiating the first and second irradiation lights at a first position and a second position at intervals in the flow direction of the specimen; 1. A common light detection means that receives each light emitted from the specimen passing through the second test position; and when the specimen passes through the first position, light having a first optical characteristic emitted from the specimen; selectively guiding light to the photodetector, and selectively guiding light having a second optical characteristic emitted from the specimen to the photodetector when the specimen passes through the second position. A specimen measuring device characterized by the following. (2) The selection means divides an optical path from each of the test positions to the photodetector into a first optical path and a second optical path, and when the sample passes through the first test position, The specimen measuring device according to claim 1, wherein one optical path is selected, and the second optical path is selected when the specimen passes through the second test position. (3) The selection means is a set of a first optical selection member and a shutter, and a second optical selection member and a shutter, which are arranged in front of the photodetector in the optical path so as to divide the optical path. When the sample particles pass through the first position, the first
(1) Control is performed to open a shutter and close the second shutter, and to close the first shutter and open the second shutter when the test particle passes the second position. 2) The specimen measuring device described above. (4) Claim (1) wherein the first and second irradiation lights are light beams from a single light source that are optically divided into two beams.
) Specimen measuring device described. (5) The analyte measuring device according to claim 1, wherein the first and second irradiation lights have different wavelengths. (6) The selection means is a first optical selection member and a second optical selection member arranged in front of the photodetector in the optical path so as to divide the optical path, and the first optical selection member is The second optical selection member has a characteristic of selecting light of a wavelength generated by the first irradiation light but not generated by the second irradiation light, and the second optical selection member selects light of a wavelength generated by the second irradiation light and not generated by the second irradiation light. The analyte measuring device according to claim 5, having a characteristic of selecting light of a wavelength that is not generated by the first irradiation light. (7) Claim (1) wherein the specimen is stained with multiple types of fluorescence.
) Specimen measuring device described. (8) A process in which each specimen in a sample is sequentially flowed, a process in which multiple irradiation lights are irradiated at intervals in the flow direction of the specimen, and light of different wavelengths emitted from the specimen passing through each irradiation position is common. A method for measuring a specimen, comprising the steps of: receiving light in time series with a photodetector;