KR20050094097A - Fluorescence activated cell sorting(facs) apparatus with laser diodes for characterizing and sorting cells and method for sorting the cells using the same - Google Patents

Abstract

The present invention relates to a fluorescence activated cell sorting apparatus for detecting and classifying biological cells using a laser diode. It consists of a cell detection continuous operation laser diode which detects the flow of the cell passing through a detection area, and a pulse operation laser diode which emits an optical pulse for a predetermined time at the time when a cell is detected. Since the fluorescent active cell sorting apparatus of the present invention uses a laser diode, it is relatively small in size, light in weight, low power consumption, and low in cost compared with the conventional fluorescent active cell sorting apparatus using an argon ion laser. Can be. In addition, the method for classifying cells according to the present invention detects the flow of cells by shining light from the cell detection continuous operation laser diode to the detection region, and the high power light pulse from the pulsed laser diode is reflected on the detected cells to Scattered light or fluorescence emitted from the light is detected, and the characteristics of the cells are analyzed and classified based on the scattered light signal or the fluorescence signal converted from the scattered light and the fluorescence.

Description

Fluorescence Activated Cell Sorting (FACS) Apparatus with Laser Diodes for Characterizing and Sorting Cells and Method For Sorting the Cells Using the Same}

BACKGROUND OF THE INVENTION 1. Field of the Invention The present invention relates to fluorescence activated cell sorting technology, and more particularly, to a fluorescence activated cell sorting apparatus using a laser diode and a method for sorting fluorescence activated cells using a laser diode.

Since observing cells through light microscopy, in order to quantitatively analyze and classify cells, "Fluorescence Activated Cells" is a combination of cell staining, optoelectronics, and techniques that allow cells to flow along microtubules. Sorting Apparatus) has been developed. In short, such a fluoroactive cell sorting device is a "fluidic system" that allows the stained cells to flow through a predetermined tube, and an "optical system" for observing cells flowing under control to the cell flow control system. optical system "and an" electronic system "which converts and processes an optical signal of an optical system into an electrical signal.

Fig. 1 is a block diagram showing a fluorescent active cell sorting apparatus using a conventional argon ion laser. For example, an argon ion laser 6 continuously emitting parallel light S1 having a wavelength of 480 nm and a light power of 100 mW. ), A focusing lens (7) receiving the parallel light (S1) at its own focus and making focused light, and a nozzle at the focus for illuminating the light focused at the focus of the focusing lens (7) on the cells; And a scattering light condenser lens 10 for collecting the light D1 scattered by the focused light as the cell passes through the focal point, and the cell flow chamber 8 for releasing the cells through the nozzle. A photodetector 11 for detecting scattered light collected by the light source 10 and converting the light into an electrical signal, a fluorescent condenser lens 9 for collecting fluorescence emitted by the light passing through the focal spot after receiving the focused light, and the fluorescent condenser lens Receive the fluorescent light collected by (9) A light filter 5 for reflecting part and transmitting part, a first light multiplier tube 4 for detecting a first reflection fluorescence reflected from the light filter 5, and the light filter 5 A dichroic light filter 3 receiving partly transmitted first fluorescence and selectively reflecting part of the fluorescence wavelength and transmitting part of the first transmitted fluorescence; and a second reflected fluorescence reflected from the dichroic light filter 3 A second optical multiplier (2), a third optical multiplier (1) for detecting the second passage fluorescence transmitted through the dichroic light filter (3), and supplying power to each element and controlling each element It consists of the power supply circuit part 12.

The cell flow chamber 8 includes a sample fluid tube 8-1 connected to receive a sample fluid containing cells from the outside, and a sheath connected to receive a sheath fluid from the outside. It consists of a fluid tube 8-2 and a nozzle 8-3 which receives the sheath liquid and the sample liquid and allows the cells to pass through the focus of the focusing lens 7.

In addition, the conventional fluorescent cell sorting apparatus using the argon ion laser is further provided with a cell sorting vessel (13) containing the cells discharged from the nozzle (8-3) and classified according to the light scattering characteristics. Here, the vessel 13, as shown in Figure 2, is composed of a plurality of inner bowl (13-1, 13-2, 13-3) that can contain cells classified according to the characteristics of the cell.

FIG. 2 is a configuration diagram showing in detail the nozzle 8-3 of the cell flow chamber 8 through which cells pass in the fluorescent active cell sorting apparatus of FIG. 1. As shown in the figure, the nozzle 8-3 is a structure having a predetermined diameter and length so that each cell is lined up one by one from the inside of the cell flow chamber toward the vessel. Here, since the light S1 focused after passing through the focusing lens 7 passes through the nozzle 8-3, the cells travel from the inside of the cell flow chamber along the nozzle 8-3 and are discharged to the outside. The cell receives the focused light S1.

In this way, with the help of the method of operating the fluorescent activated cell sorting apparatus using an argon ion laser as follows.

First, with all the components aligned, the sample fluid containing the dyed cells, such as pre-dyed with fluorescent dyes, and the sheath fluid to flow around the sample fluid, respectively, are sample fluid tubes (8-1). And enters the cell flow chamber 8 along the sheath fluid tube 8-2 and are then discharged out together through the nozzle 8-3. The sample liquid flows along the central axis of the nozzle, and the sheath liquid flows around the sample liquid. Thus, the cells surrounded by the sample liquid flow along the nozzle without departing from the central axis of the nozzle. The amount and rate of discharge of the sample and sheath fluids are controlled by the internal pressures of the sample and sheath fluid tubes.

At the same time, in order to analyze the size, shape, structure, composition, light scattering characteristics, fluorescence emission characteristics of the cells and classify the cells according to the result, the argon ion laser 6 illuminates the nozzle 803 continuously with the laser light. Allows the cells to pass through to receive laser light. Then, as shown in Figure 2, the cell material (nucleus) (8-7) of the cells (8-6) subjected to the focused argon ion laser light to produce a scattered light (D1) and at the same time stain the cell material (nucleus) The fluorescent dyes emit fluorescence (F1). The scattered light D1 is detected by the photodetector 11 via the scattered light condensing lens 10. At the same time, the fluorescence F1 is collected by the fluorescence condensing lens 9, and the focused fluorescence in the form of a bundle of light focused from the fluorescence condensing lens 9 is output.

The focused fluorescence is detected in the light multipliers 4, 2 and 1 through reflection and transmission in the light filter 5 and the dichroic light filter 3, respectively. Then, the control unit (not shown) included in the power supply circuit unit 12 processes the signals detected by the optical multipliers 4, 2 and 1, and is currently discharged from the nozzle 8-3 according to the processed result. The cell is controlled to enter any one of the predetermined inner bowl in the cell sorting vessel (13-1, 13-2, 13-2).

On the other hand, "fluorescent inactive" cells 8-5 in which the fluorescent substance does not adhere to the cell material (nucleus) due to different cell types or no nucleus, even if scattered light D1 is fluoresced even when subjected to a laser beam Since F1 is almost absent, the control unit of the power supply circuit unit 12 determines that the cells discharged from the nozzle are fluorescence inactive cells and controls to enter the inner bowl 13-2 which collects only the fluorescence inactive cells accordingly. do.

When the conventional fluorescence activated cell sorting device, which operates as described above, analyzes the characteristics of the cells passing through the nozzle and classifies them, the detection signal of the fluorescence activated cells detected by the laser beam emitted from the argon ion laser and its The relationship between power consumption will be described with reference to the drawings.

FIG. 3 (a) is a graph showing the fluorescence activated cell detection signal of the fluorescence activated cell sorting apparatus of FIG. 1, and FIG. 3 (b) is a graph showing the power consumption of the fluorescence activated cell sorting apparatus of FIG.

For example, as shown in FIG. 3 (a), among the cells classified from the cell flow chamber 8 into the cell sorting vessel 13, the fluorescently active cells 8-6 and 8-4 are time points t1. If the time passes t3 through the nozzle 8-3 and the fluorescent inactive cells 8-5 passes the time t2, the argon ion laser continuously emits the laser light having a constant light output P1. As shown in Fig. 3 (b), the photodetector 11 has time points t1 and t3 for the fluorescently active cells 8-6 and 8-4 passing through the nozzle. ) Emits a fluorescence detection signal SG1, and hardly generates a fluorescence detection signal for the fluorescent inactive cells 8-5.

However, although the fluorescence activated cell sorting device using the argon ion laser operates to sort the cells discharged from the cell chamber through the nozzle and put them in the cell bowl, the high power is always provided whether or not the cells pass through the nozzle. Since the laser light must be continuously emitted to the nozzle, its energy utilization efficiency is low. In addition, since the argon ion laser and its peripheral devices are large, heavy, and small in mobility, and very expensive, the fluorescent activated cell sorting device using the argon ion laser is also large, heavy, and occupies a large space, and thus mobility There is a problem that it is small, expensive, and thus not widely available.

Therefore, the object of the present invention is to solve the above problem, the light output is emitted from the cell by the light pulse relatively light output as necessary to characterize the cell only when the cell passes the laser light path through the nozzle Fluorescence activated cell sorting device using laser diode and fluorescent active cell sorting method having relatively small average light output, which analyzes scattered light and fluorescence and classifies the cells according to the characteristics of the cells based on the analyzed values. Is to provide.

To this end, the fluorescent active cell sorting apparatus using the laser diode according to the present invention includes a cell flow chamber 30 which discharges cells through a nozzle 30-3, and the cells passing through a first position of the nozzle 30-3. A pulse-operated laser diode 26 for illuminating an optical pulse S2 at a position thereof, and positioned before the cell passes through the first position with respect to the first position, and spaced apart from the first position by a predetermined distance. 30-3) detects the cell detection continuous operation laser diode 27 which emits continuous parallel light D0 which continuously shines the laser light at the second position of the laser beam, and scattered light or fluorescence emitted when the cell passes the continuous parallel light. A continuous light detector 33-1 for generating a detection signal, a central processing unit 34 for controlling the driving time of the pulsed laser diode based on the detection signal, and supplying and controlling power to each element; remind It is achieved by detecting the scattered light and fluorescence emitted by the stained cells by the light pulse and based on the detected signal, including a cell sorting processing unit for sorting the stained cells.

Fluorescence-activated cell sorting method using a laser diode according to the present invention comprises the steps of shining the laser light continuously from the cell detection continuous operation laser diode to the nozzle through which the cell passes, and the scattered light or fluorescence emitted by the cell passing the nozzle through the laser light Detecting the cell passing by the light source, driving a pulsed laser diode according to the detected signal, illuminating the light pulse having a relatively high light output to the cell, and the scattered light emitted by the cell receiving the light pulse; And detecting the fluorescence, and analyzing the characteristics of the cells based on the detected scattered light and the fluorescence, and classifying the cells accordingly.

When described in detail together with the fluorescence activated cell sorting apparatus using the laser diode according to the present invention.

4 is a block diagram showing a fluorescently activated cell sorting apparatus using a laser diode according to the present invention, wherein the cell flow chamber 30 sends out the cells through the nozzle 30-3, and the nozzle 30-3 receives light. A pulsed laser diode 26 for illuminating the pulse S2, a focusing lens 29 for receiving the optical pulse S2 and focusing on its own focus at the nozzle, and scattered light D2 emitted from the focal position A scattered light condenser lens 32 for collecting light, a scattered light detector 33 for detecting scattered light collected by the scattered light condenser lens 32, a fluorescent condenser lens 31 for collecting fluorescence emitted by a cell passing through the focal point, and The light concentrator 25 receives the light collected by the fluorescent condenser lens 31 and partially reflects the light, and a first light multiplier tube 24 which detects the first reflected fluorescence reflected from the light filter 25. ) And the first passage fluorescence transmitted through the light filter 25 A dichroic light filter 23 that partially reflects and partially transmits according to a wavelength, a second light multiplier 22 that detects a second reflected fluorescence reflected from the dichroic light filter 23, and A third light multiplier 21 for detecting the second pass fluorescence passing through the dichroic light filter 23, and a cell detection continuous operation laser diode 27 that emits continuous parallel light D0 having a relatively low light output. And a first path in the nozzle 30-3, wherein the optical path of the continuous parallel light D0 is positioned before the cell passes the focal point with respect to the focal point of the focal lens via the focal lens 29. A first semi-transmissive mirror 28 which is split into a position, a second semi-transmissive mirror 28-1 which changes the optical path of continuous parallel light passing through the first position, and the second semi-transmissive mirror 28-1. Continuous light detector (33-1) for detecting continuous parallel light reflected from the center, and a central unit for supplying power to each element and controlling each element It consists of the back part 34.

Here, the cell flow chamber 30 is a sample fluid pipe 30-1 connected to receive a liquid mixed with cells from the outside, and a sheath fluid pipe 30-2 connected to receive a sheath solution from the outside. And a nozzle 30-3 which receives the liquid mixed with the sheath solution and the cells and allows the cells to pass through the focal point.

In addition, the fluorescent active cell sorting apparatus according to the present invention, under the control of the central processing unit, a direction control unit (not shown) for adjusting the movement direction of the cells discharged from the nozzle 30-3, and the direction control In accordance with the adjustment of the wealth, further comprises a cell sorting vessel 35 containing the cells discharged from the nozzle (30-3). Here, the cell sorting vessel 35, as shown in Figure 5, is composed of a plurality of inner bowl (35-1, 35-2, 35-3) that can contain cells classified according to the characteristics of the cell .

FIG. 5 is a configuration diagram showing in detail the nozzle 30-3 of the cell flow chamber, through which cells pass in the fluorescence activated cell sorting apparatus of FIG. 4.

The nozzle 30-3 has a predetermined diameter and length, and the cells are reduced by the cell flow chamber 30 from the cell flow chamber 30 to the cell sorting vessel 35. In other words, the relatively slender tubular nozzle 30-3 is a fluorescent dye-coated cell material (nucleus) 30-7 and a fluorescent dye-free cell (30-4, 30-6) Fluorescently inactive cells 30-5 are lined up from cell flow chamber 30 to cell sorting vessel 35.

FIG. 6 is a detailed block diagram showing the central processing unit 34 of the fluorescent active cell sorting apparatus of FIG. 4, which controls the operation of the cell detection continuous operation laser diode 27 and outputs it from the continuous light detector 33-1. From the control unit 34-1 for controlling the operation of the pulse-operated laser diode 26 in accordance with the signal, and the scattered light detection signal from the scattered light detector 33 and the first to third optical multipliers 24, 22 and 21. Based on the fluorescence detection signal outputted, the direction control unit (not shown) is adjusted to supply a cell classification processor 34-3 for classifying cells discharged from the current nozzle 30-3 and power to each element. And a power supply circuit unit 34-2.

Here, the controller 34-1 is a cell detection continuous operation laser diode driver 34-1A for driving the cell detection continuous operation laser diode 27, and a pulse operation laser for driving the pulse operation laser diode 26. A cell detector 34-1D for inputting a diode driver 34-1B and a continuous light detection signal from the continuous light detector 33-1 to confirm that the cell is currently passing through the nozzle 30-3; And a synchronization unit 34-1E for generating a synchronization signal by determining a driving time of the pulsed laser diode 26 based on the detection signal from the cell detection unit 34-1D, and the cell detection continuous operation laser. A laser which controls the diode driver 34-1A and controls the operation of the pulsed laser diode via the pulsed laser diode driver 34-1B in accordance with a synchronization signal from the synchronizer 34-1E. die And a de control (34-1C).

The operation method of the fluorescence activated cell sorting apparatus using the laser diode according to the present invention configured as described above will be described with reference to FIGS. 7 and 8 (a) to 8 (c) and FIG. 9.

First, in a state where all the device components are optically and electrically aligned, the sample fluid and the sheath fluid mixed with the cells by appropriately fluorescent dye treatment in advance are separated from the respective sample fluid tubes 30-1 and the sheath fluid tubes 30-2. The sample liquid and the cis liquid are added to the cell flow chamber 30 along the c) and are discharged to the cell sorting vessel 35 through the nozzle 30-3 at a predetermined speed (ST1). Here, the amount and velocity of the sample fluid and the sheath fluid entering the cell flow chamber 30 along the sample fluid pipe 30-1 and the sheath fluid pipe 30-2 are equal to the sample fluid pipe 30-1 and the sheath fluid. It is controlled by the internal pressure of the pipe (30-2).

At the same time, the cell detection continuous operation laser diode 27 is operated so that the continuous parallel light shines on the cells passing through the nozzle 30-3 (ST2). Here, the fluorescently activated cells C1, the fluorescently inactive cells C2, and the fluorescently active cells C3 and C4 from the cell flow chamber 30 toward the cell sorting vessel 35 along the nozzle 30-3 are shown in Fig. 8A. It flows in turn as shown. Then, the fluorescence-activated cell C1 receives continuous parallel light D0 at a time point t1 at a first position, and has a predetermined time difference from the time point t1, and the fluorescence activity following the fluorescence-activated cell C1. Alternatively, the fluorescent inactive cells C2, C3, and C4 also receive the continuous parallel light D0 at each of the time points t2, t3, and t4.

Therefore, when the fluorescent active cell C1 receives the continuous parallel light D0 at the first position at the time point t1, it emits fluorescence D0-2 weakly with the scattered light D0-1. At this time, the continuous light detector 33-1 detects the scattered light D0-1 and sends the detection signal SG2 to the cell detection unit 34-1D as shown in Fig. 9A (ST3).

Then, the synchronizer 34-1E is based on the detection signal received through the cell detector 34-1D, and the dye cell passing through the focus and nozzle through which the optical pulse from the first position, pulsed laser diode passes, passes through the nozzle. The timing of operation of the pulsed laser diode is determined using factors such as the speed of the pulse generator, and a synchronization signal is generated and sent to the laser diode controller 34-1C.

The laser diode controller 34-1C receiving the synchronization signal controls the pulsed laser diode driver 34-1B. Then, the pulsed laser diode 26, as shown in Figure 8 (b), under the control of the pulsed laser diode drive unit, the light pulse (S2) to the fluorescent active cell (C1) and received the fluorescence received Active cells emit scattered light (D2) and fluorescence (F2) (ST4). In other words, the pulsed laser diode 26 becomes active from the inactive state at the time t1 + d to illuminate the light pulse S2 on the cell C1 for a time T.

Then, the cell sorting processor 34-3 receives the signals transmitted from the scattered light detector 33 and the first to third photomultipliers 24, 22, and 21, and passes the cell C1 currently passing through the nozzle 30-3. ) And the classification signal is output (ST5).

In accordance with the classification signal output from the cell sorting processing section 34-3, the direction control unit operates to enter the corresponding cell sorting vessel 35 into the fluorescent active cells C1 discharged from the nozzle 30-3 (ST6). ).

On the other hand, while the cell C1 is being sorted, other fluorescently inactive cells C2 passing therefrom at the time point t2 can receive the continuous parallel light, which is continuously emitted from the cell detection continuous operation laser diode 27. Then, the cell detector 34-1D receives the light scattered by the fluorescent inactive cells C2 at the time point t2 while passing through the focusing point of the continuous parallel light, and thus, as shown in FIG. 9 (a), The detection signal SG2 is generated.

Thus, the synchronizer 34-1E produces a synchronization signal. The laser diode controller 34-1C also drives the pulsed laser diode 26. Accordingly, the pulsed laser diode emits light pulses at the time point t2 + d while the fluorescently inactive cells C2 pass through the focal point, as shown in FIG. Here, since the cell (C2) does not have a fluorescent dye, the scattered light and the fluorescence signal that receive light pulses are different from those of the fluorescence-activated cell.

Then, the cell processor 34-3 receives the signals transmitted from the scattered light detector 33 and the fluorescence detectors 21, 22, and 24, and thus the characteristics of the fluorescent inactive cells C2 passing through the current nozzle 30-3. Determine and output a cell classification signal (ST5). According to the cell sorting signal output from the cell sorting processor 34-3, the direction control unit operates to enter the corresponding cell sorting vessel 35 from the fluorescent inactive cells C2 discharged from the nozzle 30-3 ( ST6).

As described above, while the fluorescent inactive cells C2 are sorted, the continuous parallel light emitted from the detection laser diode 27 illuminates other fluorescently active cells C3 at the time point t3.

The continuous parallel light received by the fluorescent active cell (C3) proceeds similarly to the continuous parallel light experienced by the fluorescent active cell (C1). At the time t3, the fluorescence-activated cells C3 receive continuous parallel light and generate scattered light D0-1 and fluorescence D0-2 as shown in Fig. 8A. Then, the continuous light detector 33-1 detects the scattered light D0-1 and transmits the detection signal SG2 to the cell detection unit 34-1D as shown in Fig. 9A (ST3). ).

The synchronizer 34-1E is configured to detect the first position, the position of the focal point through which the pulsed laser diode passes, and the speed of the fluorescent active cell passing through the nozzle based on the detection signal input through the cell detector 34-1D. The operation time of the pulsed laser diode is determined using the factors such as the above, and a synchronization signal is generated and sent to the laser diode controller 34-1C.

The laser diode controller 34-1C receiving the synchronization signal controls the pulsed laser diode driver 34-1B. Then, the pulsed laser diode 26 illuminates an optical pulse S2 on the fluorescent active cell C3 under the control of the pulsed laser diode driver, similarly to that shown in FIG. Fluorescently active cells (C3) emit scattered light (D2) and fluorescence (F2) (ST4). In other words, the pulsed laser diode 26 becomes active from the inactive state at the time point t3 + d to illuminate the light pulse S2 on the fluorescent active cell C3 for a time T.

Then, the cell sorting processor 34-3 receives the signals transmitted from the scattered light detector 33 and the first to third photomultipliers 24, 22, and 21 to pass through the current nozzles of the fluorescent active cells C3. The classification signal is output by determining the characteristic (ST5).

According to the cell sorting signal from the cell sorting processor 34-3, the direction controller is operated to enter the corresponding cell sorting vessel 35 into the fluorescent active cells C3 discharged from the nozzle 30-3 (ST6). ).

Fluorescently inactive cells C4 are also detected at the time t4 in the same manner as the fluorescent inactive cells C2 are sorted, and at the time t4 + d from the pulsed laser diode operating on the basis of the detection signal. The pulsed light emits scattered light D2 and fluorescence F2 and is classified accordingly.

Therefore, the fluorescent active cell sorting apparatus using the laser diode according to the present invention detects the flow of cells passing through the nozzle using a cell detection continuous operation laser diode, and determines the point of time at which the light pulses are reflected on the cells and at that time The pulsed laser diode is driven to illuminate the cells with light pulses, and the cells are identified and classified according to the scattered light and fluorescence which the light pulses receive.

In the embodiment of the present invention, a semi-transparent mirror is used to allow the continuous parallel light emitted by the continuous detection laser diode to pass from the continuous detection laser diode to the first position of the nozzle and to reach the photodetector from the first position. The continuous parallel light of the continuous detection laser diode may be arranged to reach the photodetector after the first position of the nozzle without using a transmission mirror.

Fluorescence activated cell sorting device is a device that efficiently classifies and collects various cells according to their characteristics. It is an important device necessary for life science (zoology, botany, microbiology, agriculture, fisheries, forestry, etc.) and medical research, but conventional fluorescent cells Lasers, such as high-powered argon ion laser systems, which are used as light sources in the sorting device, are large, heavy, power-consuming, and expensive, so that the fluorescence-activated cell sorting device using them is large, heavy, and consumes a lot of power. It was expensive, expensive, and not widely used, and the movement and installation of the device was very cumbersome and inconvenient to use. In addition, the price was very expensive and did not spread widely.

The active cell sorter using the laser diode according to the present invention, which solves such a problem, has a detection laser diode that outputs continuous light so as to accurately capture the moment when the cell passes and a predetermined time for the cell. Since a pulsed output laser diode that outputs only a high optical pulse is used, its size is small, its weight is light, and its space is small, so its mobility is large. In addition, the fluorescent active cell sorting apparatus using the laser diode according to the present invention can be easily decorated with the laser diode and its driving circuit, and therefore its value is relatively cheap compared with the apparatus using a conventional argon ion laser or the like.

Figure 1 shows the configuration of a conventional fluorescently activated cell sorting apparatus using an argon ion laser.

FIG. 2 shows in detail the nozzle of the flow chamber, through which cells pass in the fluorescence activated cell sorting apparatus of FIG. 1.

Figure 3a is a graph showing the staining cell detection signal of the fluorescent active cell sorting apparatus of FIG.

Figure 3b is a graph showing the power consumption of the fluorescent active cell sorting apparatus of FIG.

Figure 4 shows the configuration of a fluorescent active cell sorting apparatus using a laser diode according to the present invention.

Figure 5 shows in detail the nozzle of the cell flow chamber, the cells pass through in the fluorescent active cell sorting apparatus of FIG.

Figure 6 shows in detail the configuration of the central processing unit of the fluorescent active cell sorting apparatus of FIG.

Figure 7 is a flow chart illustrating a method for classifying fluorescently active cells using the fluorescently active cell sorting apparatus according to the present invention.

8a to c are diagrams illustrating a method for classifying fluorescence activated cells using a fluorescence activated cell sorting apparatus according to the present invention.

Figure 9a is a graph showing the cell detection signal of the fluorescent active cell sorting apparatus of FIG.

Figure 9b is a graph showing the power consumption of the fluorescent active cell sorting apparatus of FIG.

Description of the main symbols in the drawings

1, 2, 4, 21, 22, 24: photomultiplier

3, 23: dichroic beam-splitter

5, 25: beam-splitter

6: Argon ion laser system

7, 29: focusing lens

8, 30: flow chamber

8-1, 30-1: sample fluid pipe

8-2, 30-2: sheath fluid pipe

8-3, 30-3: nozzle

8-4, 8-6, 30-4, 30-6: nucleated cells

8-5, 30-5: non-nucleated cells

8-7, 30-7: cell nucleus

9, 31: fluorescent light collecting lens

10, 32: scattered light collecting lens

11, 33, 33-1: photodetector

12, 34-2: power supply circuit

13, 35: cell sorting vessel

13-1, 13-2, 13-3: inner bowl

26: pulse operated laser diode

27: Continuously Operated Cell Detection Laser Diode

28, 28-1: half mirror

34: central processing unit

34-1: control unit

34-3: cell sorting processing unit

Claims (9)

Cell flow chamber 30 for emitting the cells stained with the fluorescent dye through the nozzle (30-3), A pulsed laser diode 26 for illuminating an optical pulse S2 to the cell passing through the first position of the nozzle 30-3, The light is positioned before the cell passes through the first position with respect to the first position, and illuminates the continuous parallel light D0 at a second position of the nozzle 30-3, which is separated by a predetermined distance from the first position. Cell detection continuous operation laser diode 27, A continuous light detector 33-1 generating a detection signal by receiving scattered light or fluorescence of a cell passing through the continuous parallel light path; A central processing unit 34 for controlling the driving time of the pulse-operated laser diode based on the detection signal, and supplying and controlling power to each element; And a cell sorter for detecting scattered light and fluorescence that the cell receives the light pulse and classifying the cells according to optical characteristics based on the detected signal. . The method of claim 1, wherein the central processing unit 34 A control unit 34-1 for controlling the operation of the cell detection continuous operation laser diode 27 and controlling the operation of the pulse operation laser diode 26 according to a signal from the continuous light detector 33-1; A cell classification processor 34-3 for classifying the cells currently exiting the nozzle 30-3 based on the scattered light and the fluorescence according to the control signal of the controller 34-1; And a power supply circuit section (34-2) for supplying power to each element. The method of claim 2, wherein the control unit 34-1 A cell detection continuous operation laser diode driver 34-1A for driving the cell detection continuous operation laser diode 27; A pulsed laser diode driver 34-1B for driving the pulsed laser diode 26; A cell detector 34-1D for inputting a continuous light detection signal from the continuous light detector 33-1 and detecting a cell currently passing through the nozzle 30-3; A synchronizer 34-1E for generating a synchronization signal by determining a driving time of the pulsed laser diode 26 based on a detection signal from the cell detector 34-1D; Inputs a detection signal from the cell detector 34-1D, controls the cell detection continuous operation laser diode driver 34-1A, and operates the pulse according to a synchronization signal from the synchronizer 34-1E. And a laser diode controller (34-1C) for controlling the operation of the pulsed laser diode through a laser diode driver (34-1B). The method of claim 1, wherein the cell sorter Scattered light detector for detecting the scattered light that the cell receives the optical pulse and outputs the scattered light signal to the central processing unit; And a plurality of photomultipliers for detecting the fluorescence of the cells receiving the optical pulses and outputting a fluorescence signal to the central processing unit. The method of claim 4, wherein the central processing unit 34 A control unit 34-1 for controlling the operation of the cell detection continuous operation laser diode 27 and controlling the operation of the pulse operation laser diode 26 according to a signal from the continuous light detector 33-1; A cell classification processor 34-3 for classifying the cells currently exiting the nozzle 30-3 based on the scattered light and the fluorescence according to the control signal of the controller 34-1; And a power supply circuit section (34-2) for supplying power to each element. The method of claim 5, wherein the control unit 34-1 A cell detection continuous operation laser diode driver 34-1A for driving the cell detection continuous operation laser diode 27; A pulsed laser diode driver 34-1B for driving the pulsed laser diode 26; A cell detector 34-1D for inputting a continuous light detection signal from the continuous light detector 33-1 and detecting a cell currently passing through the nozzle 30-3; A synchronizer 34-1E for generating a synchronization signal by determining a driving time of the pulsed laser diode 26 based on a detection signal from the cell detector 34-1D; Inputs a detection signal from the cell detector 34-1D, controls the cell detection continuous operation laser diode driver 34-1A, and operates the pulse according to a synchronization signal from the synchronizer 34-1E. And a laser diode controller (34-1C) for controlling the operation of the pulsed laser diode through a laser diode driver (34-1B). Illuminating continuous parallel light of a cell detection continuous operation laser diode to cells flowing in a line at a predetermined speed; Detecting the continuous parallel light; Illuminating a light pulse having a relatively high light output by driving a pulsed laser diode to the cell according to the detected signal; Detecting scattered light and fluorescence emitted by the cells receiving the light pulses; And classifying the cells on the basis of the detected scattered light and fluorescence. The method of claim 7, wherein the step of illuminating the light pulse A method for classifying fluorescently active cells using a laser diode, characterized in that set by the relationship between the time point at which said continuous parallel light shines on said cell and the speed at which said cell flows. The method of claim 8, further comprising the step of setting the light pulse output time.