RU2070919C1 - Method for electrooptical analysis of cells in suspension and device for its embodiment - Google Patents

Abstract

FIELD: biotechnology, microbiology, medicine. SUBSTANCE: method for electrooptical analysis of cells in suspension includes determination of value of anisotropy of polarizability for separate fractions of cells comprising heterogeneous population used for evaluation of morphometric and physiological characteristics of these fractions. For this purpose, in course of application of radiopulse of electric field formed by generator 12 with the help of microprocessor 30 to studied cell suspension placed in electrooptic cell 1, with simultaneous passage through it of orthogonal light fluxes with the aid of pulse light sources 2 and 3, direction of vector of electric field is varied by means of electrode switch gear 15 relative to direction of light flux discretely through 90 deg with frequency F which is also varied within the range of minimum Fmin to maximum Fmax with simultaneous variation of magnetic field within the range of Emin to Emax with the help of attenuator 13 connected with paraphase amplifier 14. Values of Fmax, Fmin, Emax and Emin are determined experimentally depending on the kind of tested culture of cells. Experimental signal from tested suspension, determined as difference between light fluxes from light sources 2 and 3 is received by photodetector 9 connected with voltage converter 9 into frequency whose outputs are connected with inputs of frequency counters 10 and 11 and registered in microprocessor 30 and are used for calculation of the value of anisotropy of cell polarizability. EFFECT: higher efficiency. 11 cl, 10 dwg

Description

 The invention relates to the field of biotechnology, microbiology and medicine, and more particularly relates to a method for electro-optical analysis of a cell suspension and a device for its implementation, used to determine the morphometric and electrophysical parameters of cells and the physiological characteristics of cells associated with them.

 In the microbiological, food, medical and several other industries using biotechnological processes, the task is to determine the physiological and morphometric indicators of the state of the cells, reflecting the quality and effectiveness of the biotechnological process. Traditional microbiological methods for determining the morphometric parameters of cells by microscopy and determining physiological characteristics: viability by plating cells on nutrient media and specific growth rate by periodically counting cells in a counting chamber do not meet modern requirements due to lack of efficiency.

 Known methods for optical analysis of cell parameters, for example, based on the analysis of images obtained using a video camera (WO 89/03431). Image analysis allows you to determine the morphological characteristics of cells, but does not allow to determine the physiological characteristics of cells.

 A known method for determining the volume and concentration of cells (Cell analysis, 1982, v.1, p. 195-331), based on measuring the time dependence of the change in current flowing through a calibrated hole when the cell under study passes through it. A significant error of the method is associated with the interpretation of a signal from two or three cells closely following each other as a signal from one larger cell.

 A known method for determining the relative concentration of viable cells based on measuring the fluorescence of cells after they have been worked out by a fluorescent label (Microbiology, 1988, v. 57, No. 2, p. 347-351), requiring mandatory calibration and having a significant error due to an uncontrolled number of sites planting a fluorescent label.

 The task of quickly determining the physiological state of cells and at the same time their morphological parameters allows us to solve the method of electro-optical analysis of cells, based on the study of changes in light scattering when they are oriented in an electric field.

 The method of electro-optical analysis is based on the Kerr effect. The essence of the effect is to change the optical properties of the suspension, in particular the suspension of cells, when exposed to an electric field.

 The action of an electric field on a suspension causes the appearance of induced charges on suspended cells. Their distribution and magnitude are determined by the current polarizability mechanism (Dukhin, Electroloctics of colloids, Naukova Dumka, Kiev, 1975). In the case of the volume polarizability mechanism, induced charges appear at the interfaces between two media with different complex permittivities. The boundaries of the cell structures are the contact surfaces of the double electric layer and the cell wall, cell wall and cytoplasmic membrane, cytoplasmic membrane and cytoplasm. The magnitude of the charges induced at the interfaces is proportional to the electric field strength and depends on the ratio of the dielectric constants of the structures. An integral parameter describing the process of polarizability is the tensor a of the polarizability of the cell. The polarizability tensor of a nonspherical cell has at least two distinct components. The longitudinal component coincides with the length of the axis of the cell, and the transverse orthogonal to it. The induced charges of different signs distributed over the cell volume form a dipole. The dipole interacts with the electric field and causes the appearance of torque. A prevailing direction of cell orientation arises, which changes the equally probable distribution of cells over the orientation angles described by the Boltzmann function. The cell orientation angle is measured from the direction of the incident light flux to the direction of the long axis of the cell.

 In this case, there is a change in all optical characteristics of the suspension: birefringence, the nature of light scattering and the optical density of the suspension due to changes in the scattering cross section G (t). With an electric field strength E corresponding to a weak orientation of the cells, the dependence of the change in optical density on the square of the intensity is linear.

 Depending on the frequency of the electric field and the electrical properties of the cells, the direction of the prevailing orientation of the cells may coincide with the direction of the vector of the electric field or be orthogonal to it. Orientation of the cells along the long axis along the luminous flux will lead to an increase in the scattering cross section and a decrease in the intensity of the luminous flux, while orientation across the luminous flux will decrease it and, accordingly, increase the luminous flux.

 There is a method of electro-optical analysis of cells in suspension (SU, A, 469748), which consists in the fact that a light flux is passed through the suspension sample with simultaneous application of a fixed frequency and intensity electric field to the suspension of a radio pulse, the lines of force of which are directed along the light flux. The frequency of the electric field varies from pulse to pulse. At each frequency, the optical density of the suspension is measured in the absence and presence of an electric field. The degree of electrical orientation of the cells is determined by the change in optical density, according to which their electrophysical characteristics are judged, for example, the magnitude of their polarizability anisotropy associated with the complex permittivities of individual cell structures, and through them with the physiological characteristics of the cell.

 A device for implementing this method is known (SU, A, 469748), containing an electro-optical cell with a sample of the studied cell suspension, a light source that generates a light flux transmitted through the cell, a photodetector, a harmonic voltage generator and a device for controlling the operation of these elements. The device contains a system for supplying and discharging the test suspension.

 Using this method to analyze a suspension of cells of heterogeneous size allows one to determine only the size-average anisotropy of the polarizability of cells for the entire population and does not allow to determine this parameter for individual cell fractions with different sizes that make up this population.

 In addition, the sedimentation and flocculation of cells arising in the test suspension, as well as fluctuations in the density of the suspension, significantly reduce the sensitivity and accuracy of determining the increment of optical density, from which the polarizability anisotropy is determined. These disturbing factors are especially pronounced at a low signal level, which is typical for cell orientation in the high frequency range of the electric field. The appearance of these factors is due to the fact that the measurements are carried out in one luminous flux and the resulting density fluctuations, sedimentation and flocculation are not compensated. The measurement errors associated with the instability of the light source and the photodetector (flicker noise) are also large.

 These errors are partially eliminated by using a two-beam scheme for recording changes in the optical density of a suspension with mechanical switching of the light flux from the reference channel to the measuring one (Biotechnology, 1989, N 2, p. 214-215).

 The method of electrical analysis of cells in suspension (SU, A, 913169), which consists in the fact that the light flux is passed through two studied samples of a suspension of cells with the simultaneous application of a radio pulse of an electric field of a fixed frequency and intensity vector, allows to achieve higher accuracy and sensitivity in determining the electrical orientation of cells. which for one sample is parallel to the light flux, and for another, it is perpendicular (orthogonal) to the light flux. The frequency of the electric field, as in the previous case, is also changed from pulse to pulse. The intensity of the light flux passing through the test suspension and characterizing the optical density of the suspension simultaneously in both samples is measured. The difference in these values determines the magnitude of the anisotropy of the polarizability of the cells, according to which their electrophysical characteristics are judged.

 For cells of similar size, the change in optical density is proportional to the square of the electric field strength to a certain threshold level. This level corresponds to the transition from a weak degree of orientation of cells to a strong degree of orientation and is preferred to achieve sufficient measurement accuracy. Therefore, at the beginning of research, experimentally determine the magnitude of the tension at which the cells go from a weak to a strong degree of orientation, and then all measurements are carried out without changing the electric field strength.

 The method is implemented in a device (SU, A, 913169) containing two electro-optical cells with two flat electrodes in each cell, which are filled with the test suspension, a light source that creates two light fluxes, one of which is passed through one cell, the other through the second, and accordingly, two photodetectors with a difference amplifier for determining the difference between the intensity of the light fluxes, as well as a voltage generator. The direction of the electric field vector in one cell is parallel to the light flux in this cell, and the direction of the electric field vector in another cell is perpendicular to the light flux in this cell.

 This method can also be used only for analysis of a homogeneous suspension of cells, since it allows you to evaluate only the change in the total optical density of the suspension and, accordingly, the polarizability anisotropy averaged over the size of the cells. The use of two cells and two photodetectors in the device partially compensates for errors associated with cell sedimentation and flocculation, however, it introduces additional errors due to the non-identical design of the cells and the presence of uncompensated low-frequency noise from photodetectors.

 Thus, none of the known methods of electro-optical analysis of cells in suspension provides the possibility of reliable determination of the polarizability anisotropy of each of the uniformly sized fractions of cells that make up a heterogeneous population, i.e., it does not allow to assess the degree of heterogeneity of a population by this parameter.

 The basis of the invention is to develop a method for electro-optical analysis of cells in suspension, which allows to determine fractions with different morphometric and electrophysical parameters in heterogeneous mixtures (i.e., to assess the degree of heterogeneity of a population) by using different degrees of inertia of cells of different sizes when oriented in an electric field with changing parameters and selection of these parameters. Another task is to reduce low-frequency (flicker) noise and compensate for fluctuations in the density of the suspension, fluctuations and sedimentation of cells.

 The basis of the invention is the task of developing a device for implementing the method of electro-optical analysis of cells, allowing to analyze a heterogeneous mixture of cells.

The problem is solved in that a method of electro-optical analysis of cells in suspension is proposed, in which an electric field in the form of pulses is applied to the suspension under study, light flows are passed through it, the intensity of the light flux passing through the suspension is measured, and their difference is determined as an electro-optical signal for the subsequent determination of the frequency dependence anisotropy of the polarizability of cells, in which, according to the invention, when applying each specified pulse, the direction of the vector of electric field relative to the direction of the light flux discretely by 90 ^o with a frequency F, while the frequency F is changed in the range from F _min to F _max so that F _{i + 1} F _1/2 ^M , where i (0, j-1) , or F _i-1 F _i / 2 ^M , where i (j-1,0), M is a positive number.

в случае изменения напряженности электрического поля в сторону ее увеличения, или по формуле

в случае изменения напряженности электрического поля в сторону ее уменьшения, где i (0,j), j 1/M•log₂(F_max/F_min).To more effectively determine the fractions of small cells simultaneously with changing the frequency F of the changes in the direction of the electric field vector, the electric field strengths E are discretely changed by 90 ^° in the range from E _min to E _max so that

in case of a change in the electric field strength in the direction of its increase, or by the formula

in the case of a change in the electric field strength in the direction of its decrease, where i (0, j), j 1 / M • log ₂ (F _max / F _min ).

The values of F _min , F _max , E _min , E _max determined experimentally, depending on the type of the studied cell culture.

 The method can be implemented when programming the parameters of the radio pulses to change the frequency of changes in the direction of the vector of the electric field and electric field strength either in the direction of decreasing these values, or in the direction of increasing them.

In the case of a change in the frequency of the direction of the electric field vector decreasing, the initial maximum frequency value F _max is determined experimentally for each culture by the appearance of the electro-optical signal, and the value of F _min is determined by the disappearance of changes in the stationary value of the electro-optical signal; the value of E _{max is} determined corresponding to the threshold level of a weak degree of orientation of small cells, and the value of E _min is determined by reaching a threshold level of a weak degree of orientation of large cells.

If the frequency F increases upward, the minimum value of F _{min is} selected corresponding to the absence of changes in the stationary value of the electro-optical signal, the maximum value of frequency F _max is determined by the disappearance of the electro-optical signal, the value of E _{min is} chosen corresponding to a weak degree of orientation of large cells, and the value of E _max is determined by reaching threshold level of a weak degree of orientation of small cells.

It was experimentally determined that when implementing the method with programming the electric field supply pulses, changes in the direction of the electric field vector by 90 ^° should be performed in an amount of at least three times.

 The task of reducing low-frequency (flicker) noise and compensating for fluctuations in the density of the suspension, fluctuations and sedimentation of the cells is solved by the fact that the test suspension is taken in the form of an elementary volume (one test sample), through which two light fluxes orthogonal to each other are passed alternately.

 The tasks are solved in that the proposed device for implementing the proposed method of electro-optical analysis of cells, containing an electro-optical cell equipped with translucent windows located opposite each other and placed inside it by electrodes, a light source optically coupled to a photodetector connected to a unit for determining the difference between the intensity of transmitted through a suspension of light fluxes as an electro-optical signal and a pulse generator of electro-optical voltage, in the torus according to the invention, the electro-optical cell is made with four translucent windows and at least four electrodes in the form of rods mounted vertically between said windows, the device being provided with an additional light source also optically coupled to said photodetector, both sources being pulsed, as well as sequentially interconnected by an attenuator of electrical voltage with the ability to control the magnitude of the electrical voltage, paraphase rer and a switch electrodes connected to the generator voltage, the input of paraphase amplifier connected to the output of the attenuator and its output is connected to two diagonally arranged electrodes and input electrodes of the switch, the outputs of which are connected with the two other diagonally arranged electrodes.

 To increase the optical thickness of the suspension sample under study, which leads to an increase in the accuracy of measuring changes in the intensity of light fluxes, the electro-optical cell is made with nine electrodes mounted inside the specified cell at an equal distance from each other in three rows of three electrodes in each row and the distance between adjacent electrodes in the row is equal to the distance between adjacent rows, and the middle electrode of the middle row is central, while the extreme electrodes of the first and third rows are connected to These paraphase amplifier output, a central electrode is connected to the other output of paraphase amplifier, and the remaining pairwise opposing electrodes connected to the output electrode of the switch.

 The unit for determining the difference between the intensity of the light flux can be made in the form of a voltage to frequency converter, the output of which is connected to the inputs of two strobe frequency counters.

 The electro-optical cell can be made in the form of a parallelepiped with a square base.

The optical connection of the light sources with the photodetector can be made in the form of four mirrors, one of which is mounted on the axis of the light flux generated by one light source, the other on the axis of the light flux generated by another light source, and the other two are located between the previous mirrors and mounted relative to each other friend at an angle that ensures the fall of these light fluxes on the photodetector at an angle of 90 ^o .

Figure 1 shows the change in the magnitude of the electro-optical signal as a function of time when exposed to a test suspension of one radio pulse of electric voltage with a programmed change in the parameters of the electric field, where
a) a spatial change in the direction of the field vector by 90 ^o relative to the zero point in time with the number of changes equal to five for each frequency F, shown by arrows and time diagram;
b) a change in the field strength E during one radio pulse of the electric voltage simultaneously with a change in the frequency F of the changes in the direction of the field vector by 90 ^o with the number of changes in the direction of the vector equal to five;
c) a change in the magnitude of the electro-optical signal when exposed to a radio pulse of an electric field with a change in its parameters in accordance with graphs a) and b).

In FIG. 2 shows the dependence of the electro-optical signal on the number of changes in the direction of the electric field vector by 90 ^o ; figure 3 is a structural diagram of a device; figure 4 electro-optical cell with four electrodes in isometry; figure 5 diagram of the connection of the electrodes to the electrode switch and to the paraphase amplifier:
a) for a cell with four electrodes;
b) for a cell with nine electrodes.

Figure 6 shows the electro-optical signal from a suspension of E. coli cells with five changes in the direction of the electric field vector by 90 ^° for each of the four frequencies F of changes in the direction of the vector and intensity E (for subsequent calculations, the signal obtained after three changes in the direction of the vector is used)
a) at the frequency of filling the radio pulse f ₁ 1 kHz;
b) at a frequency of filling the radio pulse f _{2 of} 8.4 MHz.

7 shows the size distribution of E. coli cells
a) obtained as a result of processing an optical signal at a frequency f ₁ 1 kHz;
b) obtained as a result of processing micrographs of cells in an amount of 2500 pcs.

In FIG. 8 the dependence of the polarizability anisotropy on the size of E. coli cells at a frequency f ₂ filling the radio pulse normalized to the value of the polarizability anisotropy obtained at a frequency f ₁ .

In Fig. 9, the electrical signal from a suspension of Bac.subtilis cells with five changes in the direction of the electric field vector by 90 ^° for each of the four frequencies F of changes in the direction of the vector and intensity E (for subsequent calculations, the signal obtained after three changes in the direction of the vector)
a) at the filling frequency of the radio pulse f ₁ 1 kHz;
b) at the filling frequency of the radio pulse f ₂ 4.2 MHz.

In FIG. 10 shows the dependence of the polarizability anisotropy on the size of Bac.subtilis cells at the frequency f _{2 of} filling the radio pulse normalized to the polarizability anisotropy obtained at the frequency f ₁ .

It is known that the process of changing the scattering cross section G (t) of cells during exposure to an electric field and the associated change in the optical density of the suspension begins with a relaxation phase and goes into the stationary phase. For a fraction of cells with a size of b ₁ and a weight coefficient A _i it is described by the ratio:
dG _i (t) A _i • (1-exp (-6 • D _i • t)),
where A _i N _i • dQ _i • da _i ;
N _{i the} number of cells i size;
dQ _i increment of the scattering cross section of a single cell of size i after completion of its orientation;
D _i rotational diffusion coefficient for cells with size b _i ;
da _{i the} anisotropy of the polarizability of the cells of the i fraction, equal to the difference between the longitudinal and transverse components of the polarizability tensor a;
t is the duration of exposure to an electric field of constant direction, fixed intensity and frequency.

The stationary phase corresponds to the completion of the cell orientation process as t → ∞. Due to the linear dependence of the polarizability anisotropy of the cells on their volume, measurements at the threshold level of tension E _max for small cells lead to a strong degree of orientation of large cells and a distortion of their contribution to da _i (Landau L.D. Lifshits E.M. Wednesdays, M. Gostekhizdat, 1957, pp. 14-28). Due to the significant dependence of the increment of the scattering cross section of the cells on their size (not lower than cubic), performing measurements with a weak degree of orientation of large cells leads to an insignificant contribution to the total electro-optical signal of cells with small sizes, i.e., the contribution of a small number of large cells suppresses the contribution from a large number of small cells.

The essence of the invention lies in the fact that in the analysis of a cell population heterogeneous in size, methods were used to measure the strength and direction of the electric field vector with a variable frequency F within a single radio pulse of the field effect, allowing to distinguish the increment of the scattering cross section for small cells against the background of a significant change in the scattering cross section of large cells due to the difference in the nature of the optical signals from cells of various sizes when they are oriented in an electric field, the direction (vector) cat It is changed by 90 ^° with a variable frequency F. The reason for this difference is a different degree of inertia of cells of different sizes, determined by the value of the coefficient of rotational diffusion. Moreover, when changing the direction of the vector with a frequency F _max (for the case of changing the frequency F in the direction of its decrease), small cells are in a stationary state before the direction of the electric field vector changes by 90 ^o , and larger cells are in a relaxation state. A change in the electric field strength E simultaneously with a change in the frequency F of changes in the direction of the vector by 90 ^° allows us to increase and isolate the optical contribution of small cells that are stationary with a weak degree of orientation from the contribution of large cells that are stationary with a weak degree of orientation, from the contribution of large cells in a relaxed state with an average or strong degree of orientation of these cells.

 Thus, the presence of these techniques makes it possible to single out the contribution made to the total change in the optical density of each fraction of cells, and, therefore, to determine the scattering cross section for a model representation of either the polarizability anisotropy for each fraction of cells of the same size with a known size distribution, or the size distribution with a known model dependence of polarizability anisotropy on sizes.

The program of changes in the direction of the vector of the electric field, its intensity E and frequency F, changes in the direction of the vector within each radio pulse of the field effect is presented in Fig. 1, a) and b) for the case of a five-fold change in the direction of the vector by 90 ^o for any frequency F.

 With this programming of the parameters of the electric field acting on the cells, the amplitude and shape of the electro-optical signal change (Fig. 1, c), according to which, using the known model ratios for the scattering cross section, the dependence of the polarizability anisotropy of the cells on their sizes is calculated and thereby determine the electrophysical heterogeneity of the suspension.

The proposed method is as follows:
Two streams of light orthogonal to each other are passed through the suspension sample under study with a switching frequency of at least two orders of magnitude greater than the frequency F of changes in the direction of the electric field vector. The number of changes in the direction of the electric field vector by 90 ^o for each value of the frequency F is determined by the achievement of a constant signal G (t) at the stationary orientation phase. Figure 2 presents the results of modeling the effect of the number of changes in the direction of the field vector by 90 • from one to five on the shape of the electro-optical signal with an initial field strength E of zero and the subsequent supply of the field with a strength of E _max and frequency F _{max of} changes in the direction of the field vector. For clarity, the graphs of changes in the shape of the electro-optical signal as a function of time are superimposed on each other (the numbers of the graphs correspond to the number of changes in the direction of the vector). As can be seen from this figure, the influence of various initial conditions on the shape of the electro-optical signal becomes negligible after the third change in the direction of the field vector by 90 ^o .

The frequency F is varied in the range from F _max to F _min so that F _i ± ₁ F _i / 2 ^M , where i (0, j-1), (j-1,0), M is a positive number depending on the desired degree of determination of suspension heterogeneity. The number of steps for changing the frequency is defined as j 1 / M • log ₂ (F _max / F _min ). A decrease in M to 0.1 or less is accompanied by an increase in the accuracy of heterogeneity estimation, an increase in the measurement time, and a complication of the processing algorithm. Hence, the relative change in the frequency F is chosen based on a compromise between the allowable measurement time and the desired accuracy.

при изменении величины Е в сторону ее уменьшения.The magnitude of the electric field E for each value of the frequency F is kept constant. The initial value of the tension E _max at a frequency F _{max of a} change in the direction of the vector corresponds to a threshold level of a weak degree of orientation of small cells. The final value of the field strength E _min corresponds to the level of a weak degree of orientation of the fraction A _{j of} the largest cells. The electric field strength E is varied in the range from E _max to E _min so that

when changing the value of E in the direction of its decrease.

When a suspension is exposed to an electric field with an intensity E _max and frequency F _{max of} changes in the direction of the vector of the electric field, before changing the direction of the field vector, the fraction of small cells A _o with size b _o is stationary, and A _{i the} fraction of larger cells with sizes b _i , where i> 1, in the relaxation phase of orientation. An electro-optical signal after a triple change in the direction of the electric field vector by 90 ^° , defined as the difference between the intensity of the orthogonal light flux transmitted through the suspension under investigation, will consist of linear initial segments of the exponent corresponding to the i fractions and the total exponential signal from the fraction of the smallest cells. The selection of the signal from the fraction o is carried out by excluding from the total signal the linear component from the sum of the fractions starting from the second, for example, by subtracting the constant component of the differentiated signal.

The transition to the next tension E _{1 is} accompanied by a simultaneous decrease in the value of F by 2 ^M times and a decrease in the value of E ² by (E $\genfrac{}{}{0ex}{}{\text{2}}{\text{max}}$ -E $\genfrac{}{}{0ex}{}{\text{2}}{\text{min}}$ ) / j. The linear segment of the optical response from fraction A _{2 is} converted to the full exponential signal (at M = 1) or to its part (at M <1), depending on the value of M. In addition, the electro-optical signal from all fractions decreases by the amount of (E $\genfrac{}{}{0ex}{}{\text{2}}{\text{max}}$ -E $\genfrac{}{}{0ex}{}{\text{2}}{\text{min}}$ ) / j due to the quadratic dependence of the magnitude of the electro-optical signal on E.

The process of isolating the electro-optical signal from the second and subsequent fractions is iterative. At the k step, linear electro-optical signals from fractions A _i are excluded from the total signal, where i k + 1, j. The contribution of the fraction A _k is found by excluding from the stationary value of the signal the contribution of the fractions A _i , where i = 0, k-1. The calculated values of the weighting factors for these fractions with each step of the frequency measurement will decrease by (E $\genfrac{}{}{0ex}{}{\text{2}}{\text{max}}$ -E $\genfrac{}{}{0ex}{}{\text{2}}{\text{min}}$ ) / j.
The accuracy of determining the electro-optical signal for each fraction of a given size increases with a change in the direction of the vector of the electro-optical field due to the symmetry of the signal and the elimination of the error in the choice of the zero reference line, provided that the measurements are carried out on one sample of the suspension under investigation when the light fluxes are orthogonal to each other.

Performing measurements of the magnitude of the electro-optical signal during one radio pulse allows you to determine the set of values of the coefficients A _n } _i for a given frequency of filling of the radio pulse f _i . Carrying out measurements at a number of frequencies allows one to determine the sets of coefficients A _n } _i as a function of the frequency f of the electric field. Normalization of the value of A _n } _i by the value of the optical density of the suspension and the square of the electric field makes it possible to find the value of A _n } _i for each fraction of cells of the same size. Performing measurements on a set of frequencies of the electric field when applying a sequence of radio pulses with different filling frequencies allows us to determine the frequency dispersion of the quantity A _n } _i for each fraction by size.

To determine the physiological characteristics of cells, for example, their internal homeostasis, synthesis of cell structures, etc., the dependences of A _i or polarizability anisotropy on the frequency f of the electric field obtained for each fraction of cells of a certain size are used. The heterogeneity of the suspension according to the indicated physiological parameters is determined by splitting the electro-optical signal obtained for each fraction with a certain cell size into the sum of the signals corresponding to the fractions of cells with different polarization parameters. Such dependences for fractions of viable cells, damaged cells, cells at the sporulation stage, or cells susceptible to various types of influences are obtained in the process of preliminary measurements.

 A device for electro-optical analysis of cells in suspension (Fig. 3) contains an electro-optical cell 1 for the test suspension, in communication with containers for the preparation of the test sample and waste collection, not shown in the diagram, pulsed light sources 2 and 3 and mirrors 4-7, serving for information of the light fluxes from sources 2 and 3 to the photodetector 8 (i.e., to provide optical communication of light sources 2 and 3 with the photodetector 8) connected to the electric circuit with a voltage to frequency converter 9, the output of which is connected to the input E gated counters 11, 10 and frequency. The device also contains a harmonic voltage generator 12, an attenuator 13, the input of which is connected to the output of the generator 12, a paraphase amplifier 14, the input of which is connected to the output of the attenuator 13, and an electrode switch 15, the inputs of which are connected to the outputs of the paraphase amplifier 14.

 The electro-optical cell 1 is made (Fig. 4) in the form of a parallelepiped with a square base, contains four translucent walls 16 and electrodes 17-20, for the case of a four-electrode cell, or electrodes 21-29, for the case of a nine-electrode cell (Fig. 5). The electrodes 17-20 and 21-29 are made in the form of rods and serve to create an electric field in the cell 1. The electrodes 17-20 are installed vertically at the corners of the cell 1 at the same distance from its vertical axis. The electrodes 21-29 are mounted vertically in cell 1 in three rows of three electrodes in each so that the distance between adjacent electrodes in a row is equal to the distance between the electrodes, with the middle electrode 29 of the middle row installed along the vertical axis of cell 1 and is central. In this case, the electrodes 17 and 18 are connected to the outputs of the electrode switch 15, and the electrodes 19 and 20 are connected to the outputs of the paraphase amplifier 14. The electrodes 21 and 22 are connected to one output of the electrode switch 15, and the electrodes 23 and 24 are connected to the other output of the electrode switch 15, electrodes 25-28 are connected to one output of the paraphase amplifier 14, and the electrode 29 is connected to another output of the paraphase amplifier 14.

 The device is equipped with a microprocessor 30 with a common bus 31, to which the control inputs of the generator 12, attenuator 13, switch 15 of the electrodes and pulsed light sources 2 and 3 and the outputs of the gated frequency counters 10 and 11 are connected.

 The device operates as follows. In the electro-optical cell 1 is placed the investigated sample suspension. The control inputs of the pulsed light sources 2 and 3 via a common bus 31 of the microprocessor 30 are supplied with code sequences that specify the intensity of the light flux of each of the pulsed light sources 2 and 3. According to the control signals of the microprocessor 30, next with the switching frequency of the light fluxes, the pulsed light sources 2 and 3 alternately create light fluxes that pass in perpendicular directions through the electro-optical cell 1. At the same time, an electric field with variable parameters is created in cell 1.

According to the control signal from the microprocessor 30, the generator 12 generates a harmonic voltage of a given frequency in the range from 10 ³ to 10 ⁸ Hz in the form of radio pulses with a fixed filling frequency. The voltage is supplied to the control attenuator 13, in which the attenuation coefficient under the control of the microprocessor 30 changes with the transition to the next frequency of the direction of the electric field vector and thus changes the field strength in cell 1. Next, the voltage is supplied to the paraphase amplifier 14, which generates two voltages with phase shift at 180 ^o .

Two paraphase voltages of the paraphase amplifier 14 are simultaneously supplied to the electrodes 17 and 18 or 25-28 and 29 directly, and to the electrodes 19, 20 or 21 and 22, 23 and 24 through the switch 15 of the electrodes. According to the control signals from the microprocessor 30, the switch 15 provides alternately connecting to the electrodes of paraphase voltages with a frequency F changing the direction of the electric field vector, which leads to a change in the direction of the field vector in the electric cell 1 by 90 ^o .

 The electric field in cell 1 causes particle orientation, which leads to an increase in the optical density of the suspension in one direction and a decrease in the optical density in the perpendicular direction, which corresponds to a change in the intensity of the orthogonal light flux generated alternately by light sources 2 and 3, defined as an electro-optical signal. A change in the direction of the electric field vector leads to a change in the orientation of cells and a change in the nature of the change in optical density in these directions to the opposite. The system of mirrors 4-7, the light fluxes are reduced to the photodetector 8. The output voltage of the photodetector 8 is supplied to the voltage Converter 9 to the frequency. The pulses from the output of the Converter 9 are accumulated in the gated counters 10 and 11 of the frequency. The number of pulses corresponds to the intensity of the orthogonal light fluxes transmitted through cell 1. The counters 10 and 11 are read into the microprocessor 30 and, after determining their difference, is used to calculate the selective polarizability anisotropy of cells of various fractions and their morphometric parameters.

 The final signal recorded in the microprocessor 30 is the time variation of the optical properties of the cell suspension, defined as the difference in the intensities of the orthogonal light fluxes that occurs when the electric pulse is exposed to the suspension with a fixed filling frequency f and changing field parameters F and E.

 Programming the changing parameters of the electric field during each radio pulse provides the ability to determine the fractional composition of cells in suspension. In addition, a change in the direction of the vector of the electric field during the radio pulse allows to increase the accuracy of measurements of the electro-optical signal by eliminating the influence of zero-line drift.

 Determination of the difference in the intensity of orthogonal light fluxes passing through the same elementary suspension sample allows eliminating the same effect of particle sedimentation and noise of density fluctuations in the suspension.

The advantages of the proposed method and device for its implementation are illustrated by specific examples of the method:
Example 1. Determination of heterogeneity of a suspension of E. coli cells.

The culture fluid with E. coli C 600 cells obtained after cultivation on a rocking chair for 5 hours at 37 ^o C on mineral medium M9 (General bacteriology methods / edited by F. Gerhard, v. 1, M. Mir, 1984, Ch. .3) with the addition of 0.4% glucose and heated in a water bath at 60 ^° C for 10 minutes, it was filtered through a Vladipor N 5 cellulose membrane, the filter cake was washed with distilled water and resuspended in TRIS-phosphate buffer with specific conductivity of 10 ^-2 Ohm ^-1 m ^-1 . The resulting suspension with an optical density of 0.4 units placed in the electro-optical cell 1 of the above device.

They performed preliminary and basic measurements. Preliminary measurements were carried out to determine the initial parameters F _max , F _min , E _max , E _min and j for this culture. To do this, from the microprocessor 30 through a common bus 31 for supplying a signal to the control inputs of the generator 12 and received from its output a harmonic voltage with a frequency f 1,024 MHz. A harmonic voltage was applied to the electrodes 21-29 of cell 1 through a circuit consisting of a controlled attenuator 13, a paraphase amplifier 14, and an electrode switcher 15. Using the microprocessor 30, the difference in the contents of counters 10 and 11 was determined at each time point through a common bus 31 and outputted to display (not indicated in the drawings) as a function of time. The field strength in the cell E was created at 10,000 V / m by changing the attenuation value of the attenuator 13 when a signal from the microprocessor 30 was supplied to its control input via a common bus 31. The frequency value f and the voltage value E were chosen from the condition of obtaining the largest value of the electro-optical signal. By changing the frequency F of the change in the direction of the electric field vector from the value F = 4 Hz to the value F 0.031 Hz, after the microprocessor 30 determined the electro-optical signal as the difference between the contents of the counters 10 and 11, the appearance of an electro-optical signal with a stationary section at a frequency F of 0.5 Hz was selected this frequency as F _max . It was determined that the disappearance of changes in the stationary value of the electro-optical signal occurs at a frequency of 0.062 Hz, and this frequency was chosen as F _min . The direction of the vector by 90 ^{o was} changed at each frequency F five times, the same number of times at each frequency F, the electro-optical signal was measured. M was chosen equal to 1 and calculated that j = 1 / M • log ₂ (F _max / F _min ) _1/1 • log ₂ (0.5 / 0.062) 3.

We measured the electro-optical signal at a frequency f of 1024 kHz at frequencies F from F _max 0.5 Hz to F _min 0.062 Hz for each value of the voltage E when it changes from 620 V / m to 10,000 V / m with a number of steps equal to 8 and step in E ² equal to (E $\genfrac{}{}{0ex}{}{\text{2}}{\text{max}}$ -E $\genfrac{}{}{0ex}{}{\text{2}}{\text{min}}$ ) / j (10000 ² 620 ² ) / 8. We calculated the values of the weight coefficients A _o and A _j corresponding to the fractions with the smallest and largest sizes for each value of tension E, according to the iterative procedure described above. We plotted the dependence of the electro-optical signal on the squared intensity for fractions with the indicated sizes. The value of the tension E _max 10000 V / m was determined at which the deviation of the graph A _o (E) from a straight line appears. Similarly, according to the schedule A _j (E), the value of E _min 4200 V / m was determined.

The main measurements of the electro-optical signal in order to determine the heterogeneity of the cell suspension were carried out at frequencies f filling the radio pulse f ₁ 1 kHz and f ₂ 8.4 MHz with a change in F from 0.5 Hz to 0.062 Hz and a change in E from 10000 V / m to 4200 V / m at j 3 and M 1.

The frequency value f ₁ was chosen taking into account the theory of volume polarizability such that the electrophysical heterogeneity of the cells did not affect the form of the functional size dependence of the polarizability anisotropy. The frequency value f ₂ was chosen so that according to the volume polarizability model (Landau L.D. Lifshits E.M. Electrodynamics of continuous media, M. Gostekhizdat, 1957, pp. 14-28), the electrophysical heterogeneity of the internal structures of the cell caused by selective exposure to a thermal factor.

The measurement results of the electro-optical signal as a function of time and parameters f, F and E are shown in FIG. 6. When processing the available data according to the iterative procedure described above, two sets of parameters were obtained A _i } _i N _i • dQ _i • {da _i } ₁ for f ₁ 1 kHz and A _i } ₂ N _i • dQ _i • {da _i } ₂ for f ₂ 8.4 MHz, where i 0, 1, 2, 3.

 The optical response processing shown in FIG. 6, in order to determine the morphological heterogeneity of the suspension, it was made in accordance with the iterative procedure described above, and the results of this processing are presented in FIG. 7a). In FIG. 7b) shows the results of control measurements of the size distribution obtained by processing micrographs of a crushed drop of a cell suspension (Colloid Journal, 1989, v. 51, No. 5, p. 844).

 From a comparison of the data presented in FIG. 7, it is seen that the results of measuring morphometric heterogeneity obtained by processing the data presented in FIG. 6, coincide with the results of control measurements with an error not exceeding 10%, which confirms the possibility of using the proposed method to assess the morphometric heterogeneity of the studied cell population.

In FIG. Figure 8 shows the dependence of the polarizability anisotropy da _{2 of the} studied cells on their sizes at a frequency f ₂ normalized to the same dependence da ₁ at a frequency f ₁ . As can be seen from this figure, the normalized polarizability anisotropy da ₂ / da ₁ for cells with sizes greater than 3.2 μm has a smaller value than the polarizability anisotropy for cells with sizes less than 1.8 μm. This indicates the selective action of heat on cells with different sizes and the appearance of at least two fractions with different polarization parameters. Interpretation of the data using the theory of volume polarizability shows that a decrease in the magnitude of the anisotropy of polarizability in cells larger than 3.2 μm is associated with a change in the electrical conductivity of the cytoplasm due to selective heat damage of larger cells. This confirms the possibility of using the proposed method to assess the electrophysical heterogeneity of the cell population
Example 2. Determination of electrophysical heterogeneity of Bac cells. subtilis.

Culture fluid with Bac cells. subtilis at the stage of sporulation obtained after cultivation on a rocking chair at 30 ^o C for 24 hours on a medium (Beltyukova K.I. et al. Methods for the study of pathogens of bacterial diseases of plants, Kiev. Naukova Dumka, 1968), containing (in / l) enzymatic hydrolyzate of yeast (20), glucose (10), MgSO ₄ • 7H ₂ O (0.2), CaCl ₂ • H ₂ O (0.01) and the potato-glycerin mixture, was filtered through a Vladipor cellulose membrane N5 ", the filter cake was washed with distilled water and resuspended in TRIS-phosphate buffer with specific conductivity 10 ^-2 ohm ^-1 m ^-1. The resulting suspension with an optical density of 0.4 units placed in the electro-optical cell 1. Held auxiliary and basic measurements, as described in example 1 and determined F _max 0.25 Hz, F _min 0,031 Hz, E _max 6000 V / m, E _min 2100 V / m M 1 was selected and j was calculated as described in Example 1, j 3.

The main measurements were taken at the frequencies f filling the radio pulse f ₁ 1 kHz and f ₂ 4,2 MHz, selected on the basis of the same premises as described in example 1.

The measurement results of the electro-optical signal as a function of time and parameters f, F and E are shown in FIG. 9. When processing them using the iterative procedure considered above, the set of parameters A _i } ₁ N _i • dQ _i • {da _i } _1M for f ₁ 1 kHz and A _i } ₂ N _i • dQ _i • da _i } ₂ for f ₂ 4.2 MHz, where i 0, 1, 2, 3.

The results of normalizing the elements of the set A _i } ₂ to the elements of the set A _i } ₁ , which are necessary for distinguishing the dependence of the polarizability anisotropy on the cell size, are shown in FIG. 10.

The normalized polarizability anisotropy da ₂ / da ₁ according to FIG. 10 for cells with sizes> 5.2 μm is more important than for cells with sizes <3.6 μm. This indicates a change in the electrophysical parameters of cells that are at the stage of completion of spore formation in comparison with small cells that do not have spores, and the detection of at least two fractions with different morphology and electrophysical parameters. This example once again confirms the possibility of using the proposed method and device for its implementation to assess the morphological and electrophysical heterogeneity of the cell population.

Claims (11)

1. The method of electro-optical analysis of cells in suspension, including applying an alternating electric field in the form of pulses to the suspension, transmitting light flux through it, measuring the intensity of the light flux passing through the suspension and determining their difference as the magnitude of the electro-optical signal, followed by determining the dependence of the cell polarizability anisotropy on frequency the specified electric field, characterized in that when applying each specified pulse of the electric field change direction electric field vector discretely 90 ^o with a frequency F, wherein the frequency F of change F _{m i n} to F _{m a x} of formula
F _i <Mv> ± <D> ₁ = F _i / 2 ^M ,
where i (0, i-1), (j-1,0),

M is a positive number;
F _{m a x} is determined by the frequency of changing the direction of the vector of electric field strength at which the electro-optical signal appears, and F _{m i n} is determined by the frequency of changing the direction of the vector of electric field strength at which the change in the stationary value of the electro-optical signal disappears. 2. The method according to claim 1, characterized in that at the same time as the frequency F changes, the direction of the electric field intensity vector changes the electric field strength E in the range from E _{m i n} to E _{m a x} according to the formula

in case of a change in the electric field strength in the direction of its increase, or by the formula

in case of a change in the electric field strength towards its decrease,
where i = (0, i),

while E _{m i n the} magnitude of the electric field, which determines the threshold level of a weak degree of orientation of large cells;
E _{m a x is the} value of tension that determines the threshold level of a weak degree of orientation of small cells. 3. The method according to claims 1 and 2, characterized in that the indicated values of F _{m a x} , F _{m i n} , E _{m a x} and E _{m i n are} determined experimentally depending on the type of the studied cell culture. 4. The method according to p. 1, characterized in that they change the direction of the electric field vector by 90 ^° at any frequency F of changing the electric field vector at least three times.  5. The method according to claim 1, characterized in that the test suspension is taken in the form of an elementary volume through which two light fluxes orthogonal to each other are passed alternately.  6. The method according to claim 1, characterized in that an electric field is applied to the test suspension, the intensity vector of which at the initial time is parallel or orthogonal to at least one of the indicated light flux.  7. A device for electro-optical analysis of cells in suspension, containing an electro-optical cell equipped with translucent windows located opposite each other and electrodes, a light source optically coupled to a photodetector connected to a light flux difference determination unit, and an electric voltage pulse generator connected with electrodes, characterized in that the electro-optical cell contains four translucent windows and at least four electrodes in the form of rods, setting between these windows, the device is equipped with an additional light source optically connected to the photodetector, both sources being pulsed, and the device is equipped with a series-connected attenuator of electric voltage with the ability to control the magnitude of the electric voltage, a paraphase amplifier and an electrode switch connected to an electric generator voltage, while the input of the paraphase amplifier is connected to the output of the attenuator, its outputs with two diagonal the electrodes and the inputs of the electrode switch, the outputs of which are connected to two other diagonally located electrodes.  8. The device according to claim 7, characterized in that the electro-optical cell contains nine electrodes mounted in three rows of three electrodes in each row and the distance between adjacent electrodes in the row is equal to the distance between the rows, and the middle electrode of the middle row is central, while the extreme the electrodes of the first and third rows are connected to one output of the paraphase amplifier, the central electrode is connected to the other output of the paraphase amplifier, and the remaining pairwise opposite electrodes are connected to the electrode switch .  9. The device according to claim 7, characterized in that it is equipped with two gated frequency counters, the unit for determining the difference in the intensities of the light flux is made in the form of a voltage to frequency converter, the output of which is connected to the inputs of two gated frequency counters.  10. The device according to claim 7, characterized in that the electro-optical cell is made in the form of a parallelepiped with a square base. 11. The device according to claim 7, characterized in that said optical coupling of the light sources and the photodetector is made in the form of four mirrors, one of which is mounted on the axis of the light flux generated by one light source, and the other on the axis of the light flux created by another light source and the other two are located between the previous mirrors and are installed relative to each other at an angle that ensures the incidence of these light fluxes on the photodetector at an angle of 90 ^o .

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