JPS63172943A - Apparatus for analyzing and dispensing microparticle - Google Patents

Abstract

PURPOSE:To improve an always exact voltage to liquid drops by applying the specified voltage to the fine flow ejected from a flow nozzle in a period before or after the time when the fine flow changes to the liquid drops and changing the voltage in said period independently. CONSTITUTION:Sheath liquid La and microparticle suspended liquid SL are supplied to the flow nozzle 1 and are oscillated by an oscillator 2 so as to be ejected in the form of the fine flow LP from the aperture of the nozzle. The microparticles in the flow LP are detected in a photodetection part 5. A liquid drop charge control part 20 judges the kind of cells in accordance with the detected value and impresses the charge voltage corresponding to the kind of the cells to an electrode EB. The above-mentioned voltage is impressed as a specified positive or negative voltage at the time before and after each time when the liquid drops are continuously generated from the fine flow. The voltage is applied in the form of the voltage waveform, for example, staircase waveform, set discretely to each of the liquid drops. As a result, always the exact voltage is impressed to the liquid drops and, therefore, the cells, etc., are stably dispensed without missing.

Description

[Detailed Description of the Invention] [Industrial Application Field] The present invention provides a microparticle analysis and sorting device that elucidates the properties, structure, etc. of cells, etc., and sorts cells, etc. by type based on the results. In particular, the present invention relates to a microparticle analysis and sorting device that can stably separate cells and the like without dropping cells.

[Prior art] Microparticles such as cells are detected using optical detection means or electrical detection means, and the properties, structure, etc. of the microparticles are analyzed from the detection results, and microparticles with the same characteristics are identified by their type. A microparticle analysis/fractionation device that collects particles at different temperatures is generally known.

FIG. 4 shows the configuration of this microparticle analysis and fractionation apparatus.

In the figure, ■ is a flow nozzle for ejecting a jet stream (hereinafter simply referred to as a nozzle), which has a cylindrical shape with a tapered opening at the lower end and a closed upper end. A cylindrical vibrator 2 is attached to the upper end.

Further, the nozzle 1 has a through hole in its upper side surface portion, and an injection pipe 1a is connected to this through hole in a direction toward the center of the nozzle 1. The sheath liquid La is injected into the nozzle I through this injection tube 1a. Further, an electrode EB is attached to the injection tube 1a at a connecting portion with the nozzle l. This electrode EB charges the sheath liquid La via the injection tube 1a.

Furthermore, the nozzle 1 is also provided with a through hole at its upper end, and through this through hole, the injection tube lb is inserted along the central axis of the nozzle 1 so that its tip is slightly above the opening of the nozzle l. It has reached this point. The microparticle suspension SL enters the nozzle through this injection tube 1b! injected into the body. This nozzle 1
The microparticle suspension SL injected into the nozzle 1 becomes a laminar stream LP surrounded by the sheath liquid La and is ejected from the opening of the nozzle 1.

The vibrator 2 (such as an ultrasonic vibrator) applies vibration to the nozzle 1 to generate droplets (droplets are indicated by the symbol DP) from the trickle LP. The timing of droplet generation is determined by the magnitude and frequency of vibration of the vibrator 2. The excitation circuit 3 supplies a voltage that vibrates the vibrator 2, and can vary the amplitude and frequency of this output voltage. This output voltage is also supplied to the droplet charge control section 10.

Reference numeral 4 denotes a light source (such as a laser light source) that irradiates the trickle LP ejected from the nozzle 1 with light. The light emitted from this light source 4 generates microparticles (cells, etc.) in the trickle LP. Reference numeral 5 denotes a light detection unit that detects fluorescence and scattered light from cells, and two detectors 5a and 5b are arranged at different positions. The output signal from this photodetector 5 is processed by a signal processor 9
and the droplet charge control unit 10, respectively.

Reference numeral 6 denotes a continuous plate-shaped ground electrode plate, a portion of which has a through hole for allowing droplets to pass through. Further, the ground electrode plate 6 is arranged below the nozzle 1 at a predetermined distance.

7a and 7b are deflection electrode plates, respectively. These deflection electrode plates 7a and 7b are arranged below the ground electrode plate 6 at a predetermined distance, and are opposed to each other with their lower sides open in a V-shape at a predetermined interval. Further, a positive voltage is applied to the deflection electrode plate 7a from the power source Ba, and the deflection electrode plate 7b
A negative voltage is applied from the power source Bb.

8a, 8b, and 8c are containers that receive droplets dropped from the nozzle l, respectively. These containers 8a.

8b and 8c are arranged below the deflection electrode plates 7a and 7b at a predetermined distance apart, and at positions corresponding to the angle at which the droplet is deflected by the deflection electrode plate 7a17b.

Next, the signal processing section 9 analyzes the properties, structure, etc. of the cells based on the detection signal output from the photodetection section 5, and
The output is displayed as a cytogram or histogram on the (cathode ray tube). Further, the signal processing unit 9 can manually or automatically set the number of cells to be subjected to signal processing in advance, and when this set value is reached, the signal processing is stopped and a stop signal is supplied to the droplet charge control unit IO. do. This signal processing section 9 usually uses a microcomputer or the like.

The droplet charge control unit 10 determines the presence and type of cells based on the detection signal supplied from the light detection unit 5, and sorts the detected cells according to the type based on the determination result. It generates a positive or negative charge or voltage. At the same time, the number of positively charged droplets and the number of negatively charged droplets are supplied to the signal processing unit 9.

When charging a cell, the droplet charge control unit IO accurately sets the charge based on the determination result to the droplet containing the cell, since there is a time delay between when the cell is detected and when the cell is generated. In order to charge the cell, the timing of generating the charging voltage is set to the time immediately before the droplet containing the cell is generated, based on the flow velocity of the trickle LP and the distance between the cell detection point and the droplet generation point. ing.

Further, the droplet charging control unit IO stops generating the charging voltage by receiving a stop signal from the signal processing unit 9.

Next, the operation of this circuit will be explained. In FIG. 4, when a voltage having a constant frequency and amplitude is supplied from the excitation circuit 3 to the vibrator 2, the vibrator 2 starts vibrating and causes the nozzle 1 to vibrate. At the same time, the sheath liquid La and the microparticle suspension SL are injected into the nozzle through injection tubes 1a and Ib, respectively. It is supplied into the atmosphere and is ejected into the atmosphere from the opening of the nozzle l as a laminar stream LP. Then, this trickle LP becomes a droplet at a position shown by point B in the figure at a timing synchronized with the vibration of the nozzle l. Until the trickle LP becomes a droplet, the light source 4 and the light detection unit 5 detect cells present in the trickle LP at the position indicated by point A in the figure. Then, the detection signal output from the photodetector 5 is supplied to the droplet charge control unit IO, and based on this detection signal, the droplet charge control unit 10 determines the type of cell. A corresponding charging voltage is supplied to the electrode EB. At the same time, the detection signal output from the photodetection section 5 is also supplied to the signal processing section 9, and based on this detection signal, the signal processing section 9
analyzes the properties, structure, etc. of cells and outputs and displays them on a CRT as a cytogram or histogram.

Next, the droplets falling in the atmosphere pass through the through holes of the ground electrode plate 6, and then reach the deflection electrode plates 7a and 7b. At this time, the droplet passing between the deflection electrode plates 7a and 7b is deflected according to its charged state. The droplet is then dropped into one of the containers 8a, 8b, and 8c arranged in the deflection direction. In this case, if the droplet is positively charged, when it passes between the deflection electrode plates 7a and 7b, a repulsive force is generated from the deflection electrode plate 7a to which a positive voltage is applied.
b receives an attractive force and bends toward the deflection electrode plate 7b.

As a result, the droplets fall into the container 8a. Further, if the droplet is negatively charged, contrary to the above, it receives an attractive force from the deflection electrode plate 7a and a repulsive force from the deflection electrode plate 7b, and bends toward the deflection electrode plate 7a. As a result, the droplets fall into the container 8c.

Moreover, if the droplet is not charged, the deflection electrode plate 7a,
The liquid is directly dropped into the container 8b without being affected by the liquid 7b.

Through the process described above, the droplets are separated into the respective containers 8a, 8b, and 8c.

[Problems to be Solved by the Invention] When charging droplets containing microparticles, there is a time delay between the detection of the microparticles and the generation of droplets containing the microparticles, so the charging voltage As mentioned above, the timing of generation is delayed by the previous time delay from the time when the microparticle is detected. However, there is a probability that the microparticles will be included in the droplets before and after the droplet that is charged due to the actual time delay with respect to the set time delay. From this, in order to separate microparticles with a high recovery rate, the droplets before and after the droplet expected to contain the microparticles are charged with the same polarity voltage; Sorting in the same direction is routinely performed. In this case, conventional microparticle analysis and fractionation devices charge droplets to be continuously charged with a continuously increasing charging voltage. This means that the charge of the next generated droplet is influenced by the charge of the previously generated and charged droplet.
The problem is that the x amount decreases and the deflection electrode plate 7a.

This is to prevent problems such as when droplets successively pass between 7b and the droplets that are dropping first are subjected to strong air resistance, resulting in a decrease in the droplet speed and an increase in deflection. there were.

For example, the waveform of the charging voltage when three droplets are charged in succession is shown in FIG. 5(b). In addition, FIG. 3A is a diagram showing the timing at which droplets are generated from the trickle LP, and the droplets are generated in the order of 1→2...→9→1O.

In the same figure (b), due to the charging voltage waveform rising at time ta, charging voltage va is applied to the second droplet generated at time t+ (t+ > ta), and charging voltage va is applied to the third droplet. A charging voltage vb is applied to the fourth droplet at time t, and a charging voltage v'c is applied to the fourth droplet at time t3.

By the way, in the conventional microparticle analysis/preparation circuit described above,
Since the continuously generated droplets were charged with a continuous charging voltage waveform, it was not always possible to apply an accurate charging voltage to the droplets due to the following points.

■ It is difficult to determine the slope of the curve of the charging voltage waveform by calculation, and when determining the charging voltage waveform by experiment, it is difficult and time-consuming because various waveforms must be created.

(2) The charging voltage waveform determined in section (2) is usually a complex function that is not a -dimensional number or an exponential function, and therefore it is difficult to electrically realize this.

(2) Furthermore, in the charging voltage waveform determined in section (2), if the timing at which a droplet is generated and the timing at which the charging voltage rises deviate, the relative value of the charging voltage applied to each droplet will change. This situation will be explained with reference to the charging voltage waveform shown in FIG.

This figure shows the timing at which droplets are generated on the time axis and the charging voltage waveform CV. The droplet is numbered l → 2
→3-4→5 occurs in the order. Further, the amount of charge on each droplet corresponds to the charging voltage when the droplet is generated.

Now, when the charging voltage waveform CV rises at time ta, the charging voltage ratio of each droplet 2 to 4 is I:1.
6:2.3. Assuming that the deflection amounts of droplets 2 to 4 are equal in this state, if the timing of the rise of the charging voltage waveform Cv shifts from time ta to time tb, the charging voltage ratio will be 1:l.

5:2. It becomes O.

If the charging voltage ratio changes in this way, the amount of deflection of each droplet will differ when each droplet passes through the deflection electrode plates 7a and 7b (see Figure 4), so some droplets may , container 8
There is a risk that it will protrude from a, 8b, and 8c.

Note that the reason why the charging voltage ratio changes due to a shift in the rise timing of the charging voltage waveform Cv described above is that it is almost impossible for the charging voltage waveform to be an exponential function.

As described above, the conventional microparticle analysis and separation apparatus cannot apply an accurate and stable charging voltage to each droplet when separating the droplets.

This invention was made against this background, and its purpose is to easily determine each charging voltage to be applied to each droplet when charging droplets successively, and to prevent the generation of droplets. An object of the present invention is to provide a microparticle analysis/fractionation device that can always apply a constant charging voltage to droplets even if the timing and the timing of the rise of the charging voltage are different. [Means for Solving the Problems] The present invention includes a flow nozzle that spouts a suspension of microparticles into the atmosphere as a trickle, and a method for vibrating the flow nozzle to form droplets from the trickle. a vibration means, a detection means for detecting the microparticles present in the rivulet, and a charging means for controlling whether or not to apply a voltage to the rivulet and the polarity of the applied voltage based on a detection signal from the detection means;
In a microparticle analysis and preparative device having a deflection means disposed below the flow nozzle for generating an electric field and deflecting the droplets, there are It is characterized in that means are provided for applying a constant positive or negative voltage to the trickle over time and for applying an individually set voltage value to each droplet.

[Function] According to the above means, a constant voltage can be applied to the trickle during a period before and after the time when the trickle ejected from the flow nozzle becomes a droplet, and the voltage value within this period can be changed independently. . This ensures that an accurate voltage is always applied to the droplet.

[Embodiment] An embodiment of the present invention will be described below with reference to the drawings.

FIG. 1 is a block diagram showing the configuration of a microparticle analysis and separation apparatus which is an embodiment of the present invention. In this figure, parts corresponding to those in FIG. 4 described above are given the same reference numerals, and their explanation will be omitted.

In the figure, 20 is a droplet charge control section, and a photodetection section 5
The presence and type of cells are determined based on the detection signal supplied from the detector, and a charging voltage is generated for sorting the detected cells according to their types based on the determination results. At the same time, the number of positively charged droplets and the number of negatively charged droplets among the droplets containing cells are supplied to the signal processing section 9 .

The timing at which the droplet charging control unit 20 generates the charging voltage is determined immediately before the droplet containing the cell is generated, based on the flow velocity of the fine KLP and the distance between the cell detection point and the droplet generation point. A constant charging voltage is generated from this time until immediately after the droplet is dropped.

In this case, details of the charging voltage output from the droplet charging control section 20 will be explained with reference to FIG.

FIG. 5A is a diagram showing the timing at which droplets are generated from the trickle LP, and the droplets are generated in the order of 1→2...→9→1O. FIG. 3B is a diagram showing a charging voltage waveform for charging a droplet. This charging voltage waveform is made into a step-like shape each time a droplet drops from the trickle LP. For example, when three droplets are charged in succession, a charging voltage va is applied to the second droplet from time t before the droplet is generated to time t immediately after the droplet is generated. A charging voltage vb that is a predetermined voltage increase with respect to the second charging voltage va is applied to the third droplet over time tx t3, and a charging voltage that is a predetermined voltage increase with respect to the charging voltage vb is applied to the fourth droplet in the same manner. Voltage VC is applied over time t3-t4.

As described above, if the continuously generated droplets are charged stepwise each time they are dropped, this charging voltage waveform can be easily achieved. Further, by using a stepped charging voltage waveform, the value of the charging voltage applied to each droplet does not change even if the rising timing of the charging voltage is shifted.

Note that the waveform of the droplet charging voltage output from the droplet charging control section 20 in the above embodiment may be a rectangular waveform as shown in FIG. 3(b).

In the figure (b), the charging voltage waveform is a rectangular wave in which a predetermined voltage is generated before and after the timing at which droplets are generated from the trickle LP.

For example, when three droplets are charged in succession, the charging voltage va is applied from time t,' before the second droplet is generated to time ty' immediately after the second droplet is generated. Thereafter, similarly, the charging voltage vb is applied to the third droplet from time t3' to tt', and the charging voltage vb is applied to the fourth droplet from time ts' to tt'.

A charging voltage vc is applied over .

Furthermore, in the embodiments of the present invention, a microparticle analysis and sorting device that uses optical detection means to detect cells has been described.
Needless to say, the present invention can be similarly used in a microparticle analysis/fractionation device using electrical detection means.

[Effects of the Invention] As explained above, according to the present invention, there is provided a flow nozzle that spouts a suspension of microparticles into the atmosphere as a rivulet, and a flow nozzle that vibrates to generate droplets from the rivulet. vibrating means for forming a microparticle, a detection means for detecting the microparticles present in the rivulet, and controlling whether or not to apply a voltage to the rivulet and the polarity of the applied voltage based on a detection signal from the detection means. In a microparticle analysis and separation apparatus having a charging means and a deflection means disposed below the flow nozzle and deflecting droplets by generating an electric field, each occurrence time when droplets are successively generated from the trickle. Since a means is provided to apply a constant positive or negative voltage to the trickle over the time before and after the droplet, and to apply a voltage value set individually to each droplet, when charging the droplet, the droplet Since it is sufficient to apply a constant charging voltage before and after the time at which the charging voltage occurs, the charging voltage characteristics can be easily determined.

Furthermore, when charging droplets successively, the charging voltage value applied to each droplet can be changed within a certain range without affecting others.

Further, it is possible to easily determine the relative value of the charging voltage when each droplet is generated, that is, the charging voltage value of the next generated droplet corresponding to the charging voltage value of the first charged droplet.

Therefore, the relative value of the charging voltage can be easily changed when changing the ejection state of the trickle ejected from the flow nozzle.

Furthermore, by applying a constant charging voltage before and after the droplet generation time, even if the timing of droplet generation and the timing of the rise of the charging voltage are different, the value of the charging voltage applied to each droplet can be The advantage is that it does not change.

[Brief explanation of the drawing]

FIG. 1 is a block diagram showing the configuration of a microparticle analysis and separation apparatus according to an embodiment of the present invention, FIGS. 2 and 3 are waveform diagrams for explaining the operation of the same embodiment, and FIG. 4 is a conventional FIGS. 5 and 6 are waveform diagrams for explaining the operation of the conventional microparticle analysis and separation apparatus. SL... ...Small particle suspension, l... ...
Flow nozzle, 2... ... Vibrator, 3... ・
・・Excitation circuit (2 and 3 are excitation means), 4・・・・
Light source, 5...--Photodetection unit (4, 5 are detection means), 20...Droplet charge control unit, EB...
... Electrode plate (20, EB is charging means), 7a, 7b
... Deflection electrode plate, Ba5Bb... Power supply (7a17b and Ba, Bb are deflection means).

Claims (1)

[Claims] A flow nozzle that spouts a suspension of microparticles into the atmosphere as a rivulet, a vibration means for vibrating the flow nozzle to form droplets from the rivulet, and detecting the microparticles present in the rivulet. a charging means for controlling whether or not to apply a voltage to the trickle and the polarity of the applied voltage based on a detection signal from the detection means; in a microparticle analysis and preparative device having a deflection means for deflecting a droplet, applying a constant positive or negative voltage to the rivulet over a period of time before and after each generation time when droplets are successively generated from the rivulet; A microparticle analysis/fractionation device, further comprising means for applying an individually set voltage value to each droplet.