JPH0510946A - Cell analyzer - Google Patents

Abstract

(57) [Summary] [Purpose] To enhance the measurement sensitivity of light intensity and reproducibility of measurement in a wide range from weak fluorescence to strong fluorescence emitted from cells upon receiving irradiation light. [Structure] As a fluorescence detector in a measurement optical system, a multi-channel photodetector capable of measuring fluorescence intensity from the sum of photon-counted values of light intensities of respective channels was used. Moreover, in order to measure the fluorescence amount exceeding the measurement range of the multi-channel photodetector, an ordinary photomultiplier tube is used together. With such a configuration, even if the fluorescence amount is dispersed in each window of the multi-channel photodetector, the measurement S / N ratio does not decrease, and it is impossible to measure the fluorescence amount due to the extremely weak fluorescence amount. It is possible to perform photon counting up to a strong fluorescence amount and enhance the reproducibility of measurement. Further, the one using the ordinary photomultiplier tube together has a small load on the measuring circuit, and the dynamic range of the measurable fluorescence amount is expanded.

Description

Detailed Description of the Invention

[0001]

BACKGROUND OF THE INVENTION 1. Field of the Invention The present invention relates to an apparatus for irradiating a fluid to be measured with a light beam and measuring fluorescence or scattered light from cells contained in the fluid to identify and sort cells.

[0002]

2. Description of the Related Art With the development of biotechnology, there is an increasing demand for a device for automatic analysis and sorting of cells in a wide range of fields such as medicine and biology. A flow cytometer is known as a typical one of such cell analyzers.

FIG. 4 is a schematic diagram for explaining the construction of the main part of a general cell analyzer (flow cytometer) and its operation. In FIG. 4, a sample liquid 1 containing cells in a container is guided to a flow cell 4 by an air pump 3 together with a sheath liquid 2 in another container, and inside the flow cell 4, the sheath liquid 2 collects the sample liquid 1. A sheath-shaped sheath flow that wraps in a cylindrical shape is formed. This sheath flow is formed as a means for accurately flowing cells one by one along the central axis of the flow cell 4. The laser light 7 emitted from the laser light source 5 and narrowed down by the focusing lens 6 is applied to the lower portion of the flow cell 4. In many cases, the cells contained in the sample liquid 1 are fluorescently labeled with a fluorescent substance such as a fluorescent dye or a fluorescently labeled monoclonal antibody, and when the cells pass through the laser beam 7, scattered light and fluorescence are generated. The scattered light passes through a condensing optical system including a condensing lens 8 and a beam block 9, and is detected by a photodetector 10 such as a photodiode. On the other hand, regarding the fluorescence, the red fluorescence is the condenser lens 1
1, half mirror 12, condenser lens 13, filter 14
Is collected by the condensing optical system consisting of and is detected by the photodetector 15, and the green fluorescence is on a different path from the half mirror 1
The light is collected by the condenser lens 16 and the filter 17 from 2 and detected by the photodetector 18. Usually, a fluorescence detector 15,
An electron multiplier that can detect weak light is used as 18. Photodetector 10 for detecting scattered light, photodetector 15 for detecting red fluorescence, and photodetector 18 for detecting green fluorescence
The signals from are sent to a signal processing circuit 19, respectively, where the cells are identified by analyzing the intensity of scattered light and fluorescence.

In the above example, the laser light source 5 is used as the light source, but there is also an apparatus using a cheaper mercury lamp or xenon lamp. There is also an apparatus for measuring fluorescence by subdividing it into three or four colors, and an apparatus for measuring multi-angle components of scattered light. In addition to the cell identification function, there is also a device equipped with a mechanism for sorting and sorting the identified cells,
This is called a cell sorter.

[0005]

In the above-mentioned cell analysis device, the accuracy of cell identification is required, and in order to meet this, the detection sensitivity of fluorescence and scattered light, that is, S / N At the same time that the ratio is high, the light intensity must be measured with good reproducibility. Especially in the case of fluorescence measurement, since the intensity of the fluorescent label differs depending on the type of fluorescent substance and the type of cell, the fluorescence intensity generated may be extremely weak in the case of a combination of a specific fluorescent substance and cells. A device with higher detection sensitivity is required. Further, when such weak fluorescence is measured, there is a problem that the reproducibility is extremely deteriorated, so it is an important problem to improve the reproducibility of the measurement of the weak fluorescence.

The reason why the reproducibility deteriorates when measuring weak fluorescence is as follows. 5A and 5B show the waveform of the output signal from the detector, that is, the photomultiplier tube. In the case of normal measurement, the output signal is as shown in FIG.
Such an analog waveform is obtained, and the fluorescence intensity can be obtained from the peak value of the analog waveform. However, when the fluorescence intensity becomes extremely weak, the output signal becomes a group of discrete pulses corresponding to photons as shown in FIG. 5B, and the peak value thereof does not correspond to the fluorescence intensity. In this case, it is conceivable to use a charge integrating type preamplifier to integrate the charges of the discrete pulse, but the discrete pulse width is a few n.
Since the pulse width is s and the pulse width is short, accurate integration by an electric circuit is difficult. Further, even if the charge integration of discrete pulses is accurately performed, there is a certain limit to the reproducibility of measurement.

FIG. 6 is a schematic diagram for explaining the operating principle of the headphone type photomultiplier tube. In FIG. 6, the photomultiplier tube contains a photocathode 21 for converting light into electrons, a focusing electrode 22, an electron multiplier 23 having n dynodes, and an anode 24 for collecting electrons in a vacuum container 20. When one photon 25 is incident on the photocathode 21, one photoelectron is emitted with a probability of quantum efficiency η. The emitted photoelectrons (primary electrons) enter the first dynode 23a and emit secondary electrons. Since these secondary electrons are about several discrete quantities, they are usually regarded as Poisson distribution, and have the same dispersion p as the average electron number p. The same thing is performed for the dynodes after the second dynode 23b one after another, and the final electron group is output from the anode 24 through the n-stage stochastic multiplication process. In FIG. 6, the multiplication process of photoelectrons is represented by an arrow line. However, because the non-uniformity of the multiplication factor due to the difference in the position where the photoelectrons collide with each dynode and the electrons (thermoelectrons) that do not go through the original multiplication process as shown by the arrow in FIG. The peak value is not constant, but has variations.
FIG. 7 shows a distribution diagram of this pulse crest value. Due to the above phenomenon, even if these pulses can be accurately integrated using the charge integrating type preamplifier as described above, the fluorescence measurement values will vary. Then, this variation becomes larger as the fluorescence amount is weaker.

When detecting weak light, there is a method called photon counting as one of the measuring methods that does not cause the above-mentioned variations in the fluorescence measurement values. In the photon counting method, the discrete output signal pulses of the photomultiplier tube are counted as pulses and the pulse count value is evaluated as the amount of light, so there is no measurement error due to variations in pulse peak value. FIG. 8 is a pulse wave height diagram in which a light-derived signal component and a noise signal are divided according to the upper and lower limits of measurement. As shown in FIG. 8, the light-derived pulses to be counted are passed through a discriminator to determine the peak value. If the upper and lower limits are set, noise components such as the dark current pulse (pulse with low wave height due to thermoelectrons) and cosmic ray pulse (pulse with high wave height) shown in Fig. 8 can be easily removed, and the above analog The S / N ratio can be set higher than that of the conventional measurement. As described above, the photon counting method has the advantages of a high S / N ratio and good quantification, but when it is applied to a flow cytometer, the cells emit fluorescence in a short time (several μs to several tens of tens). μs) is high-speed and the dynamic range of measurement must be wide,
The following problems occur.

There is an upper limit to the amount of light that can be measured by photon counting. That is, to the extent that no more than two photoelectrons exist within the time resolution (pulse width) of the photomultiplier tube,
Measurement is possible only in a discrete range of the output pulse, in other words, only when the light quantity per unit time is as small as this. The limit depends on the characteristics of the photomultiplier tube,
The photoelectron pulse density is about 10 ⁷ pulses / second or less.
On the other hand, although the upper limit of the fluorescence intensity to be measured by the flow cytometer depends on the fluorescent substance to be measured and the type of cell, the amount of light is stronger by 2 to 4 digits. When photon counting up to such a strong light intensity, it is common to use an optical filter to reduce the light intensity.In the case of a flow cytometer, however, the time when cells fluoresce is as described above. Instantaneous time (several μs to tens of μs)
Therefore, it cannot be dimmed. For example, when measuring the amount of fluorescence at a photoelectron density of 10 ⁶ to 10 ⁹ / sec, the actual number of photoelectrons is 10 to 10 ⁴ if the emission time is 10 μs, and 1/10 is required for photon counting. When 100 is dimmed, the number of photoelectrons is 0.1-1.
It is reduced to 0 ² . A region with one photoelectron or less cannot be measured, and the reproducibility of the measurement deteriorates when the number of photoelectrons decreases. Therefore, for the measurement of weak fluorescence in a flow cytometer, the high S / The problem is that the advantage of good quantitativeness in N ratio cannot be utilized.

Therefore, as a simple method of applying photon counting to a flow cytometer, a photon counting optical system is added as a new fluorescence measuring optical system in addition to the conventional fluorescence measuring optical system, and when the fluorescence intensity is weak. It is also possible to perform photon counting and perform conventional pulse height measurement when the fluorescence intensity is strong. However, this method does not lead to an essential solution.

Similar to the above, considering the measurement of the amount of fluorescence at a photoelectron density of 10 ⁶ to 10 ⁹ pieces / sec, in this case, 10 ⁶ to
Photon counting is performed in the fluorescence range of 10 ⁷ pieces / second, and measurement of the fluorescence range of 10 ⁷ to 10 ⁹ pieces / second is performed by the conventional pulse wave height measurement. When this time is 10μs light emission time, 10 to 10 ^two in the substantial number of photoelectrons range photon counting, by conventional methods 10 ²
-10 ⁴ pieces. The problem here is the weak fluorescence region of about 10 ² within the range of the number of photoelectrons of the conventional method, and with this amount of fluorescence, the output signal pulse is still in a discrete state. , Measurement reproducibility is not improved. That is, although using photon counting in combination with the conventional method is effective in improving the detection sensitivity, it does not improve the reproducibility, so that cell identification and classification cannot be performed effectively. Of.

The present invention has been made in view of the above points, and an object thereof is to provide a cell analysis device capable of highly sensitively and reproducibly measuring a wide range from weak fluorescence to strong fluorescence. It is in.

[0013]

In order to solve the above-mentioned problems, the cell analysis device of the present invention, as a fluorescence detector in the measurement optical system, uses the total of the photon-counted values of the light intensity of each channel as the fluorescence. It is equipped with a multi-channel photodetector that measures the intensity, and as a second measurement optical system, add a photomultiplier tube that measures the fluorescence amount exceeding the measurement range of the multichannel photodetector. You can also

[0014]

When a multi-channel photodetector is used as the fluorescence detector and the fluorescent light flux is expanded by a lens or the like and is incident, the total amount of fluorescence is dispersed and entered into each window of the multichannel photodetector. The amount of fluorescence in the window becomes small. In general, the fluorescence intensity measured by a flow cytometer is weak, and the fluorescence intensity is further dispersed in each window. Therefore, if the number of windows is large, the fluorescence amount of each window is reduced to a range where photon counting can be easily performed. Therefore, after photon counting the signal output of each window and calculating the fluorescence amount of each window, the fluorescence amount (count value) of each window is summed up to enable photon counting even for a certain amount of strong fluorescence. Become.

At this time, in the case of measuring a very weak fluorescence such as a few photons per one emission time, since the fluorescent light flux is incident on each window of the detector so as to be dispersed, the energy amount of each window is Less than one photon. However, this does not make it difficult to generate photoelectrons. The phenomenon that photoelectrons are emitted from the photocathode is a stochastic process due to quantum theory, so the emission probability of photoelectrons in each window is very small. This is because it will be the same as the photodetector.

As described above, even if the fluorescence amount is dispersed in each window of the multi-channel photodetector, it is impossible to measure the fluorescence amount from the extremely weak fluorescence amount without lowering the S / N ratio of the measurement. In addition, photon counting can be performed up to a strong fluorescence amount, and reproducibility of measurement can be improved while taking advantage of the high S / N ratio of the photon counting method.

[0017]

EXAMPLES The present invention will be described below based on examples. FIG. 1 is a schematic diagram showing the configuration of the cell analysis device of the present invention.
The parts common to FIG. 5 of FIG. 1 are represented by the same reference numerals,
The liquid sending system, the flow cell, the light projecting system, and the scattered light detection unit in FIG. 1 are the same as those in FIG. 5, and therefore their explanations are omitted here. The main difference between FIG. 1 and FIG. 5 is the photodetector 1 of FIG.
Instead of 5, 18, a multi-channel photodetector is used in FIG. A multi-channel photodetector is a photodetector that has a plurality of light incident parts and measures and outputs the amount of light incident on each window (channel). There are double tubes and image intensifiers. In FIG. 1, fluorescence from cells is photoelectrically converted by a multi-anode photomultiplier tube 26 which is a multi-channel photodetector.

Here, the multi-anode photomultiplier tube 26 will be described once. 2A and 2B are schematic views for explaining the structure, FIG. 2A is a side view showing the structure, and FIG. 2B is a partially cutaway perspective view of a microchannel plate.
In FIG. 2A, when the photon 27 is incident on the photocathode 28, photoelectrons 29 are emitted, and the photoelectrons 29 whose luminous flux has been expanded by the electron lens 30 are transferred to the microchannel plate 3
It enters 1 and is electronically amplified. As shown in FIG. 2B, the micro channel plate 31 is a bundle of many thin glass tube channels 32.
Is coated with a secondary electron emission material, and each of the channels 32 is an independent continuous secondary electron amplifier. The electron group multiplied by each channel 32 of the micro channel plate 31 is output to the closest anode of the multi-anode 33 [FIG. 2 (a)] composed of a large number of anodes arranged at the end. The number of the anodes can be arbitrarily made from several to several hundred. Multi-anode photomultiplier tube 26
Is optimal as a multi-channel photodetector used in the present invention, and a signal pulse (electron group) corresponding to photoelectrons 29 can be distributed to a multi-anode 33 composed of a large number of anodes.

Next, the explanation will be continued with reference to FIG. 1 again. The output signal pulse of each anode of the multi-anode photomultiplier tube 26 is supplied to the preamplifier 34, the discriminator 35,
It can be measured by a photon counting system 38 surrounded by a dotted line, which is composed of a gate circuit 36 and a counter 37. In the gate circuit 36, the gate of the gate circuit 36 is opened only while the cell is passing the laser beam, by looking at the output of the forward scattered light signal of the comparator 39. 40 is a preamplifier. The photon counting values of the respective anodes are summed by the adder 41 to obtain the final fluorescence intensity,
It is output to the signal processing circuit 19, and in the signal processing circuit 19,
The cells can be identified from the scattered light intensity and the fluorescence intensity.

In the apparatus of the present invention, as a multi-channel photodetector, a bundle of a plurality of photomultiplier tubes may be used instead of the expensive multi-anode photomultiplier tube 26. However, in this case, since the number of detector channels (the number of photomultiplier tubes) cannot be extremely increased, the measurable amount of light is limited, but the same effect as described above can be easily realized. it can.

Further, according to the present invention, the first measuring optical system using a multi-channel photodetector and the second measuring optical system using a single-channel photodetector are opposed to each other at 180 ° with a flow cell interposed therebetween. It is also effective to arrange. FIG. 3 is a schematic diagram showing the configuration, and the same reference numerals are used for the common parts with FIGS. However, the liquid delivery system is not shown in FIG. As shown in FIG. 3, this device has a multi-anode photomultiplier tube 2 centered around a flow cell 4.
A photomultiplier tube 42 is provided at a position symmetrical with 6, and the peak value of the signal is output as the fluorescence amount. Signal processing circuit 1
Regarding the fluorescence amount measurement value sent to 9, the signal output of the multi-anode photomultiplier tube 26 which is the first measurement optical system is prioritized, but when the fluorescence amount becomes large and an error signal is output to the photon counting system 38. , An error signal is sent to the signal selector 43, and the signal selector 43 receives the signal processing circuit 19
The signal output of the photomultiplier tube 42, which is the second measurement optical system, is adopted as the fluorescence amount measurement value to be sent to.

Among the devices of the present invention, in a device using a detector in which a multi-anode photomultiplier tube 26 or a plurality of photomultiplier tubes instead of this is bundled, the larger the number of channels of these detectors, the larger the number of channels. The upper limit of the measurable fluorescence amount becomes large. However, each detector channel must be individually photon-counted, so as the number of channels increases, the pulse measurement circuit for photon counting becomes large, and it is difficult to ensure a wide dynamic range due to circuit limitations. Become.
On the other hand, as described above, the device using the second measurement optical system in combination uses a multi-channel photodetector only in a weak fluorescence range that could not be sufficiently measured conventionally, and the fluorescence amount is large. In the region, the conventional detection method is switched to, and the measuring means is selectively used depending on the fluorescence amount, so that the number of channels of the multi-channel photodetector can be suppressed to the necessary minimum. Therefore, comparing the two devices in Fig. 1 and Fig. 3, the device in Fig. 1 has many channels and the measurement circuit is complicated, but the device in Fig. 3 can do that easily. It has the advantages of a small load and a wide dynamic range of measurable fluorescence.

[0023]

EFFECTS OF THE INVENTION When a fluid containing cells passes through a flow cell, a conventional cell analysis device (flow cytometer) is used as an optical photodetector for irradiating the cell with a laser beam and measuring the fluorescence emitted from the cell. A photomultiplier tube is used,
The output signal waveform becomes a group of discrete pulses for weak fluorescence measurement, and the peak value is not constant, so the reproducibility of the light intensity measurement is poor. This is because even if the photon counting method is used, the S / N ratio is Although higher, the reproducibility of the measurement is not essentially improved. On the other hand, the present invention configures a cell analysis device using a multi-channel photodetector as a photodetector of an optical system for fluorescence measurement, and determines the light intensity of each channel of the multichannel photodetector. Since photon counting is performed and the fluorescence intensity is measured from the total value of the photon counting values of each channel, the fluorescence intensity is measured with good reproducibility while taking advantage of the high S / N ratio characteristic of the photon counting method. Therefore, the two important problems of detection sensitivity and reproducibility necessary for the measurement of the cell analyzer were solved at the same time, and the accuracy of cell identification and classification was greatly improved. Further, by using an ordinary photomultiplier tube together, there is an advantage that the load on the measurement circuit is small and the measurable dynamic range of the fluorescence amount is expanded.

[Brief description of drawings]

FIG. 1 is a schematic diagram showing the configuration of a cell analysis device of the present invention.

2A is a schematic side view showing the structure of a multi-anode photomultiplier tube, and FIG. 2B is a partially cutaway perspective view of the same microchannel plate.

FIG. 3 is a schematic diagram showing a configuration of an apparatus in which a second measurement optical system of the present invention is arranged.

FIG. 4 is a schematic diagram for explaining a configuration and an operation of a main part of a flow cytometer.

5 (a) and 5 (b) are waveform diagrams of output signals from the photomultiplier tube.

FIG. 6 is a schematic diagram showing a photoelectron multiplication process of a headphone type photomultiplier tube.

FIG. 7 is a distribution diagram of output pulse peak values from the photomultiplier tube.

FIG. 8 is a pulse wave height diagram in which a signal component derived from light and a noise signal are divided into upper and lower limits of measurement.

[Explanation of symbols]

1 Sample Liquid 2 Sheath Liquid 3 Air Pump 4 Flow Cell 5 Laser Light Source 6 Focusing Lens 7 Laser Light 8 Focusing Lens 9 Beam Block 10 Photodetector 11 Focusing Lens 12 Half Mirror 13 Focusing Lens 14 Filter 15 Photodetector 16 Focusing Lens 17 Filter 18 Photodetector 19 Signal processing circuit 20 Vacuum container 21 Photoelectric surface 22 Focusing electrode 23 Electron multiplier 23a First dynode 23b Second dynode 24 Anode 25 Photon 26 Multi-anode photomultiplier tube 27 Photon 28 Photoelectric surface 29 photoelectrons 30 electron lens 31 microchannel plate 32 channel 33 multi-anode 34 preamplifier 35 discriminator 36 gate circuit 37 the counter 38 photon counting system 39 comparator 40 preamplifier 41 adder 42 photomultiplier 43 Shin Selector

   ─────────────────────────────────────────────────── ─── Continued front page    (72) Inventor Hiroshi Hoshikawa             1-1 Tanabe Nitta, Kawasaki-ku, Kawasaki-shi, Kanagawa             Within Fuji Electric Co., Ltd.

Claims (5)

[Claims] 1. A liquid sending system for sending a fluid containing cells to a flow cell, a projection optical system for irradiating the fluid passing through the flow cell with a light beam, and a light emitting system for receiving the light beam. A cell analysis device comprising a measurement optical system that measures at least one of scattered light and fluorescence, and a cell analysis device that identifies the cells from the fluorescence intensity measured by the measurement optical system, as a fluorescence detector of the measurement optical system. A cell analysis device using a multi-channel photodetector that measures fluorescence intensity from the sum of photon-counted values of light intensity of each channel. 2. The cell analysis device according to claim 1, wherein the multi-channel photodetector is a multi-anode photomultiplier tube. 3. The cell analysis device according to claim 1, wherein the multi-channel photodetector is a bundle of a plurality of photomultiplier tubes. 4. The apparatus according to claim 1, further comprising, in addition to the multi-channel photodetector, a second photomultiplier tube for measuring the fluorescence amount exceeding the measurement range of the multi-channel photodetector. A cell analyzer equipped as a measurement optical system. 5. The cell analysis device according to claim 1, wherein the second measurement optical system is located at a position symmetrical to the multichannel photodetector with respect to the flow cell.