JPH04188044A - Specimen measuring apparatus - Google Patents

Abstract

PURPOSE:To detect fluorescence and scattered light with the same photodetector in time series regardless of the levels of the generated amounts of the fluorescence and the scattered light by providing a level switching means which changes the detecting levels. CONSTITUTION:Passing-optical-amount adjusting means (ND filters 26a, 26b, 36a and 36b) are provided in response to the light emitting amount of fluorescence used. The difference between the amounts of the lights which are cast into photodetectors is made small. Thus, it is not necessary to use expensive photodetectors having the large dynamic ranges. The amplitude of each photodetector is switched based on the kind of the measuring light. A circuit for changing the gain of the amplifier in synchronization with the passing of particles through 1a and 1b when the outputs of the photodetectors 25 and 35 are latched in a memory operating circuit 161 is provided. The detecting sensitivity is changed in response to the amount of light emission. The gain of the amplification is determined in response to the measuring condition inputted from an input setting means.

Description

DETAILED DESCRIPTION OF THE INVENTION [Field of Industrial Application] The present invention relates to the field of specimen measurement, in which specimens are analyzed by irradiating each specimen with light and performing optical measurements.

[Prior Art] Flow cytometers have been known as an example of sample testing devices, and are widely used in the biological and medical fields.

A typical configuration of this flow cytometer is shown in FIG. A sample liquid such as blood is pretreated by staining with a fluorescent reagent or the like and adjusted to an appropriate reaction time and dilution concentration. Then, put this into the sample liquid container 115. 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 thin flow principle, the sample liquid is wrapped in the sheath liquid in the flow cell 104 and converged into a thin flow, and the sample liquid is
It passes through approximately the center of the flow section in 4.

At this time, individual test particles (cells,
(microorganisms, carrier particles, etc.), that is, the specimen is separated and sequentially flows one particle 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, and irradiate the flow of the particles to be detected. 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 the optical path in front of the condenser lens 105, so that the direct light from the irradiation light source, And the transmitted light that has passed through the test particles is removed. This makes it possible to photometer only the scattered light from the test particles.

Among the scattered lights, the lights emitted in the lateral directions perpendicular to the laser optical axis and the flow of the particles to be detected are collected by 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 bandpass filter 121 that selectively transmits light (488 nm in the case of an Ar+ laser). In addition, when the test particles are fluorescently dyed, in order to photometer the multiple colors of fluorescence generated together with the scattered light, among the fluorescence that is collected by the condenser lens 107 and passed through the dichroic mirror 108, the dichroic mirror 109 , a bandpass filter 122 for green fluorescence wavelength (near 530 nm), and a photodetector 112, and the total reflection mirror 110 detects the green fluorescence.
．． Red fluorescence is detected by a bandpass filter 123 for red fluorescence wavelength (near 570 nm) and a photodetector 113. The signals of the photodetectors 106, 111, 112, 113 are each input to an arithmetic circuit 116, and the arithmetic circuit 11
In step 6, 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.

Therefore, in Japanese Patent Application No. 1-325001, the applicant proposed a device that can obtain quantitative measurement parameters of the photodetector by detecting multiple types of light in time series using a common photodetector. did. The present invention aims at further improvements to the proposed device.

[Means for Achieving the Object] A sample measuring device of the present invention that achieves the above object includes an irradiation means for irradiating each sample with first and second irradiation light in time series, , a light detection means for detecting each light emitted from the specimen with the second irradiation light in time series using the same photodetector; and a first optical detection means emitted from the specimen when irradiated with the first irradiation light; a first optical means that selectively guides light having a characteristic to the photodetector, and when irradiating with the second irradiation light, selectively guides light having a second optical characteristic emitted from the specimen to the photodetector; It is characterized by having a second optical means for guiding the light to the container, and a level switching means for switching the detection level between when detecting the light having the first optical characteristic and when detecting the light having the second optical characteristic. shall be.

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

FIGS. 1 and 2 show configuration diagrams of the first embodiment. 1st
The figure is a basic configuration diagram of the embodiment, and represents the entire system including a front optical system, a fluid system, a control system, etc. In the same figure, 1 is a flow cell for forming a so-called sheath flow in which test particles (for example, biological cells or carrier particles) in a sample liquid are separated one by one so as to be wrapped in a sheath liquid and flowed in order. The particles to be tested flow through the internal flow section from above to below the page. The fluid system for forming a sheath flow includes a sample liquid container 150 that accumulates sample liquid such as a blood sample or immune reaction liquid, a pump 152 that pressurizes the container, and an electric regulator that adjusts the flow rate of the sample liquid. 154, a sheath liquid container 151 for accumulating a sheath liquid such as physiological saline, a pump 153 for pressurizing the sheath liquid, an electric regulator 155 for adjusting the flow rate of the sheath liquid, and for fluidly connecting these and guiding them into the flow cell l. It consists of a tube. Sample liquid container 150
The sample liquid is pushed out by pressurizing the inside of the sheath liquid container 151 with the pump 152, and the sheath liquid is pushed out by pressurizing the inside of the sheath liquid container 151 with the pump 153. The flow speed, flow interval of individual particles, etc. are set.

In addition to the above-mentioned pressurizing mechanism using a pump and regulator, it is also possible to adopt a configuration using a syringe as shown in Japanese Patent Application Laid-Open No. 2-213744 and adjust the extrusion speed of the syringe. good.

Now, the front optical system will be explained next. 2゜3 is a laser light source with different wavelengths, which is commonly used in this field.
Lasers such as an r+ laser, a He-Ne laser, a dye laser, and a semiconductor laser, as well as various light sources other than lasers, can be used. As shown in Figure 5(D), the laser light source is
If more than one light source is prepared and the laser beam from each light source is selectively guided to the irradiation optical path according to the measurement conditions, a highly versatile system capable of performing even more versatile measurements can be obtained. In FIG. 5(D), 74 is a total reflection mirror, 75 is a half mirror, and 176 is an optical shutter.

Returning to FIGS. 1 and 2, 4a and 4b are condenser lenses that image the irradiation light beam onto the test area of the flow cell section,
Laser beams from laser light sources 2 and 3 are imaged at positions la and lb 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 about 100 μm, which is larger than the size of the particles to be measured.
This is sufficiently shorter than the flow interval of particles flowing sequentially. Members 5a and 5b disposed in the straight beam direction are light stoppers for blocking the laser beam from the laser light source 2.3 and forming a dark field optical system, and 6 is a condenser for condensing forward scattered light. Optical lens, 7 is field diaphragm, la,
The open lower a is located at the conjugate position corresponding to the position of lb.

7b is provided. 8a and 8b are photodetectors that detect forward scattered light from the la and lb positions.

Note that it is not necessarily necessary to prepare two laser light sources to form two laser beams. For example, Figure 5 (A)
(B) A beam from a single laser source as in (C),
It may be branched into two by a Mira member. At this time, if a configuration as shown in FIGS. 5(B) and 5(C) is adopted, a plurality of beams with different wavelengths can be obtained. FIG. 5A shows an optical system in which a laser beam from a short wavelength single mode laser is generated into two beams of the same wavelength by a half mirror 71 and a total reflection mirror 72. Further, in FIG. 5(B), the laser beam is split into two beams by a half mirror 71 and a total reflection mirror 72, and each branched beam is sent to a wavelength converting member 772.7.
7b (nonlinear optical element, AO, etc.) is an optical system that generates two beams of different wavelengths by converting the wavelength. Furthermore, FIG. 5(C) shows an optical system that splits a laser beam from a multimode laser having a plurality of wavelengths into two beams of different wavelengths using a dichroic mirror 73 to produce two beams of beams of different wavelengths.

In the configuration shown in FIG. 1, laser light emitted from the laser light source 2 is focused by a condenser lens 4a and irradiated onto the test area 1a. When a test particle passes through the test region 1a, light scattering occurs due to the test particle, and at this time, if the test particle is 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 the condensing lens 6 and the aperture 7a of the field stop 7, and then passes through the photodetector 8a.
The intensity is detected and a first forward scattering signal is obtained. Similarly, the laser light emitted from the laser light source 3 is focused by the condenser lens 4b and irradiated onto the test area 1b.

When the test particles pass through the test region 1b, the forward scattered light passes through the opening 7b, and the intensity is detected by the photodetector 8b to obtain a second forward scattered signal.

