JPS61270639A - Flow sight meter - Google Patents

Abstract

PURPOSE:To measure the same sample on the best condition for the detection of respective necessary pieces of light information by irradiating a sample flow with laser beams which differ in shape and kind by using plural irradiation luminous flux systems. CONSTITUTION:Two irradiation luminous flux systems 8 and 9 are provided and a laser beam from a light source is converged to illuminate different positions A and B of the sample flow. Photodetection systems 10 and 11 are provided corresponding to the laser beam irradiated positions A and B. Scattered light generated at the position A is measured and fluorescent light generated at the position B is measured. The time difference between both measurements corresponds to the time that the sample flow requires to flow between the points A and B. A signal fetch/arithmetic processing part 16 for the fluorescent light is operated a specific time after the peak of the scattered light to integrate fluorescent light pulses. Consequently, both pieces of light information on the same sample are measured while made to correspond to each other accurately.

Description

DETAILED DESCRIPTION OF THE INVENTION OBJECTS OF THE INVENTION Industrial Field of Application The present invention relates to a flow cytometer for automatically measuring the characteristic sieve of a sample of microparticles in a continuously flowing suspension. The present invention relates to a flow cytometer that enables measurements under optimal conditions according to each characteristic.

Conventional flow cytometers analyze and measure cells at a rate of several dozen or more cells per second, and can quickly determine the properties of cells based on the analysis results, making them useful in the fields of disease diagnosis and gene biology. Demand is increasing.

In general, a flow cytometer V [, a flow cell section in which an inert fluid (sheath liquid) is passed through a capillary tube as a laminar flow, and a sample such as a cell suspension is caused to flow in the center of the flow of the sheath liquid;
An irradiation beam system that irradiates this sample flow with a laser beam,
It is equipped with a photodetection system that detects scattered light, fluorescence, etc. from Sansol caused by laser beam irradiation, and a processing surface water system that electrically processes signals from the photodetection system to obtain necessary optical information.

Fig. 1 schematically shows a part of a typical front flow cell.
4 schematically shows the laser beam irradiated onto the sample flow 1 as seen from the irradiation direction, a is the beam width,
b is the beam thickness.

Conventionally, one laser beam is irradiated onto the sample stream, and the scattered light, fluorescence, etc. from the same irradiated sample are detected at the same time through processing 1. Therefore, for example, the temporal relationship between the two detected signals is as shown in Figure 2. It becomes as shown in . In other words, in Figure 5, the X-axis is time (t), the y-axis is relative intensity (voltage V), and 5 is the electric signal pulse (hereinafter referred to as "pulse J")% due to scattered light; 5 and 6 are detected and measured in a temporally overlapping relationship.

Problems to be Solved by the Invention However, when a conventional flow cytometer is used to perform measurements in the event of a disaster, the shape of the laser beam that irradiates the sample flow does not have a significant effect on the measurement results. For example, (1) If you want to obtain data with a small coefficient of variation (LV value) and good reproducibility, it is desirable to widen the beam width a in Figure 1; (2) For samples that emit weak fluorescence To measure with high sensitivity, it is necessary to narrow the beam width and increase the beam energy density; (3
) When measuring the length and size of the sample from the pulse width, it is necessary to make the beam thickness as small as possible compared to the target particle.

In other words, when obtaining multiple pieces of optical information from the same sample, the optimal beam shape is different for each, so conventional equipment either optimizes only one condition at the expense of others, or compromises between each condition. I had no choice but to do it. For example, when measuring forward scattered light and fluorescent light as shown in Fig. 2, intermediate conditions had to be used, and the spectral intensity of both had to be sacrificed to some extent.

Also, when treating optical information not as the pulse height but as the time integral value of the pulse, for example, the most commonly used method is to detect the arrival of a particle using scattered light/9 lass. During that time, the fluorescence pulse is integrated, but in this case, the fluorescence pulse is integrated only while the scattered light pulse exceeds one level. , the oblique part is the integral region.

