JP2749928B2 - Sample measuring method and sample measuring device - Google Patents

Description

DETAILED DESCRIPTION OF THE INVENTION [Industrial Application Field] The present invention irradiates an individual specimen in a sample with light and performs optical measurement such as scattered light or fluorescence to analyze the specimen or perform antigen-antibody reaction. The present invention relates to a sample test method and device for performing measurement and the like.

[Prior Art] A flow cytometer is known as an example of a conventional sample testing apparatus. This involves fluorescently labeling specimens such as blood cells in a sample solution, and flowing these specimens one by one to a target to be irradiated with a light beam. By sorting, these parameters are measured for each individual sample, and the sample is analyzed based on the statistical tendency of the measurement parameters for a large number of samples. This makes it possible to perform cell DNA analysis and search for surface antigens.

[Problems to be solved by the invention] However, in general, when light is applied to a fluorescently labeled specimen,
Not only the target fluorescence generated by excitation of the labeled fluorescent dye, but also the autofluorescence of the specimen cells themselves, and the autofluorescence of the suspended matter such as dust and the fluid itself are generated at the same time. Since this auto-fluorescence is generated over a wide wavelength range, auto-fluorescence in the same wavelength region as the target fluorescence emitted from the labeled fluorescent dye cannot be optically wavelength-selected. The mixed fluorescence intensity will be measured.

When a multi-oscillation laser light source or white light is used instead of a single-wavelength laser light source, the scattered light in the same wavelength region as the target fluorescence contained in the irradiation light becomes miscellaneous light and becomes the target fluorescence. There is also a fear that it will be mixed into the photometric value of

That is, in the conventional apparatus, there is a problem in that the intensity of the fluorescence photometric light includes miscellaneous light as an error, and the S / N ratio of the obtained fluorescence measurement value deteriorates.

The present invention has been made to solve the above problems, and an object of the present invention is to provide a method and an apparatus capable of purely detecting the intensity of target fluorescence without being affected by light interference.

[Means for Solving the Problems] The present invention for solving the above-mentioned problems includes a means for irradiating light to an irradiation position, a means for sequentially passing individual fluorescently labeled specimens to the irradiation position, First means for measuring the light from the sample at the irradiation position; and second means for measuring the fluorescence from the sample at a predetermined position downstream of the irradiation position, based on the light measurement result of the first means. A sample measuring device for controlling a time point of photometry of the second means.

[Example] Hereinafter, an example of the present invention will be described in detail with reference to the drawings.

1 and 2 show an optical arrangement according to an embodiment of the present invention. A sample solution such as blood or latex suspension, which is adjusted to an appropriate reaction time and concentration and further labeled with a fluorescent reagent, and a sheath solution such as physiological saline or distilled water are prepared. The liquid is pressurized and guided to the flow cell 3, and the sample liquid is wrapped in the sheath liquid in the flow cell 3, converged into a thin flow, and passes through the flow section in the flow cell 3 according to the sheath flow principle. At this time, test particles such as individual cells and latex, which are specimens contained in the sample liquid, are separated and flow one by one or one lump downward from the top of the paper. In response to the flow of the test particles, the laser light emitted from the laser light source 1 (in this embodiment, an ultraviolet laser having a wavelength of 300 nm) emits two light beams each having a generatrix direction orthogonal to the flow portion direction and the flow portion direction. The light is converged into an arbitrary elliptical shape by the imaging optical system 2 composed of a cylindrical lens and is irradiated. In general, the shape of the light beam applied to the test particles is preferably an elliptical shape having a major axis in a direction perpendicular to the flow. This is because even if the position of the flow of each test particle slightly fluctuates in the fluid, the test particle is irradiated with the light beam with uniform intensity.

When the test particle is irradiated with a light beam, scattered light is generated.
Of the generated scattered light, the forward scattered light emitted in the forward direction of the optical path is measured by the condenser lens 6, the aperture 7, and the light detection unit 8. The opening of the aperture 7 is arranged conjugate with the light irradiation position 4 so that only light from the irradiation position 4 is guided to the photodetector 8. In order to prevent the irradiating strong light beam from directly entering the photodetector 8, a light-absorbing minute stopper 5 is provided in front of the converging lens 6 in the optical path. In addition, transmitted light that has passed through the test particles is removed. Thereby, only the light scattered from the light irradiation position 4 can be measured.
In this configuration, the light intensity is measured together with the forward scattered light and the fluorescent light. However, there is no problem since the fluorescent light intensity is generally extremely weak as compared with the forward scattered light intensity. Of course, a band-pass filter that selectively transmits the scattered light wavelength may be arranged in front of the photodetector 8.

Further, of the scattered light, the light emitted in the lateral direction orthogonal to the laser optical axis and the flow of the test particle, respectively, is a condensing lens 9.
The light is focused. The collected light is a dichroic mirror 10
Reflected by the wavelength of the scattered light, that is, the wavelength of the laser light (300 nm
(Nearby) through the band-pass filter 11, the condenser lens 13, and the aperture 15 to measure the side scattered light through the photodetector 17. The aperture 15 is provided with an opening 15a at the center of the optical axis as shown in the figure. The opening 15a has a conjugate relationship with the light irradiation position 4, and only the scattered light emitted from the light irradiation position 4 is transmitted to the photodetector 17. Is detected.

Further, in order to measure the fluorescence having a long fluorescence lifetime generated from the test particles together with the scattered light, the fluorescence collected by the condenser lens 9 and passed through the dichroic mirror 10 is subjected to a band-pass filter for the fluorescence wavelength (around 610 nm). The wavelength is selected at 12, and fluorescence of a specific wavelength is detected by a set of the condenser lens 14, the aperture 16, and the photodetector 18. The aperture 16 has an opening 16a slightly above the center as shown in the figure. That is, the opening 16a is conjugated to a position slightly downstream of the light irradiation plate 4 and guides only the fluorescence of a specific wavelength from the non-irradiation position downstream of the light irradiation position 4 to the photodetector 18. ing. That is, light is not simultaneously incident on the photodetector 17 that measures the scattered light and the photodetector 18 that measures the fluorescence, and the output pulse of each photodetector is obtained in a time series with time. Has become.

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 converted into a pulsed electrical signal as a trigger circuit 21.
Is input to the analog processing circuit 22. The side scattered light output of the light detector 17 and the fluorescence output of the light detector 18 are input to analog processing circuits 23 and 24, respectively. Each analog processing circuit detects a peak value or an integrated value of a signal pulse. When the forward scattered light signal inputted to the trigger circuit 21 exceeds a certain level, that is, when the test particle comes to the light irradiation position, a trigger signal is generated for a certain period, and the signal is generated by an analog processing circuit. 22 and the analog processing circuit 23. Each analog processing circuit has a sample-and-hold function, and an input signal is processed only during a period in which a trigger signal is generated. The trigger signal from the trigger circuit 21 is input to the fluorescent trigger generating circuit 25, fluorescent for trigger generating circuit 25 will generate only between the trigger signal time t ₁ of t _2, analog processing circuit the signal 24 Is input to Between t ₁ to t ₂ after which the particles are passed through the optical irradiation position 4, the fluorescence light detector 18
Can be sampled. The output of each analog processing circuit 22, 23, 24 is converted to a digital value by the A / D conversion circuit 26, and the forward scatter signal, side scatter signal,
It is stored in the memory 27 as a fluorescence signal.

The memory 27 obtained for a large number of specimens as described above
The calculation of the sample analysis is performed in the arithmetic circuit 28 based on the measurement parameters stored in the storage unit, and the result is output to the output unit 29 such as a CRT or a printer.

Next, the measurement principle of the present invention will be described with reference to FIG. In the present invention, the fluorescent dye used has a longer fluorescence lifetime than ordinary autofluorescence. FIG. 4 is a graph showing a lifetime curve of fluorescence excited by light irradiation. The abscissa represents the elapsed time, and the instant at which the test particles are irradiated with light at the test site is defined as time 0. The vertical axis is the amount of generated fluorescence. In the figure, a curve 19 is based on a commonly used fluorescent dye or autofluorescence possessed by a cell or dust, and the fluorescence lifetime from excitation to when the fluorescence intensity becomes 0 is about 100 nsec. . On the other hand, a curve 20 is a life curve of a fluorescent dye having a longer life than usual used in the present invention. For example, a curve using an Eu ³⁺ chelating substance as a fluorescent dye is used. The fluorescence lifetime in this case is
It is more than 1000nsec. Note that this fluorescence lifetime curve has substantially the same shape if the intensity of the laser light for exciting the fluorescence is constant.

Assuming that the particles are irradiated at the moment of time 0, the generation of scattered light ends instantaneously, and the amount of fluorescent light generated by time t ₁ is normal fluorescent light with a short fluorescence lifetime and auto-fluorescent light emitted by cells and dust. Becomes 0. The opening 16 of the aperture 16 shown in FIG.
a is arranged conjugate with a non-irradiation position slightly downstream of the light irradiation position 4 where the light spot hits, and guides only light from this portion to the photodetector. In other words, assuming that the particle flow velocity is constant, the time t ₁ to t _{2 in} FIG.
It measures only the light in the shaded area between the two. As is apparent from FIG. 4, only fluorescence of the Eu ³⁺ chelate substance having a long fluorescence lifetime is generated between t ₁ and t ₂ .
The intensity of the target fluorescence can be measured purely without being affected by the mixed light other than the target fluorescence, such as autofluorescence, light from other fluorescent dyes, or scattered light.

Value Although fluorescence intensity for metering in between t ₁ of t ₂ is not a peak value, fluorescence lifetime curves are curves of substantially the same shape as long as the laser beam intensity to excite fluorescence is constant, proportional to the peak output Is not a particular problem because If necessary, a peak output can be predicted by giving a correction coefficient to the photometric output based on the shape of this curve and the times t ₁ and t ₂ . This correction may be performed even if a detection level changer is provided when photometry is performed by the photodetector 18, or may be corrected by performing an arithmetic process based on the measurement value once stored in the memory 27. Is also good.