Reference numeral 160 denotes an input setting means for inputting and setting various measurement conditions such as various modes of the system, flow rate and passing interval of particles to be detected, type of fluorescent agent to be used, and measurement items. 161
is a storage arithmetic circuit, and the outputs of photodetectors 8a and 8b and the outputs of photodetectors 25 and 23 of the side optical system, which will be described later, are connected to a gain-switchable amplification amplifier, a peak hold circuit, an integration circuit, and /D converter etc., and digital data of peak values and integral values is stored in a storage means. Particle analysis calculations are later performed based on this data, and the results are output to output means such as a CRT or printer.

As for the analysis method, statistical processing using histogram or cytodulum is generally used, but detailed explanation will be omitted here. The storage/calculation means 161 also has a function of calculating the flow velocity from the detection output timing of the two forward scattered lights, canceling the acquisition of erroneous measurement data, etc. 16
Reference numeral 2 denotes a control means that performs comprehensive control of various operating means of the system. Specifically, pump 1
52, 153 drive, electric regulator 154, 15
5 adjustment, each ON10 F F of laser light source 2.3
control, switching of the fluorescence detection level, etc., which will be described later.

Next, the lateral optical system will be explained using FIG. Second
The figure is a side view of FIG. 1 and shows the side optical system in detail. In the figure, reference numeral 11 denotes a condensing lens, which condenses light emitted from the laser light sources 2 and 3 in a lateral direction perpendicular to the rectilinear direction of the irradiated laser beams. 13 is the field aperture,
An opening 13a is provided at the conjugate position corresponding to both the la and lb positions.
, 13b are provided. 14a and 14b are parallel flat plates, 1
5a.

15 is a lens, and the combination of both members makes it 1a1°1b
Each light beam from the is converted into a parallel beam of light.

21a, 21b, 31a, and 31b are dichroic mirrors 22a, 22b, and 32a for color-separating the side scattered light and fluorescence generated by the test particles;

Reference numeral 32b represents a bandpass filter that selects the wavelength of each fluorescence, and the wavelength of each detection light is selected by the combination of this dichroic mirror and the bandpass filter. 26a, 26b, 36a.

36b are ND filters each having a predetermined transmittance. 24.34 is a lens, 25.35 is a photodetector for detecting side scattered light and fluorescence, and a photomultiplier with high detection sensitivity is suitable as the photodetector. The photodetector 25° 35 is connected to a storage/arithmetic means 61. Here, the light emitted from position la is at opening 13a.
Once the image is formed, dichroic mirrors 21a, 31a,
The photodetector 25.2 passes through the optical path of the bandpass filters 22a, 32a, and the ND filters 26a, 36a.
5 and reimage here. On the other hand, the light emitted from 1b is imaged once at the aperture 13b, dichroic mirrors 21b, 31b, bandpass filters 22b, 32b,
The photodetector 2 passes through the optical path of the ND filters 26b and 36b.
5.25 and reimage.

FIG. 3 shows a modification of the above-mentioned side optical system, and is a view of FIG. 1 viewed from above. Note that the front optical system is omitted in the figure. In the figures, the same reference numerals as in the previous figures represent the same or equivalent members.

The light emitted laterally from the laser irradiation position [1a] from the laser light source 2 is once routed to the upper optical path in the figure, passes through a reflection member 99 such as a corner cube or a porroprism, and is once condensed at the aperture stop 13a. The light is imaged, re-imaged by a photodetector 25.35, and then incident. On the other hand, the light emitted laterally from the laser irradiation position 1b from the laser light source 3 is guided to the optical path in the lower part of the figure, is once imaged by the aperture stop 13b, and is focused on the common photodetector 25.
It is re-imaged and detected at 35. This example is basically the same as the optical system shown in FIG. 2 above, but is characterized by the optical arrangement in which the optical path for detecting the fluorescence from la and the fluorescence from 1b are clearly separated.

FIG. 4 is an example of a detection pulse obtained by each photodetector when a particle to be detected passes through. Figure 4 (A), Figure 4 (B)
are the detection outputs of the forward scattered light obtained by the photodetectors 8a and 8b, respectively, FIG. 4(C) and FIG. 4(F) are the timing pulses created by comparing them with a predetermined threshold, and FIG.
E) and FIG. 4(F) respectively show the fluorescence detection outputs obtained by the photodetectors 25 and 35. Fluorescence detection outputs from each photodetector 25.35 are obtained in time series when the test particle passes through the la and lb positions, and these are acquired separately in time series using the previous timing pulse. It's crowded.

As described above, in this embodiment, four channels can be detected using the two side detectors, and two forward scattered lights are added to this, making it possible to detect a total of six types of light with different optical characteristics.

In addition, although the above description has shown an example in which the number of side photodetectors is two, the number of detectors is not limited to this. If there is only one, two-color fluorescence can be obtained with a single photodetector, similar to the conventional example.
- The device configuration becomes extremely simple. Furthermore, if the number of photodetectors is three or more, even more parameters can be obtained.In addition, the side photodetectors detect not only fluorescence but also side scattered light. You can do it like this.

Now, the basic form of the system has been explained above, and next, the characteristic functions of the system of the embodiment will be explained.

(1) 1 Rainbow Laser When using the system of this example for multiple purposes, two laser light wavelengths may not necessarily be necessary, and only one may be sufficient depending on the measurement purpose and the type of fluorescent agent used. . Alternatively, if a configuration is adopted in which laser beams from three or more types of light sources are selectively irradiated as shown in FIG. 11(D), unused laser beam wavelengths are unnecessary.

Therefore, in the system of this embodiment, multiple measurement modes are switched depending on the intended use, and the laser light source that is not in use is turned off.
It has the function of Specifically, it switches between a first mode in which two laser beams are irradiated simultaneously to perform time-series measurements, and a second mode in which one of the laser beams is irradiated and the other is deactivated. be able to. This depends on the measurement conditions set in the input setting means 160.
The control means 162 controls the irradiation of each laser light source to 0N1.
0FF is performed independently to put the unused laser light source into sleeve mode or cut off the power to the laser light source. This results in lower system power consumption and longer laser life.

(2) 11 Since the distance between the two laser irradiation positions 1a and lb is a constant value (approximately 100 μm), the forward scattered light detector 8
Figures 4 (C) and 4 (D) are created from the outputs of a and 8b.
) It is possible to compare the generation timings of the two timing pulses shown in FIG. This timing comparison and calculation of the flow velocity are performed by the memory calculation circuit 161, and the control means 16
In step 2, this flow rate is constantly fed back to adjust the electric regulators 154 and 155 so that the flow rate is set by the input setting means 160.

This allows the set flow rate to be maintained accurately at all times, improving stability and measurement accuracy.

Note that the pressurizing mechanism is not limited to a pump and an electric regulator, and a pressurizing mechanism using a syringe may be used. In that case, adjust the extrusion speed of the syringe.

(3) = data cancellation The flow interval of the test particles flowing one after another is set to be sufficiently larger than the interval (about 100 μm) between the two irradiation positions 1a and lb, but in rare cases the interval is too large. In some cases, the particles may flow without being placed, and each particle may approach the repositioning location almost simultaneously. In this case, scattered light and fluorescence are generated from the respective positions and enter the common photodetector in a mixed manner, resulting in mixed data being detected.

Therefore, in order to prevent this, in this embodiment, la.

A means is provided for detecting that the specimens have passed through the position 1b at almost the same time and canceling the data from the detection means and not capturing it if it is determined that the specimens have passed at the same time. Specifically, in the storage calculation means 161, the fourth
If the generation timings of the timing pulses shown in FIG. 4(C) and FIG. 4(D) match or are very close to each other, la.

It is determined that two particles to be detected have passed almost simultaneously to the repositioned position 1b, and the data of each photodetector at this time is canceled and not captured.

Alternatively, the following method may be used.

It is considered that the time required for particles to move from 1a to 1b is approximately constant. There, a detection pulse (output of photodetector 8a) is generated when a particle passes through 1a, and then again during a predetermined period of time during which the particle moves to and passes through 1b.
When a detection pulse is generated from a (output of photodetector 8a), it is determined that particles have continued to flow, and data acquisition is canceled.

By providing the above means, there is no possibility of capturing erroneous data, so more reliable measurements can be performed.

(4) Level 1 In general, the intensity levels of fluorescence and scattered light are very different, and
The amount of fluorescent light generated also varies greatly depending on the type of fluorescent dye. Therefore, in order to detect these in time series with a common photodetector, a detector with a very wide dynamic range is required.

Therefore, in this embodiment, in each optical path leading to the photodetector, there are passing light amount adjusting means (ND filters 26a, 26b) for adjusting the amount of passing light according to the amount of fluorescent light used.

36a and 36b) are provided, respectively, so that the difference in the amount of light incident on the photodetector is small. This eliminates the need to use an expensive photodetector with a large dynamic range, resulting in a lower cost system.

