JPS6225989B2 - - Google Patents

Description

DETAILED DESCRIPTION OF THE INVENTION The present invention relates to an apparatus for measuring the electrophoretic velocity of suspended biological cells.

When leukocytes and lymphocytes are suspended in a suitable solution and an electric field is applied, these cells undergo electrophoresis, and diagnostic tests can be performed by measuring their electrophoretic mobility, that is, (electrophoretic velocity)/(electric field strength). You can get various information. For this reason, several cell electrophoresis devices have been proposed in the past. These conventional devices apply a direct current electric field to a cell suspension to cause cells to electrophorese in one direction, and measure the state of cell migration using various optical methods. In conventional methods like this, cells move in one direction, so the optical device only briefly observes individual cells passing through its field of view once, making it impossible to track and observe individual cells over a long period of time. I couldn't do it. The electrophoresis speed is (passing distance)/(time), and the time can be set relatively accurately, so the measurement accuracy is determined by the measurement accuracy of the passing distance, and the longer the measuring length is, the more accurate the passing distance will be. easy. However, since cells in solution precipitate, it has not been possible to measure cell electrophoresis over an arbitrarily long period of time. Therefore, it can be said that in the past, long-term observation of a small number of cells was replaced by simultaneous observation of a large number of cells.

The present invention applies an alternating current electric field to a cell suspension, causes the cells to perform electrophoretic reciprocating motion, that is, vibration, and detects the characteristic values of the vibrational motion of particles, thereby making it possible to substantially track cells for a long time. It is something.
Here, the characteristic value of the vibrational motion of the particle refers to the displacement velocity or amplitude value due to the vibration of the particle near the vibrational displacement O, and since the cell is reciprocating within a relatively narrow range, the observation field of view is The object of the present invention is to provide a cell electrophoresis measuring device which is characterized in that it can be made longer in the vertical direction than in the horizontal direction and can be observed for a long time without considering precipitation. The present invention will be explained below with reference to Examples.

FIG. 1 shows an embodiment of the invention. Reference numeral 1 denotes an electrophoresis tank, which is a horizontally flat rectangular tank whose depth is larger than its width in the left and right directions shown in the figure, and parallel plate electrodes 2 and 3 are inserted to face each other on the left and right. 4
is a buffer solution isotonic with blood, and a sample injection nozzle 5 stands in the center of the tank. A biological cell suspension is injected from the nozzle 5. A slit 6 that is long in the vertical direction and has a width L is arranged on the front outside of the electrophoresis tank 1, and the liquid in the electrophoresis tank 1 is strongly illuminated from below with a light beam that has a width L from side to side. A voltage of V is applied between electrodes 2 and 3 while reversing the polarity at regular time intervals. In short, a positive and negative rectangular wave voltage having a peak value V is applied between the electrodes 2 and 3. One cell is injected into the liquid 4 through the nozzle 5. The injected cells move back and forth from side to side due to the action of an electric field that reverses their direction from side to side as they settle. When this cell is viewed through the slit 6, it is illuminated from below and is observed as a light spot, passing through a beam of light having a width L for a certain time T. When a light receiving element P is placed behind the slit 6 and the time T during which the element receives light is measured, the electrophoretic velocity of the cell is given by L/T. The electric field strength E is given by V/D, where D is the distance between the electrodes 2 and 3, and the electrophoretic mobility σ is given by LD/VT. Light receiving element P
The photoelectric output of is a pulse with a time width T, and this pulse is counted by a counter K, and on the other hand, the output pulse of a pulse generator G, which outputs a pulse with a period shorter than T through a gate A opened by the same pulse, is Count with counter C. The above counting operation continues while the cell descends to the height of the slit 6, and the calculation circuit S divides the count of the counter C by the count of the counter K minus 1. Find the speed. The counting of counters K and C is started by the set output of flip-flop F, which is set at the rising edge of photodetector P, and is triggered by the falling edge of the output of P, and continues for a considerably longer time than the expected period of K. At the fall of the output of timer circuit t which produces an output of , F is reset and the count outputs of counters C and K are input to arithmetic circuit S. Counters C and K are reset when injecting the next cell.

FIG. 2 shows another embodiment of the invention. In the above-mentioned embodiment, it is necessary to aspirate the cells one by one into a capillary tube under a microscope and inject them into the tank 1. Although the amount of sample can be small, the operation is troublesome. This example allows the electrophoretic velocity of a large number of cells to be measured at once. The structure of the electrophoresis tank is the same as the embodiment shown in FIG. An alternating voltage is applied between the electrodes 2 and 3, and an appropriately diluted cell suspension is injected into the liquid 4 from the nozzle 5. The cells sediment while reciprocating from side to side. Since the voltage applied between electrodes 2 and 3 is an AC voltage, the cell is
It sinks while performing a sinusoidal motion with a maximum displacement at a phase shift of . Therefore, the liquid 4 is illuminated from below only while the voltage applied between the electrodes 2 and 3 is ±Δ (Δ is smaller than the applied AC voltage and has a phase angle of about ±10°). In other words, when the AC voltage connected between electrodes 2 and 3 is converted into a trapezoidal wave by a limiter whose slice level is set to ±Δ and differentiated by a differentiating circuit, the AC voltage becomes ±Δ.
Since a pulse signal having a constant value within the range of is obtained, the positive direction pulse of this signal opens the switch and causes the light source l to emit light. In this way, the cells are illuminated near the maximum displacement, and electrophoresis of the cells almost stops in that vicinity. The cell suspension injected into Solution 4 is initially in the form of cloudy small lumps resembling smoke, but during electrophoresis in one direction, it gradually expands laterally due to the distribution of migration speed, and then in the return direction. In electrophoresis, cells that move far in one direction move faster and therefore return faster, and when the applied electric field strength becomes 0, all cells gather near the perpendicular line to the injection position, and eventually disperse in the opposite direction. I'll go. Although it performs such a reciprocating movement,
When such a group of cells is illuminated only near the maximum displacement phase, it is observed as a cloud with a certain concentration distribution of brightness in the lateral direction. This concentration distribution represents the distribution of migration speed of the cell group. Therefore, the tank 1 is compressed from the side in the vertical direction through a cylindrical lens B1 and an ordinary convex lens B2, an image of turbidity caused by floating cells is projected onto a one-dimensional solid-state image sensor I, and the photoelectric output is transferred to each of the elements. It accumulates bit by bit. The output of the solid-state image pickup device I is read out and stored in the readout memory M by a timer set in advance for a period of time long enough for the cells to settle out of the field of view of the lens systems B1 and B2 after one sample injection. This memory can be retrieved later to draw a distribution curve of electrophoretic velocity.

The embodiment shown in Fig. 2 is easy to operate since it is not necessary to separate and measure each cell, and the measurement of the velocity distribution is not only done once, but is repeated many times, resulting in a concentration distribution corresponding to the velocity distribution. Since the cell suspension is stacked, an accurate velocity distribution curve can be obtained even if the cell suspension is dilute.

The cell electrophoresis measurement device of the present invention has the above-described configuration, and is designed to repeat measurements on individual cells many times while applying an alternating electric field to floating cells and making them perform reciprocating motion. The same level of measurement accuracy as electrophoresis can be obtained, and since the cells are only moving within a certain range, a device with a long vertical field of view allows for long-term measurement without considering cell sedimentation. It has the advantage of being able to perform measurements and requiring a smaller amount of sample than short-time measurements.

[Brief explanation of the drawing]

FIG. 1 is a front view and a block diagram of a circuit portion of an apparatus according to one embodiment of the present invention, and FIG. 2 is a block diagram showing the configuration of another embodiment of the apparatus according to the present invention. 1... Electrophoresis tank, 2, 3... Electrode, 4... Solution, 5
...Nozzle, 6...Slit, I...Solid-state image sensor, M
...Memory, l...Light source.

Claims (1)

[Claims] 1 Place two flat plate electrodes facing each other with their surfaces perpendicular to each other in an electrophoresis tank, place a sample injection nozzle between the two electrodes, and cut a slit on the side of the electrophoresis tank so that the space between the two electrodes can be seen through. Arranges an optical element such as a lens system and a light-receiving element, applies an alternating voltage between the two electrodes, causes the sample particles injected by the nozzle to perform vibrational motion, and detects the characteristic value of the vibrational motion. A cell electrophoresis measurement device characterized by: