JPS6239702B2 - - Google Patents

Description

DETAILED DESCRIPTION OF THE INVENTION The present invention relates to a method for measuring the electrophoretic velocity of visible particles, such as cells, in electrophoresis in solution.

Special medical information can be obtained by measuring the electrophoresis of leukocytes in a solution. For this reason, devices for measuring the electrophoresis of floating cells and other visible floating particles have already been proposed. The principle of this previously proposed device is as follows. A sample solution containing suspended particles is placed in an electrophoresis tube, an electric field is applied, and the suspended particles are illuminated with strong light, forming a shining image of the suspended particles on a grid. The image of the suspended particles on the grid moves according to the electrophoresis of the suspended particles. The grating has grid lines extending perpendicular to the moving direction of the floating particle image, and many of these grid lines are arranged in parallel in the moving direction of the floating particle image.The light receiving element is placed behind this grating. Then, when the bright image of the floating particles crosses the grid lines, the amount of light transmitted through the grid decreases, so the output of the light-receiving element fluctuates periodically as the floating particle image moves on the grid. Therefore, by frequency-analyzing the output of this light-receiving element, the electrophoretic mobility of suspended particles can be determined. In this method, if there are particles other than the target particles mixed in with the suspended particles and their mobility is close to that of the target particles, it is not possible to distinguish between the two, and cells such as white blood cells are large Therefore, since the image on the grating is formed across several grating lines, the amplitude of periodic fluctuations in the grating transmitted light due to the movement of the image becomes small, and the S/N ratio decreases. The present invention has been made for the purpose of providing a solution to such problems.

In the present invention, suspended particles are irradiated with excitation light, and the fluorescent light emitted from the suspended particles forms an image of the fluorescent portion of the suspended particles on a grating, slit, etc. For example, cells have a nucleus, and the nucleus contains nucleic acids, which have fluorescence.
Therefore, the image of the fluorescent light emitted from the nucleic acid in the nucleus is smaller than the image of the entire cell irradiated with visible light, and can be easily distinguished from other particles that do not have fluorescent properties. The present invention will be explained below with reference to Examples.

FIG. 1 shows an embodiment of the present invention. Reference numeral 1 denotes an electrophoresis tube with electrodes 2 and 3 inserted at both ends to apply an electric field to the sample liquid within the tube. Reference numeral 4 denotes an ultraviolet light source that irradiates a suitable location within the electrophoresis tube 1 with ultraviolet light using a condensing lens 5. 6 is a projection lens having an optical axis perpendicular to the condenser lens 5, and 7 is a filter installed behind the lens 6 to remove ultraviolet rays and transmit visible light, that is, in this case, fluorescent light emitted from floating particles. It is made of transparent glass. Reference numeral 8 is a grating on which a fluorescent image of suspended particles is formed. Reference numeral 9 denotes a light receiving element placed behind the grating 8, and its output is applied to the signal processing circuit 10.

FIG. 2 shows another embodiment of the invention. 1 is an electric permanent tube, 4 is a light source, 6 is a projection lens,
Other parts corresponding to the respective parts of the structure shown in FIG. 1 are given the same numbers as in FIG. Reference numeral 11 denotes an entrance slit of the spectrometer, the length direction of which is parallel to the moving direction of suspended particles in the electrophoresis tube 1, and the lens 6
An image of the suspended particles is formed on this slit. If the width of the slit 11 is too narrow, there may be a situation where there is no image of floating particles on the slit, but on the other hand, the image of floating particles is small (specifically, 0.01
~0.1 mm), so each image itself can be considered as the entrance slit of the spectrometer, so the width of the slit 11 is made wider than in the case of a normal spectrometer. Reference numeral 12 denotes a concave reflection diffraction grating, and the floating particle image on the slit 11 is separated into spectra by the diffraction grating, and is divided into excitation light and fluorescent light, which are respectively formed on gratings 8 and 8'. These lattices are made up of lattice lines extending parallel to the width direction of the slit 11 and arranged in the length direction of the slit 11. Since the cell particle diffraction image of each wavelength moves on the gratings 8, 8' in accordance with the movement of the suspended particles on the slit 11, the diffraction images of the cell particles at each wavelength move on the gratings 8, 8', and are therefore A change in the amount of light transmitted through 8 and 8' is detected and sent to the signal processing circuit 10.

A modification of the embodiment shown in FIG. 2 will be described.
In this case, the length direction of the entrance slit of the spectrometer is perpendicular to the moving direction of the floating particle image on the slit.
A light receiving element is placed at each wavelength position of excitation light and fluorescence in the diffraction spectrum by the diffraction grating. An optical path polarizer is disposed between the projection lens and the entrance slit of the spectrometer to swing the optical axis of the projection optical system back and forth in a direction perpendicular to the entrance slit of the spectrometer. Although this device is a mirror that can be oscillated within a small angle range, an element such as an electro-optic deflection element that changes the direction of light passing through the element depending on an applied voltage may also be used. With the above configuration, the image of the suspended particles moves over the entrance slit of the spectrometer in accordance with the deflection of the projection optical path. Since light is incident on the spectrometer only when the particle image passes over the entrance slit, the output of the light receiving element has a waveform as shown in Figure 3a in one turn of the projection optical path, and the waveform in the next turn is as follows. During the swing, the particle image moves by the amount of electrophoresis compared to the previous swing, so the output of the light receiving element becomes as shown in Figure 3b. When this is done, the electrophoretic velocity of suspended particles can be determined. That is, the second
If the waveform in Figure b is superimposed on the waveform in Figure 3 and shifted to the left little by little, the waveforms in Figure 3 a and b become the overall shape when shifted to the left by a length corresponding to the electrophoretic movement of the particle image. Therefore, the electrophoresis speed can be determined from the shift amount of the waveform b when the best overlap between the waveforms a and b is obtained.

FIG. 4 shows another embodiment of the present invention. In this embodiment, a filter is used instead of a spectrometer to form a fluorescent image and a scattered light image of floating particles separately on gratings 8 and 8', and 7 and 7' are filters and 6 , 6' are projection lenses for fluorescent images and scattered light images, and other parts are given the same numbers as the respective parts in the embodiments of FIGS. 1 and 2.

In the embodiments described above, images of suspended particles are formed on a grating or slit, but a one-dimensional array detector may be used instead of the grating. A one-dimensional array detector arranges minute unit light-receiving elements in a line, scans the unit elements from one end to extract the output of each unit element, and forms a video signal of the image projected onto the element. be. Therefore, it is also possible to read out the output from the array element at regular time intervals to form a video signal of floating particles, and perform calculations to determine the correlation function between the video signals of one readout and the next readout.

The electrophoresis measurement device of the present invention has the above-mentioned configuration and uses an image of fluorescence emitted by suspended particles, so when the target is a cell, the grid has the same effect as if the cell were reduced to the size of a nucleus. When used, the S/N ratio when determining the electrophoretic velocity is improved, and when target particles that emit fluorescence and non-fluorescent particles are mixed, it is possible to distinguish between the two and determine the electrophoretic velocity. Become.

[Brief explanation of the drawing]

FIG. 1 is a perspective view of essential parts of an apparatus according to an embodiment of the present invention;
FIG. 2 is a perspective view of essential parts of another embodiment of the device of the present invention;
FIG. 3 is a waveform diagram of a video signal of a floating particle image, and FIG. 4 is a perspective view of a main part of still another embodiment of the present invention. 1... Electrophoresis tube, 2, 3... Electrode, 4... Light source, 6... Projection lens, 7... Filter, 8...
Grating, 9... light receiving element, 10... signal processing device,
11... Spectrometer entrance slit.

Claims (1)

[Claims] 1. A device for illuminating the electrophoresis tube, a projection lens system for forming an image of suspended particles in the electrophoresis tube on a grid, slit, or light receiving element, etc., and a device installed before or after the electrophoresis tube and the projection lens system. The spectroscopic means separates the light coming from the suspended particles in the electrophoresis tube into scattered light by the suspended particles and fluorescence emitted from the suspended particles. A cell suspension solution characterized in that an image of a part of the suspended particles that emits fluorescence is formed on a grid, a slit, a light-receiving element, etc., and the velocity of the suspended particles is determined based on the fluorescence detection signal generated by the movement of this image. A method for measuring the electrophoretic velocity of cells in a cell.