One level of the bird distinguishes between electrical noise and signal pulses.
This is an introductory value and is set slightly higher than the noise level*
tic 2 If the pulse of the scattered light is sufficiently large compared to the noise, such as 5 in Figure 2 (a) or 5L in Figure 2 (b), there is almost no problem with this, but 5 in Figure 2 (b)
When the scattered light/4' Lux is small as in 8 (for example, when the particles are small), the time during which the pulse exceeds the trigger level becomes short, and the integrated value of fluorescence becomes smaller than the expected value.

Structure of the Invention Means for Solving the Problems In order to solve this problem in the prior art, the flow cytometer according to the present invention allows an inert fluid to pass through a capillary tube in a laminar flow state as a sheath liquid. , a flow cell section that causes a sample such as a cell suspension to flow through the center of the flow of the sheath liquid; a plurality of irradiation beam systems that irradiate at least two laser beams to different parts of the sample flow in the flow cell section; A photodetection system is installed corresponding to each laser beam irradiation position to detect optical information such as scattered light and fluorescence from the sample by laser beam irradiation, and based on the signal from the photodetection system, the sample is detected. It is characterized by being equipped with a processing display system that electrically processes and obtains multiple pieces of necessary optical information regarding the same sample while taking into account the time difference between the laser beam irradiation positions of the flow, and is optimally suited for the required optical information. This makes it possible to perform measurements under various conditions, and also ensures that sufficient integration time is always available for fluorescence and 9 lus.

An example of the present invention will be described in detail below with reference to FIGS. 3 and 4. Note that parts corresponding to those in FIG. 1.2 are indicated by the same numbers.

FIG. 3 is a block diagram showing the configuration of a flow cytometer according to the present invention, in which 6 is a flow cell section, 8 and 9 are an irradiation beam system, In, I1 is a photodetection system, and 12 is a processing display system.

The flow cell section 6 has the same configuration as the conventional one, and as shown in FIG. Flow cell part 6 while maintaining the flow state
The flow in this flow cell section forms the sensing region.

In the illustrated example, the irradiation optical system is the first and second irradiation beam system 8.
, 9 are provided, and a laser beam from a laser light source (not shown) is focused through an appropriate optical system [7] and directed to different points A and H of the sample flow flowing through the flow cell 3. irradiated. The optical system of each irradiation beam system 8.9 can be adjusted independently so that the shape of the focused beam can be changed arbitrarily, and the beam shape is optimal for detecting the necessary optical information. Although a single laser light source may be used, different light sources may be used depending on the optical information to be measured, and laser beams of different shapes and types may be irradiated.

The photodetection systems 111, ], 1 correspond to the above laser beam irradiation positions A and B, respectively (1, is set at V1, and in the illustrated example, 10 is for measuring forward scattered light of the sample, 11i is for
This is for fluorescence measurement of i samples. Each detection system 10.11 is equipped with a spectrometer, a detector, an amplifier, etc., and an electric signal corresponding to the intensity of scattered light and fluorescent light is emitted from each detector-amplifier. Since these photodetection systems have well-known configurations, their explanations will be omitted here.

When Sansol is flowed in a one-meter configuration and the sample flow is irradiated with two systems of laser beams, if we look at the same Sansol, first the scattered light generated at the irradiation position itA is measured, and then the scattered light generated at the irradiation position [B] is measured. The time difference Δt between the six measurements where the light is measured corresponds to the time required for the sample flow to flow between AB. In other words, if this time difference is known, it is possible to correctly obtain a plurality of pieces of optical information regarding the same sample during processing, even if measurements are performed on the same Sansol at different positions. If two signals related to the same sample obtained from the two-party detection system of the device of the present invention are shown in this way, the fourth
It will look like the figure. As in Figure 2, the y-axis is time (t),
The y-axis is the relative intensity (voltage V), Fl' is the scattered light pulse,
Reference numeral 6' denotes a fluorescent pulse, and the two pulses are separated by a constant time difference Δt.

Signals from both party detection systems 10 and 11 are acquired by signal acquisition/
The light enters the arithmetic processing unit IF>, 16, is led to the data processing system 18 via the data interface 17, and is subjected to electrical processing for displaying and recording the intensity of the scattered light and fluorescent light by a well-known method. In the figure, reference numeral 19 denotes one circuit, and 20 denotes a delay circuit, which respectively provide a delay time corresponding to the time difference Δt between one level of the scattering light processing circuit and the two pulses of the same sunsol. In other words, by operating the fluorescence signal acquisition/arithmetic processing unit 16 with a certain period of time delay from the peak of the scattered light and integrating the fluorescence i4 pulse 6', both light information regarding the same sample can be correctly correlated. measured.

In addition, it is possible to easily know by flowing Sansol particles, which are known in advance by a one-time difference Δt, and electrically measuring the time difference between the first pulse peak and the second pulse peak, and at the same time automatically. You can also set a delay time. Of course, it is also possible to visually measure the time difference between the two and the gurus using a multi-phenomenon oscilloscope.

The integration time d' is the i4 Lus width when the above-mentioned known sample particles are flowed, but since it varies somewhat depending on the size of the particles to be measured, it should be set in advance to be slightly longer than the Lus width. This is sufficient for practical use. This integration time d
' can be easily set automatically by electrically measuring the pulse width, and of course can also be measured visually using an oscilloscope or the like.

Effects of the Invention As described above, according to the present invention, a plurality of irradiation beam systems are used to irradiate different shapes and types of laser beams at different points in the sample flow (lO), so that the required For example, when measuring scattered light and fluorescent light, it is necessary to use intermediate conditions as in the past. Since the laser beam can be irradiated with the optimal beam shape for both, good optical signals can be obtained for both.

Further, in the present invention, both spectra are associated with each other based on the time difference between them, and the integration time can be set arbitrarily, so that further improvement in fluorescence measurement is possible.

In addition, in a flow cytometer, the flow rate has a significant effect on the time constant of the photodetection system and subsequent electrical processing system, the stability of the integral value, and even the stability of the sample flow. By constantly monitoring the time difference between the first and second peaks, it is possible to know the flow velocity, and it becomes possible to use systems such as controlling the flow velocity and correcting data fluctuations due to the facilitation of the flow velocity. There are also advantages.

[Brief explanation of the drawing]

Figure 1 is an explanatory diagram of the flow cell part of a flow cytometer.
FIGS. 2(a) and 2(b) are diagrams showing an example of the relationship between scattered light and fluorescent Al irradiation using a conventional device, and FIG. 3 is a block diagram showing the configuration of an example of a flow cytometer according to the present invention. FIG. 4 is a diagram showing an example of the relationship between scattered light, fluorescent light, and I-laser by a flow cytometer according to the present invention. l... Sample flow, 2... Sheath flow, 4... Laser beam, 6... Flow cell i, 8.9... [It
i Bundle system, In, 11... Light detection system, 12... Processing display system, 13... Sheath liquid, 14... Sample.

Claims (1)

[Claims] A flow cell part in which an inert fluid is passed through a capillary tube in a laminar flow state as a sheath liquid, and a sample such as a cell suspension is caused to flow in the center of the flow of the sheath liquid, and at least two laser beams are applied to the sample in the flow cell part. Multiple irradiation beam systems that irradiate different parts of the flow, and photodetectors installed corresponding to each laser beam irradiation position to detect optical information such as scattered light and fluorescence from the sample due to each laser beam irradiation. system, and a processing display system that electrically processes and obtains a plurality of necessary optical information regarding the same sample based on the signal from the photodetection system and taking into account the time difference between the laser beam irradiation positions of the sample stream. A flow cytometer equipped with a plurality of irradiation beams, characterized by comprising: