In microbiology, monitoring the growth of any microorganism in culture is important for studying and optimizing the growth kinetics, the biomass and the metabolite production. In this work, we show that laser speckle imaging is a reliable technique that can be used to perform real-time monitoring of bacteria growth kinetic in liquid culture media. Speckle parameters, specifically speckle grain size and the spatial contrast of the speckle images, and standard analytical parameters (optical density, pH and colony forming units) were measured during the culture of different strains of Bacillus thuringiensis. Our results show that both speckle grain size and spatial contrast decrease with bacterial growth. Furthermore, speckle parameters are sensitive to the fermentation conditions. Statistical analysis revealed a relatively high correlation between speckle and analytical parameters.

Real-time monitoring of bacterial growth kinetics in suspensions using laser speckle imaging.

Theory of the speckle dynamics arises in the image plane of the object as a result of deterministic and random displacements of point scattering centers are considered. It was shown that time-average radiation intensity and the correlation coefficient of the speckle image segment depend on the average value, variance, and the correlation time of displacement difference of the scattering center pairs. The sensitivity, accuracy, and calibration of the method are considered. Application of the technique in studies of micro-and macroprocesses in high-cycle fatigue of metals and in a monolayer of live cells is given.

Speckle metrology of dynamic macro- and microprocesses in deformable media

Previously, a speckle interferometry technique and a device that allows quantitative evaluation of the metabolic activity of cultured cells were theoretically grounded and successfully tested. A speckle time-averaging technique was proposed to separate and study the processes occurring in cells at various velocities. The objective of the present research was comparing the parameters of speckle dynamics used to evaluate cell metabolism and averaged in areas of various size. Areas inside the speckle image of a cell as well as areas of the image plane with various numbers of cells were averaging areas. The target of the research was cells from L-41 culture placed onto a special optical tray with a nutrient solution immediately after defrosting. Time-average value T of digital radiation intensity in the TV camera pixels I (1) and the correlation coefficient of two digital images η (2) were used as speckle field change parameters. The digital images corresponded to a single frame area at two time moments. It is shown that in the area containing hundreds of cells dependence η(t) levels off, which indicate stationarity of random value I~ . Features of η(t) dependences obtained by averaging over large and small cell numbers image are discussed. Using the data obtained, we formulated recommendations on selecting area sizes for averaging physical values to study various processes occurring in cells.

Applicaton of speckle dynamics for studies of cell metabolism

Relation between the phase dynamics of the waves sounding thin biological object and the dynamics of the speckles in the object image plane was theoretically detected using a model dealing with interference of multiple waves with random phases. Formulas determining the dependence of time-average intensity ~ I and temporal autocorrelation function η 1⁄4 ηðtÞ of this intensity at a point of the image plane with mean value 〈x〉, mean square deviation σu, and correlation time τ0 of the difference between the optical paths ∆u of the wave pairs in the neighborhood of a conjugate point of the object plane were obtained. A relation between a normalized temporal spectral function of stationary process ∆uðtÞ and a temporal spectral radiation intensity fluctuation function was substantiated. An optical device relevant to the model used in the theory was developed. Good quantitative coincidence between the theory and the experiment was shown by means of dosed random variation of path difference ∆u. The calibration procedure for the device determining σu was developed; errors and the sensitivity limit of the technique were assessed. Application of value σu as a cell activity parameter on biological objects, namely, a monolayer of live cells on a transparent substrate in a thin cuvette with the nutrient solution was substantiated. It was demonstrated that the technique allows determination of herpes virus in the cells as early as 10 min from the experiment start. A necessity to continue upgrading of the technique was pointed out as well as its prospects for studying the cell reaction to toxic substances, bacteria, and viruses considered.

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Dynamic Speckle Interferometry of Thin Biological Objects: Theory, Experiments, and Practical Perspectives

The paper briefly considers the theory of the dynamic variant of optical speckle interferometry and its application to study in real time macroscopic and microscopic processes in deformable media. In the theoretical part, it is shown that it is convenient to study the macroscopic translational displacement, rotation and deformation of bodies by the movement of the whole picture of speckles and the shift of interference fringes of the two speckle fields, while the microscopic phenomena are conveniently studied by the change of the structure of speckle images. The paper presents some applications of original techniques developed by the author to determine rotations and strains of bodies under elastic and plastic deformations, to detect ultrasonic waves and to study the characteristics of crack initiation in high-cycle fatigue. Practical application of the developed techniques for testing macro- and microprocesses in living cells is shown.

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Dynamic Speckle Interferometry of Microscopic and Macroscopic Processes in Deformable Media

Speckle dynamics in a thin biological object illuminated through a diffusor box is studied both theoretically and experimentally. A model that allows for random time variations in the phase difference Δφ of pairs of waves transmitted through the object is used in the theoretical part of this study. Formulas for the time-averaged intensity of the radiation at a certain point of observation and a temporal autocorrelation function of the radiation intensity are deduced. The relationship between the characteristics of the random variable Δφ and the parameters of the speckle dynamics is discussed. The case of occurrence of a quasiperiodic variation in the radiation intensity is separately considered. The theoretical results are used for interpretation of the data obtained in the experiments on studying the metabolic activity of cells cultivated on a glass substrate.

Dynamic Speckle-Interferometry of Microscopic Processes in Thin Biological Objects

The dynamics of speckles in the image plane of a monolayer of cells cultivated on a glass substrate has been recorded. Cell cultures HEL-3, L-41, and Vero were selected as the objects of research. The digitized value of the radiation intensity I in one pixel and the parameter η characterizing the change in the intensity distribution on an 10 × 10 pixel area was recorded for 24 hours. The multiple determinacy coefficient of three cell cultures, which was obtained from the time dependences of η, was equal to 0.94.

Using Speckle Dynamics for Comparison of the Metabolic Activity of Different Cell Cultures

The report considers the results of the experiments in which digital values of light intensity I and the image area correlation index η values were recorded on a real-time basis for one or two days. Three cell cultures with viruses along with intact cultures were investigated. High correlation of dependence of η values on time t values was demonstrated for three cultures. The η=η(t) and I=I(t) dependences for cells with and without viruses differ considerably. It was shown that the presence of viruses could be determined as early as ten minutes after measurements were started.

Application of speckle dynamics for studying metabolic activity of cell cultures with herpes virus

This work is aimed at creating the theoretical basis for the method allowing one to layer-by-layer reconstruct the parameters which characterize the processes occurring in small regions of living cells. The problem of the speckle dynamics in the object-image plane, which is caused by random variations in the optical paths of the waves in various layers inside the cell, is theoretically solved for a model of the cell in the form of a thin phase object located near the phase screen. On the assumption that the random variations in the difference Δu of the optical paths of the wave pairs are independent in these layers, expressions for the time-averaged radiation intensity Ĩ at an arbitrary point of the image plane and for the time autocorrelation function η(t1, t2) of this intensity are obtained. The formulas relating the parameters which characterize the occurring processes (the cell functions) to the quantities Ĩ and η are obtained. In the formula for Ĩ, such parameters are the average value and variance of the quantity Δu, which are obtained by averaging over the time and the region whose sizes are equal to the linear resolution of the lens and the layer thickness. The average values and variances of the quantity Δu at the time instants t1 and t2, as well as the time autocorrelation function η(t1, t2) of the quantity Δu, which are additionally averaged over the ensemble of objects, are the functional parameters of the regions in the formula for η(t1, t2). It is theoretically shown that the above-mentioned parameters, which are obtained by averaging over the cell thickness, are sums of the similar parameters in the cell layers. The correspondence of the formula for η(t1, t2) to the physical processes occurring in the cells is shown using living cells, which are located on a glass substrate after defrosting. Assuming that four processes, which independently change the wave phases, are observed in the cells, a very good agreement between the theory and experiment is obtained.

Speckle Tomography of the Living-Cell Functions

Earlier we reported developing a speckle interferometry technique and a device designed to assess the metabolic activity of a cell monolayer cultivated on a glass substrate. This paper aimed at upgrading the technique and studying its potential for real-time assessment of herpes virus development process. Speckle dynamics was recorded in the image plane of intact and virus-infected cell monolayer. HLE-3, L-41 and Vero cells were chosen as research targets. Herpes simplex virus-1-(HSV-1)- infected cell cultures were studied. For 24 h we recorded the digital value of optical signal I in one pixel and parameter η characterizing change in the distribution of the optical signal on 10 × 10-pixel areas. The coefficient of multiple determination calculated by η time dependences for three intact cell cultures equals 0.94. It was demonstrated that the activity parameters are significantly different for intact and virus-infected cells. The difference of η value for intact and HSV-1-infected cells is detectable 10 minutes from the experiment start.

A. P. Vladimirov

Papers

Dynamic Speckle-Interferometry of Microscopic Processes in Thin Biological Objects

Using Speckle Dynamics for Comparison of the Metabolic Activity of Different Cell Cultures

Application of speckle dynamics for studying metabolic activity of cell cultures with herpes virus

Speckle Tomography of the Living-Cell Functions

Application of speckle image correlation for real-time assessment of metabolic activity in herpes virus-infected cells