Note that the amount of passing light may be adjusted not only by the ND filter but also by an optical mask that limits the light-blocking area of the optical path.

Furthermore, if a mechanism for replacing the ND filter or a mechanism for freely adjusting the amount of light passing through is provided, and this is adjusted according to the fluorescent dye used, the system becomes more versatile.

(5) Level -2 Regarding the switching of the intensity level, this embodiment includes, in addition to the ND filter, a circuit that switches the amplification factor of the photodetector depending on the type of measurement light. This is the memory calculation circuit 16
When receiving the output of the photodetector 25.35 at 1, a circuit is provided to switch the gain of the amplifier in synchronization with the passage of particles 1a and 1b, and the detection sensitivity is switched according to the amount of light emitted. ing. This gain can be adjusted freely to accommodate a wide range of measurements, and the input setting means 1
The amplification gain is determined according to the measurement conditions input from 60.

Now, although the above two embodiments are examples of the basic configuration of the present invention, some more specific usage examples will be explained below.

It goes without saying that the types and combinations of fluorescent dyes that can be used are not limited to these.

Messenger ``L-what''.

In this example, a specimen is simultaneously stained with three types of fluorescent dyes, and four channels are detected using two photodetectors in a side optical system.

In Figures 1 and 2, laser light sources 2 and 3 have a wavelength of 4
Two 88 nm -Ar+ laser light sources are used. In addition, instead of preparing two laser light sources, the laser beam from a single light source can be optically split into two beams using a half mirror and a total reflection mirror as shown in Figure 11 (A). good. At this time, it is more preferable to change the intensity ratio of the two laser beams so that the intensity is suitable for the excitation efficiency of each fluorescent agent.

As the type of fluorescent dye that stains the test particles, fluorescence excited by excitation light with a wavelength of 488 nm is selected. For example, FITC (
Assuming that staining is performed using three types of fluorescence: 530 nm), PE (570 nm), and DC extraction (610 nm), 488 nm and 530 nm from the test particles.

Light of four different wavelengths, 570 nm and 610 nm, is generated simultaneously. Note that the DC is not directly excited by the wavelength of 488 nm, but has a stepwise excitation process in which the DC is excited by the fluorescence (570 nm) generated when PE is excited.

After staining the specimen with the three types of fluorescence described above as pretreatment, measurement is performed using the apparatus of this example. Here, the light separation wavelengths of the dichroic mirrors 21a, 21b, 31a, and 31b are 510 nm and 590 nm, respectively.

Band pass filters 22 a, 22 b, 32 a, 32 are set to about 450 nm and 650 nm.
b as 530 nm, 488 nm, and 570 nm, respectively.
.

FITC is used in each optical system using a material that selectively transmits light with a wavelength around 610 nm.

Intensity detection of SS (side scattered light), PE, and DC is performed.

When the test particle passes through the position la, the four types of light mentioned above are generated. In the device 35, the fluorescence intensity of the PE selected by the bandpass filter 32a is detected. Next, when the same test particle passes through the position 1b, the SS intensity selected by the bandpass filter 22b is detected in the detector 25, and the SS intensity selected by the bandpass filter 32b is detected in the detector 35. The fluorescence intensity of the selected DC is detected.

In this way, measurements of light with four different optical characteristics can be obtained using two detectors. In this, photodetectors 8a and 8b
By adding the two types of forward scattered light intensities obtained in , a total of six types of measurement parameters can be obtained.

Next, we will explain an example of use in which 6-channel detection is possible, in which specimens stained simultaneously with four types of fluorescent dyes can be measured using a lateral optical system.

In an optical system having three photodetectors by adding another side detection system to the configuration shown in Fig. 2, the laser light source 2 is
An Ar+ laser light source with a wavelength of 488 nm is used as the laser light source 3, and a dye laser light source with a wavelength of 600 nm is used as the laser light source 3. Note that if the configuration shown in FIG. 5(B) or FIG. 5(C) is adopted, the device will be even simpler.

The type of fluorescent dye that stains the test particles has a wavelength of 488 nm.
Fluorescence excited by the excitation light of 600 nm and fluorescence excited at a wavelength of 600 nm are selected. For example, FITC (530nm) and PE (570nm) are suitable for 488nm.
TR (610 nm) is suitable for 600 nm.
Stain with a total of four types of fluorescence using APC (660 nm) and APC (660 nm).

After staining the specimen with the four types of fluorescence described above as pretreatment, measurement is performed using the apparatus of this example. Here, the light separation wavelengths of the dichroic mirrors 21a, 21b, 31a, and 31b are 570 nm and 605 nm, respectively.

Band pass filters 22 a, 22 b, 32 a, 32 are set to about 550 nm and 630 nm.
b, 42 a.

42b is 488 nm and 600 nm, respectively.

A material having a characteristic of selectively transmitting light having wavelengths around 530 nm, 610 nm, 570 nm, and 660 nm is selected.

When the test particles pass through position 1a where Ar+ laser light is irradiated, FITC is performed with 488 nm irradiation light.

PE is excited, 488nm, 530nm, 570nm
Three types of light are generated. At this time, the SS (488 nm) is detected by the detector 25, and the detector 35
FITC is detected by the detector 45, 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, it is TRed with 600 nm irradiation light.

APC is excited, 600nm, 610nm, 660n
Three types of light are generated. At this point, the quantity t=shi←
--°'- SS (600 nm) at detector 25, T at detector 35
R and APC are detected by the detector 45, respectively.

As described above, in this example, it is possible to detect a total of 6 channels, 2 channels of side scattered light and 4 channels of fluorescent light, with the 3 side detectors, and a total of 7 types of different optical characteristics including forward scattered light. light becomes detectable.

Highlighting "Stool" Similar to the above usage example 2, another usage example will be described in which a specimen simultaneously stained with four types of fluorescent dyes can be measured using a side optical system, and 6-channel detection is possible.

A He-Ne laser light source with a wavelength of 633 nm was used as the laser light source 2, and an Ar+ laser light source with a wavelength of 488 nm was used as the light source 3, and the types of fluorescent dyes used to stain the test particles were 6.
APC (660n) is suitable for 33nm excitation light.
m), UL; = iJ 烰碍 (695 nm),
FITC (530n) is suitable for 8nm excitation light.
m), select PI (620 nm) and multiplex stain the specimen with these four types of fluorescent dyes.

and dichroic mirrors 21a and 21b.

The wavelength on the optical side of 31a and 31b is 640 nm, respectively.

Band pass filters 22 a, 22 b, 32 a are set to about 500 nm, 670 nm, and 550 nm.
.

32b, 42a, and 42b are each 633 nm.

488nm, 660nm, 520nm, 695nm.

A material with a characteristic of selectively transmitting light with a wavelength around 620 nm is used.

As a result, similarly to Usage Example 2, six channels can be detected with the side optical system having three photodetectors.

[Effects of the Invention] As described above, according to the present invention, fluorescence and scattered light can be detected in time series with the same photodetector regardless of the level of the generated light, so it is versatile and can be used for various measurements. High quality equipment can be provided at low cost.

[Brief explanation of the drawing]

Fig. 1 is a configuration diagram of an embodiment of the present invention, Fig. 2 is a configuration diagram of a side optical system of the embodiment, Fig. 3 is a diagram of a modification of the lateral optical system, and Fig. 4 is an embodiment. Figure 5 is a diagram of a modified example of the irradiation optical system, Figure 6 is a configuration diagram of a conventional example, and the main symbols in the diagram are: 1...flow cell, 2.3. ...Laser light source, 8...Photodetector, 22.32...Band pass filter, 25.35.
...Photodetector, 26.36...ND filter, Fluorescence (35) Fuda 5 Ward

Claims (3)

[Claims] (1) An irradiation means for irradiating each specimen with first and second irradiation lights in time series; and a same photodetector for each of the lights emitted from the specimen with the first and second irradiation lights. a light detection means for detecting the light in time series, and a first light detection means for selectively guiding light having a first optical characteristic emitted from the specimen to the light detector during irradiation with the first irradiation light.
an optical means for guiding light having a second optical characteristic emitted from the specimen to the photodetector in the case of irradiation with the second irradiation light;
and a level switching means for switching a detection level between when detecting light having the first optical characteristic and when detecting light having the second optical characteristic. measuring device. (2) The level switching means has a light passage restricting member disposed in the optical path of the first and second optical means (
1) The specimen measuring device described above. (3) The sample measuring device according to claim 1, wherein the level switching means includes means for switching the detection sensitivity of the photodetector.