As an example of the measurement by the above-described apparatus, a method for preparing a reagent for testing a surface antigen of blood cells using a Eu ³⁺ chelating substance as a fluorescent dye having a long fluorescence life of a predetermined period or longer, and measurement using this reagent The procedure of is described below.

Eu ³⁺ chelate is excited by light of wavelength around 300 nm, 610
It has the property of generating red fluorescence of a wavelength near nm for a long time. First, a monoclonal antibody that specifically binds to the desired surface antigen on the cell surface is prepared and biotinylated, and streptavidin is labeled with Eu3 ⁺ chelate, and by binding these biotin and streptavidin, Eu is obtained. A monoclonal antibody reagent labeled with a ³⁺ chelate can be obtained. The reagent is mixed with a blood sample diluted to an appropriate concentration and reacted for a certain period of time to perform cell staining, and the reaction sample solution is measured by the above-described device, and the cell intensity is determined from the fluorescence intensity of the obtained Eu ³⁺ chelate. Qualitative or quantitative measurement of the surface antigen can be performed.

Next, another embodiment of the present invention in which more parameters can be obtained with a smaller number of photodetectors will be described. Since the structure is similar to that of FIG. 2, the description will be made with reference to FIG.

In this embodiment, the bandpass filter 12 shown in FIG. 2 is omitted, and an aperture 30 having a plurality of openings as shown in FIG. 6 is arranged instead of the aperture 16.
In FIG. 6, two openings 30a and 30b are provided in the center of the light-shielding aperture 30 and in the vicinity thereof. Bandpass filters 31, 32 are attached to the openings 30a, 30b, respectively. In order to simultaneously excite a plurality of types of fluorescent dyes, a multi-oscillation laser light source may be used if necessary.

If it is assumed that the test particles are labeled with two types of fluorescence, that is, a red fluorescent dye having a long fluorescence lifetime, such as Eu ³⁺ chelate, and a green fluorescent dye having a normal fluorescence lifetime, the aperture 30
As the characteristics of the bandpass filters 31 and 32, those having characteristics of selectively transmitting green fluorescence and red fluorescence, respectively, are selected. In such a configuration, each time the test particle passes through the light irradiation position, red fluorescence and green fluorescence are selectively incident on the photodetector 18 in time series, respectively.
Two different fluorescences can be measured with one photodetector. At this time, red fluorescence due to Eu ³⁺ chelate having a long emission lifetime is measured in the time range t _{1 to} t ₂ where normal fluorescence does not occur,
Accurate measured values can be obtained without any contamination of light.

If the characteristics of the bandpass filter 31 are such that the scattered light wavelength is selectively transmitted, two different parameters of the side scattered light intensity and the fluorescence intensity can be obtained by the same photodetector 18. The 17 optical systems become unnecessary. In this case, since the intensity difference between the scattered light and the fluorescent light is large, it is preferable to use a wide dynamic lens for the photodetector 18 or a high optical attenuation for the bandpass filter 31.

As described above, in the present embodiment, similarly to the previous embodiment, the fluorescence intensity measured by using the time delay is highly accurate without any influence of the light interference, and furthermore, the fluorescence intensity is higher than that of the photodetector. Can be obtained, which contributes to miniaturization and cost reduction of the apparatus.

In the above embodiment, the number of photodetectors and the number of apertures are not limited to two, and more parameters may be obtained by further increasing the number. In this case, prepare multiple types of fluorescent light with a long fluorescence lifetime, label them at the same time on the test particles, select the wavelength of these fluorescent light after the occurrence of the light, and detect them individually to reduce the influence of the light. The fluorescence intensity of a plurality of parameters that are not affected can be obtained by a single measurement.

[Effects of the Invention] According to the present invention as described above,
The fluorescence intensity can be measured more accurately. As a result, highly accurate sample measurement can be performed.

[Brief description of the 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 unit of the device of the embodiment, FIG. 4 is a graph showing a fluorescence lifetime curve, FIG. 5 is a diagram showing the relationship between the shape of the aperture and the measurement time, and FIG. 6 is a diagram showing the configuration of the aperture according to another embodiment, where the main symbols in the figure 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, 16, 30… Aperture, 15a, 16a, 30a, 30b …… Aperture opening, 31, 32 …… Band pass filter,

Claims (3)

(57) [Claims] A means for irradiating the irradiation position with light; a means for sequentially passing individual fluorescently labeled specimens to the irradiation position; a first means for measuring light from the specimen at the irradiation position; And a second means for measuring the fluorescence from the specimen at a predetermined position downstream of the irradiation position, wherein the time of light measurement by the second means is controlled based on the result of light measurement by the first means. Sample measurement device. 2. The sample measuring apparatus according to claim 1, wherein the fluorescent dye to be labeled has a fluorescence lifetime longer than the autofluorescence of the sample itself. 3. The sample measuring apparatus according to claim 1, further comprising means for correcting the photometric intensity of the fluorescence based on a fluorescence lifetime curve.