RU2335763C1 - Chamber for simultaneous experiments set using microelectrophoresis method - Google Patents

Abstract

FIELD: physics.

SUBSTANCE: chamber for simultaneous experiments set using by electrophoresis method contains number integrated electrophoretic cells each of which has a platform, electrodes, covers and slides, temperature-sensitive element and heaters. Platforms are provided with stepped holes for slides with covers frames on the surface. At that the platform has pockets that are electrode boxes in which electrodes are placed. Electrode boxes are covered with condensing lids. Stepped holes grooves contain attached frames with fixed semipermeable membranes. Besides, the chamber is supplied with holding jigs for fixation on microscope stage and allowing for motion of electrophoretic cells relative to microscope objective.

EFFECT: higher testing accuracy and improved performance.

12 cl, 7 dwg

Description

The invention relates to medicine, as well as to veterinary medicine and microbiology, and is intended for biological studies of cell suspensions and biopsy samples.

The well-known chamber for microelectrophoresis design N. Abramson 1929 (see S. Kharamonenko, A. A. Rakityanskaya "Microelectrophoresis of blood cells in normal and pathological conditions. Minsk." Belarus ", 1974). The known device contains a capillary of rectangular cross section, connected with two compartments in which the electrodes are located. A suspension of cells is placed in a capillary and exposed to it by a constant electric field, causing unidirectional movement of cells having an electric charge.

The disadvantage of this camera is its large volume. In addition, the design of the chamber leads to the formation of pronounced convection fluid flows, causing cell movement. There is no thermal stabilization. The capabilities of the chamber are limited due to the peculiarities of its filling, which allow working only with suspensions of certain types of cells. The design of the camera does not allow for several studies at the same time.

A known camera for microelectrophoresis (Patent RU 2271734 C2), taken as a prototype. The camera has a housing into which a retractable platform with several pairs of electrodes is inserted. The camera has a temperature sensor and a heating device. The platform is equipped with a side that prevents the spreading of liquid. There are stops that fix the distance between the slide and the cover slip.

A disadvantage of the known chamber is that its design does not eliminate liquid evaporation, which leads to a change in current parameters (voltage, current, resistance) and a change in the concentration of substances in the incubation liquid. Uneven evaporation of liquid from the chamber leads to the appearance of unidirectional movement of cells, not associated with the influence of an electric field and interfering with the experiment. The rapid evaporation of liquid from the chamber, especially when operating in thermostabilization mode, makes it impossible to experiment for more than 5-7 minutes without adding new portions of liquid. The accumulation of bubbles of gaseous products of electrolysis between the slide and the cover glass leads to a change in the direction of the electric field lines and a change in the direction of movement of the cells. In addition, bubbles can lead to the disappearance of the contact between the electrode and the incubation fluid located between the coverslip and the glass slide (i.e., to break the electrical circuit) and the camera to stop functioning.

It is not possible to reconfigure the distance between the coverslip and the slide during operation, depending on the needs and conditions of the study. There is no possibility of conducting several experiments in parallel, which makes the camera unsuitable for the purpose of screening studies.

The camera does not allow working with cells with high adhesive properties (for example, with cells of some lymphosarcomas and leukemia, fibroblasts, cells of certain epithelia).

The possibility of replacing the electrodes is not provided.

The objective of the claimed invention is the creation of a reliable device that improves the accuracy of research conducted with its help and has advanced functionality.

The problem is solved due to the fact that in the chamber for simultaneous series of experiments by electrophoresis, there are several electrophoretic cells combined in one block, each of which has a platform, electrode compartments, electrodes, coverslips and slides, a temperature sensor and a heating device; stepped openings are made in the platforms, in which slides are mounted, and frames with coverslips are mounted on them; recesses are made in the platform, which are electrode compartments in which the electrodes are placed; electrode compartments are equipped with condensing covers; frames with fixed semipermeable membranes are fixed in the slots of the stepped holes, in addition, the camera is equipped with a device that secures it to the microscope stage and allows the electrophoretic cells to be moved relative to the microscope objective.

At least 2 variants of the camera device are possible: 1) electrophoretic cells can be arranged in parallel in a row. 2) electrophoretic cells can be located radially around a common axis of rotation.

The camera has 2 systems for supplying current to electrophoretic cells, which allows it to be supplied either to all electrophoretic cells of the camera, or to some of them, or only to that electrophoretic cell, which is currently located under the microscope objective.

The use of the claimed invention improves the accuracy of the research, as well as several times increase productivity.

The combination of several electrophoretic cells in one block, alternately at certain intervals supplied under the microscope objective, allows a series of experiments to be carried out simultaneously. Due to the presence of electrode compartments filled with liquid and the presence of condensing caps, the effect of evaporation is reduced. The fluid level in the electrode compartments is above the fluid level between the slide and the cover glass. Even if some of the liquid from the electrode compartments evaporates, this will not affect the volume of liquid located between the slide and the cover glass, the contact of the liquid with the electrodes, or the constancy of the current parameters in the chamber. Due to the fact that there is a sufficiently large volume of liquid in the electrode compartments, the evaporation of a certain amount of it will not lead to a significant change in the concentrations of substances that make up the incubation medium. The electrode compartments have the same shape and size, the same capacities of the heating elements contained in them, the areas from which the liquid evaporates are equal, therefore, the evaporation of liquid from them occurs identically and does not lead to the appearance of flows and the appearance of cell movement unrelated to the influence of an electric field. The simultaneous filling of the electrode compartments with equal volumes of liquid does not lead to the appearance of fluid flows at this moment. The peculiarities of the arrangement of the electrodes do not allow bubbles of gaseous products of electrolysis to accumulate between the slide and the cover glass and interrupt the electrical circuit. Using microscrews allows you to precisely adjust the gap between the slide and the cover slip and, if necessary, change it during the experiment. The location of part of the heating elements directly in the liquid (in the tanks) contributes to more effective thermal stabilization. Due to the presence in the slide of the holes in which the tubes are fixed, the possibility of additional introduction of various solutions and suspensions of cells into the chamber is achieved.

When using slides and coverslips made of materials capable of transmitting light to the desired part of the spectrum, it becomes possible to conduct studies using fluorescence microscopy using various labels with different emission spectra.

The manufacture of slides and coverslips from materials with low sorption ability will allow to avoid losses (due to adsorption) of substances used in experimental conditions in low concentrations.

The use of glass slides and coverslips made of materials with low adhesive properties makes it possible to work with cells that have high adhesion to glass and other materials.

The implementation of slides and coverslips removable makes them easily replaceable and dramatically expands the possibilities of using the camera, and if necessary, greatly simplifies its repair.

The use of replaceable sets of electrodes of various shapes and sizes, made of materials having the necessary properties, allows you to expand the possibilities of using the camera and simplifies its repair.

The use of electric resistance regulators makes it possible to create the same (or specified by the experimental conditions) current strength in electrophoretic cells, which is important in some cases. In addition, when several electrophoretic cells are connected in parallel, the total electrical resistance of the system turns out to be several times lower than that of a single electrophoretic cell, while an increase in the current strength will lead to failure of the current source designed to work with one cell. Therefore, the regulation of electrical resistance is necessary.

Variants are possible when each electrophoretic cell of a chamber can have not one, but two or more pairs of electrodes. In this case, the current is supplied alternately between each pair of electrodes. Such a solution allows studies on the effect of cytoskeleton structures on the amplitude of vibrations of nuclei and sections of cell cytolemma immobilized between the subject and cover glasses of the electrophoretic cell.

All of the above contributes to increasing the reliability of the device, expanding the possibilities of its use in the above areas, increasing reproducibility and comparability of research results.

The claimed device is illustrated by drawings, where:

figure 1 presents a longitudinal section of a camera with a linear arrangement of electrophoretic cells, mounted in a slide device;

figure 2 presents a top view of a camera with a linear arrangement of electrophoretic cells, mounted in a slide device;

figure 3 presents a longitudinal section of a slide device;

figure 4 presents a top view of the slide device;

figure 5 presents a side view in section of a chamber with a radial arrangement of electrophoretic cells;

figure 6 presents a top view of a camera with a radial arrangement of electrophoretic cells, mounted on a microscope stage;

in Fig.7 presents a side view in section of a camera with a radial arrangement of electrophoretic cells, mounted on a microscope stage.

The claimed device contains integrated into a single block platform 1 (1, 5), in each of which there are electrode compartments 2, in which the electrodes are located 3. The platform has a step opening 4, on the step 5 of which a glass slide 6. is mounted on which is fixed thermal sensor 7 (figure 2, 6). Top frame 4 is inserted into the hole 4 (Figs. 1, 5) with a cover glass 9 mounted on it.

The side walls of the frame 8 are tightly attached to the surface of the hole 4 of the platform 1, which makes it impossible to get liquid between the walls of the frame and the protrusions of the housing, and also prevents the floating of the frame 8 with the cover glass 9 when filling the chamber with liquid. If necessary, the frame 8 with a cover glass 9 can be rigidly fixed using the fixing screws 10 (Fig.2, 6). Microscrews 11 are mounted in the frame 8 (FIGS. 2, 6), which regulate the distance between the cover glass 9 (FIGS. 1, 5) and the slide 6. The electrode compartments 2 are closed by condensing covers 12 having one or more holes 13 designed for yield of gaseous products of electrolysis. In figure 2, 6, the condensation caps are conventionally not shown. The heating elements 14 (figures 1, 2, 5) are located in the tanks 2 and under the slide 6. Through the holes in the platform 1 to the electrodes 3 are suitable contact screws 15 (figure 2, 6), which also provide fastening of the electrodes. Power supply of the heating elements 14 (Figs. 1, 2, 5) and wires coming from the temperature sensor 7 (Figs. 2, 6), as well as wires going to the contact screws 15, are not conventionally shown in the drawings. In the slide 6 (FIGS. 1, 5) there are openings 16 in which tubes 17 are fixed, through which additional solutions and suspensions of cells can be introduced into the chamber during operation if necessary. The electrode compartments 2 are separated from the space between the slide and the cover glass by semi-permeable membranes 18, fixed in removable frames 19. The presence of semi-permeable membranes prevents cells from entering the electrode zones from the working area and allows only molecules (or ions) to pass, the size of which does not exceed the pore diameter of the membrane .

A variant of the device is possible when each electrophoretic cell can have not one but several pairs of electrodes, each of which is located in its own electrode compartment.

To supply current only to the electrophoretic cell, which is currently under the microscope objective, additional contacts 20 are mounted below on the platform 1 and connected to the electrodes 3 (Figs. 1, 5). In the embodiment with a radial arrangement of electrophoretic cells, the additional contacts 20, when the camera receives the corresponding positions, come into contact with the contact pads 21 (Fig. 7), mounted on the microscope stage and connected by wires 22 to the current source. In the embodiment with a linear arrangement of electrophoretic cells (Fig. 1), additional contacts 20, when the camera receives the corresponding positions, come into contact with the contact pads 23 (Figs. 1, 3, 4) located on the slide device 24 and connected to the current source by wires 25. The slide device 24, mounted on a microscope stage, has an opening 26 (FIGS. 3, 4) through which light from the microscope illuminator passes.

The current is supplied simultaneously to all electrophoretic cells of the camera in a variant with their radial arrangement through a switching device 27 (Fig. 7) mounted on an axis 28 around which the entire chamber rotates (Figs. 5, 7). The axis 28 itself is mounted in the hole 29 (Fig. 7) made on the microscope stage. The rotation of the chamber around the axis 28 (Fig. 5) is provided by bearings 30. The switching device 27 (Fig. 7) consists of contact bearings 31 (Fig. 5), insulating spacers 32, insulating washers 33 and contact bushings 34 to which wires 35 are connected coming from a current source. On the axis 28, the switching device 27 is fixed with a nut 36. The switching device is externally covered by a protective casing 37 having holes 38 through which the contact screws 39 pass. Resistors 40 are mounted on the camera platforms 1 (Figs. 5, 6). The wires going from the contact screws 39 to the resistors 40 and from them to the electrodes 3 are conventionally not shown in the drawing.

In a variant with a linear arrangement of electrophoretic cells, the current supply from the source to all electrophoretic cells of the chamber is ensured by connecting them in parallel using wires that must be attached to the contact screws 15 and are not conventionally indicated in figure 2. Adjustment of electrical resistance in each of the electrophoretic cells can also be carried out using resistors or rheostats, additionally introduced into the electrical circuit of the camera. In figure 1, 2 to simplify the drawing, they are not shown.

The cell electrophoresis chamber operates as follows.

In electrophoretic cells with removed condensing covers 12 (Figs. 1, 5) and frames 8 with coverslips 9, the required volumes of cell suspension or the necessary volumes of liquid are placed into the slide 6 with a pipette or syringe into which samples of biopsy material are placed. It is advisable to preheat this liquid or suspension to the temperature at which the study will be conducted. Then, frames 8 with cover glasses 9 are inserted into the slots of the stepped holes from above. In this case, the liquid should completely fill the space between the slide 6 and the cover glasses 9, a sign of which is the entry of minimal amounts of liquid into the tanks 2 (if necessary, this small excess can be easily removed ) The distance at which the coverslip 9 can be brought closer to the slide 6 is set by microscrews 11 (FIGS. 2, 6) before starting work, but can be changed if necessary after filling the chamber with liquid during operation. Frames 8 (Figs. 1, 5) with coverslips 9 can be rigidly fixed relative to the platform using fixing screws 10 (Figs. 2, 6). In the electrode compartments 2, frames 19 (Figs. 1, 5) with membranes 18 are installed.

Then at the same time strictly identical volumes of liquid are introduced into all electrode compartments 2 of the electrophoretic cell. Tanks 2 are closed on top with condensing caps 12. It is advisable that the liquid introduced into the electrode compartments be preliminarily brought to the required temperature.

The camera with the studied objects is placed on the microscope stage and connected to a current source, for example, the Biotest device from the Cyto-Expert microelectrophoresis device included in the register of medical devices (RF certificate dated 14.06.05 No. FS 022a 2005, 174405 ), or another similar device.

Depending on the objectives of the experiment, a current supply mode is selected, which can be supplied either to all electrophoretic cells of the camera, or to some of them, or only to that electrophoretic cell, which is currently under the microscope objective. To supply current to all electrophoretic cells of the chamber with their radial arrangement using wires, the contact screws 15 (Fig. 6) (adjacent to the electrodes 3) are connected by wires to the contact screws 39 (Fig. 5) of the switching device 27 (Fig. 7), which in turn, wires 35 (figure 5) is connected to a current source. To supply current to the chamber with a linear arrangement of electrophoretic cells, the latter using wires are connected to each other in parallel through the contact screws 15 (figure 2), after which it is connected to a current source. If a current supply is not needed in one or several cells under the conditions of this experiment, the wires going to the corresponding electrodes are disconnected (in both considered variants of the device). As already mentioned above, resistors (rheostats) can be introduced into the electrical circuit.

When applying current only to the electrophoretic cell, which is currently under the microscope objective, in the version of the camera with a linear arrangement of electrophoretic cells, the current from the source is supplied via wires 25 (Fig. 4) to the contact pads 23 of the slide device 24, or, in variant with a radial arrangement of electrophoretic cells, the current is supplied via wires 22 (Fig.7) to the contact pads 21 located on the microscope stage.

When using a variant of the camera, the electrophoretic cells of which have more than one pair of electrodes, the current is supplied between the pairs of electrodes alternately in the order specified by the Biotest device settings from the Cytoexpert microelectrophoresis device complex (RF certificate dated 14.06.05 No. FS 022a 2005 , 174405) or other similar device.

After connecting the camera to the current source, the electric voltage between the electrodes is measured (for this it is necessary to remove the condensing covers). The value of the applied voltage is adjusted.

If necessary, using resistors 40 (Fig.6) achieve the same (or necessary for the purposes of the experiment) electrical resistance in all electrophoretic cells of the chamber. It is advisable to carry out the adjustment in a preliminary experiment with filling the cells with appropriate solutions before starting the main experiment.

Then the thermostabilization mode is turned on and the temperature of the liquid in the chamber is brought to the necessary.

Monitoring the movement of objects in the camera can be done through a microscope visually, or video recording or other data recording can be carried out. After the observation (video shooting) is completed, in one electrophoretic cell, the next electrophoretic cell is fed under the lens and the process is repeated. The movement is achieved by turning the device to the required angle if the radial version of the camera is used, or by moving the device according to the slide mechanism, if the linear version of the device is used. Thus, during the experiment, the same camera will visit the microscope several times. This is convenient because it will allow you to conduct a series of observations (videos) at the required intervals and track the process in dynamics. Without deterioration in the quality of the results, it becomes possible to simultaneously conduct a series of experiments by observing (visual or video) cells located in different cells.

If necessary, various solutions or cell suspensions can be additionally introduced into a particular electrophoretic cell through tubes 17 (FIGS. 1, 5). In some studies, it may be necessary to change the distance between the slide 6 and the cover glass 9. For this, the current supply to the electrodes of the chamber is stopped, then by turning the screws 10 (FIGS. 2, 6), the frame 8 with the cover glass 9 is released (FIG. 1, 5) from the rigid fixation and using the microscrews 11 (FIGS. 2, 6), the desired distance is established between the slide 6 (FIGS. 1, 5) and the cover glass 9. After that, the frame 8 with the cover glass 9 is again fixed with screws 10 (Fig.2, 6). Then, in order to standardize the results, the changed resistances are brought by the resistors 40 (Fig.6) to the desired value, after which the experiment can be continued. After the end of the study, the camera is disconnected from the current source, removed from the microscope stage, the condensing covers 12 are removed (Figs. 1, 5), the frames 8 with coverslips 9 are released from the screws 10 (Fig. 2) and removed from the platform 1 (Fig. .1, 5), are removed from the electrode compartments 2 of the frame 19 with semipermeable membranes 18. Then, the chamber and all its removable parts are washed with water and detergents. Then they are disinfected by placing chlorhexidine or another disinfectant in a solution that does not damage the chamber material for the time specified in the instructions for its use. After disinfection, the chamber is washed several times with distilled water and dried. In this case, drying in a stream of warm air is allowed.

Claims (12)

1. A chamber for conducting a series of experiments by electrophoresis, containing several electrophoretic cells combined in one unit, each of which has a platform, electrodes, coverslips and glass slides, a temperature sensor and heating devices, characterized in that stepwise openings are made in the platforms, in which slides are installed, and frames with coverslips are mounted on them, and, in the platform, recesses are made, which are electrode compartments in which the electrodes are placed The electrode compartments are equipped with condensing caps, frames with fixed semipermeable membranes are fixed in the slots of the stepped holes, in addition, the camera is equipped with a device that secures it to the microscope stage and allows the electrophoretic cells to be moved relative to the microscope objective. 2. The camera according to claim 1, characterized in that the platforms are arranged in parallel in a row. 3. The camera according to claim 1, characterized in that the platforms are located radially around a common axis of rotation. 4. The camera according to claim 1, characterized in that microscrews are installed in the frames of the coverslips to regulate the distance between the slide and the coverslips. 5. The camera according to claim 1, characterized in that the slides and coverslips are made of materials that transmit light to the desired part of the spectrum, for example, ultraviolet radiation. 6. The camera according to claim 1, characterized in that the slide has holes with tubes fixed to them for additional administration of solutions and suspensions of cells. 7. The chamber according to claim 1, characterized in that the slides and coverslips can be made of a material having low adsorption properties, for example polypropylene. 8. The chamber according to claim 1, characterized in that the slides and coverslips can be made of a material having low adhesive properties. 9. The camera according to claim 1, characterized in that the slides and coverslips are removable. 10. The chamber according to claim 1, characterized in that the electrodes are interchangeable and can be made of materials with different properties and have different shapes and sizes. 11. The camera according to claim 1, characterized in that it is equipped with a system for adjusting the electrical resistance of each electrophoretic cell. 12. The camera according to claim 1, characterized in that it has 2 current supply systems that allow it to be supplied either to all electrophoretic cells of the camera, or to some of them, or only to that electrophoretic cell, which is currently located under the microscope objective.

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