A brief summary of the basic principles of the dynamic light scattering-photon correlation spectroscopy technique is given. Experiments on DNA in both dilute and nondilute solutions are used to illustrate both the types of information that may be obtained from DLS-PCS as well as some of its limitations.

A comparative dynamic light scattering study of BSA and lysozyme is presented. A backscatter fiber optic system and a conventional light scattering spectrometer are used to measure the diameter of proteins in the dilute regime, that is, below concentrations of 10 mg/ml. The fiber optic system operating with a power level of 2 mW at a wavelength of 632.8 nm compares favorably with a conventional system operating with a power level of 40 mW at a wavelength of 514.5 nm. Quasi-elastic light scattering measurements taken at several concentrations demonstrate the utility of a backscatter fiber optic probe for sizing of small molecular weight proteins. The fiber probe, comprising two optical fibers, is about 3 mm in diameter and can be positioned either inside or outside the scattering cell.

Dynamic light scattering was applied to study aggregation phenomena in mucin, the glycoprotein responsible for the visco-elastic properties of mucus which is found as a lining on most epithelial cell surfaces. Intensity autocorrelation functions measured on purified mucin solutions under varying experimental conditions were analyzed by Laplace inversion methods. The results showed that at low pH (below 4) solutions of gastric mucin contain very large supra-molecular aggregates, with diffusion constants 100 times slower than those of the 2 X 10⁶ molecular weight glycoprotein of mucin. Similar methods were used to investigate the interaction of gall bladder mucin with cholesterol-phospholipid vesicles. Repeated measurements of the intensity correlation functions after adding mucin to a suspension of vesicles showed a two-fold increase in the hydrodynamic radius of the vesicles over a period of three hours after which the vesicle size stayed constant. Control experiments with latex particles in mucin and vesicles in other proteins showed no change in size, implying that the fusion of vesicles is due to vesicle-mucin interactions.

A theory for the dynamics of semi-rigid polymers, based on the wormlike chain, has been extended to include hydrodynamic interactions. The hydrodynamic interactions are treated in the preaveraged form using the kernel of Yamakawa and Fujii [Macromolecules 6:407(1973)]. The dynamic equation is solved by perturbation theory to second order for the eigenvalues and to first order for the eigenfunctions. The solutions to a Fokker-Planck equation are used to compute the polarized dynamic light scattering correlation function for a dilute system of wormlike chains. The electric field correlation function decays with dynamics due to translational diffusion, end-over-end tumbling of an elongation axis, and flexing degrees of freedom.

Laser Raman scattering is the method of choice for probing macromolecular structures and interactions in complex biological assemblies. The Raman technique is a valuable complement to high-resolution structure methods applied to biological molecules in the crystal (x-ray crystallography) and to static and dynamic light scattering methods applicable to solutions. Raman spectroscopy is particularly well suited to investigating assembly mechanisms of viruses and conformations of their nucleic acid and protein constituents. Here we describe the use of digital difference Raman methods to probe structures of the double-stranded (ds) DNA genome of the icosahedral virus P22 in packaged and unpackaged states. The findings are compared with structural changes in protein subunits of the P22 viral capsid, attendant with capsid expansion and DNA packaging. We find no evidence in the Raman spectrum of specific intermolecular interactions involving capsid protein and major groove sites of the packaged DNA. The present results suggest a simple model for organization of condensed dsDNA chromosomes in icosahedral viruses.

Physicochemical properties of monodisperse type I collagen were studied using the dynamic light scattering and analytical ultra-centrifugation techniques. The measured osmotic second virial coefficient A₂ is much smaller than the excluded volume limit calculated from the length L, diameter b and molecular weight M of collagen. The strength k_D of the concentration dependence of the mutual diffusivity ^*D_G of collagen is negative in the sign and large in the magnitude. The relationship k_D equals 2A₂M - v - k_(zetz ) does not hold for collagen, while it holds for fd virus and poly((gamma) - benzyl L-glutamate), where v is the partial specific volume, and k_(zetz ) the strength of the concentration dependence of the friction coefficient (zetz) . We conclude from these three lines of evidence that water is a poor solvent of collagen and that there must be very weak attractive interactions among collagen filaments which can easily be destroyed by external perturbations.

Five composite liquid solutions (CL1-5) were prepared consisting of silica spheres and the rigid rod polymer poly(γ-benzyl α,L-glutamate) (PBLG) in the polar organic solvent dimethylformamide. The sizes of the macromolecular components in each solution were chosen such that the sphere radius/rod length ratio decreased from CL1 to CL5. The sphere diffusion constants in the composite liquids were measured as a function of the rod concentration by dynamic light scattering. Microviscosities (η_μ ) as low as 1/2 of the solution viscosity (η) were found. The sphere diffusion constant data was fit to a stretched exponential of the form D/D_o equals exp(-αc^v) which is predicted by several models for the diffusion of spheres in polymer solutions. From these fits, we conclude (1) the stretched exponential fit is worst if η_μ < η, (2) models predicting a stretched exponential offer no explanation for η_μ < η, and (3) the experimentally determined values for v compare well to other experimental data but not to theoretical predictions.

Quasi-elastic light scattering measurements of the normalized time-dependent density correlation function, g(t), have been carried out on fibrin networks formed at T equals 25 degree(s)C from fibrinogen solutions. Fibrinogen is a protein present in vertebrate body fluids, which is capable of forming gels because of an initial activation and a successive polymerization process. The correlation function g(t) has been systematically measured as a function of the fibrinogen concentration in solutions at a fixed pH 8, activated by 400 nM of thrombin. As the fibrinogen concentration increases from 84.5 nM to 880 nM, g(t) exhibits pronounced nonexponential relaxation. The stretched exponential function e^{-(t/τ) β} has been shown to fit the g(t) function over a four decade time range of measurement, for q values ranging between 1.32 X 10⁵ cm^-1 and 2.45 X 10⁵ cm^-1 stretched exponent (beta) equals 0.65 +/- 0.05, commonly observed near the glass transition point, has been obtained. The average decay rate (lambda), accounting for short time decay rates of g(t), decreases as concentration increases showing a minimum at p equals 400 +/- 50 nM, while the relaxation time τ shows a maximum at the same concentration.

Quasi-elastic light scattering studies of model membranes have yielded much information on their structure and dynamics. Unusually, the q dependence of the dynamic structure factor S(q,(omega) ) carries no significant structural information. Instead the information on membrane structure has derived from changes in the scattered intensity as the membrane is passed through a phase transition or other change, or from fluctuations in the dynamic part of the scattering factor in situations where the signal is non-stationary. We concentrate here on the latter aspect, and consider the information on the structure of various model membrane systems which has been gathered.

A simple mathematical model is analyzed to investigate how the `non-classical' migration of photons within a multiply scattering medium might affect the characteristics of signals obtained in laser Doppler blood flowmetry measurements.

The stimulated Brillouin scattering (SBS) excitation and hypersound generation experiments in collagen structures are described. Peculiar features of such structures explaining energy characteristics of SBS are considered. Potential biology and medicine applications of hypersound generated by SBS are discussed.

We have developed optical methods to indicate when micro-organisms are present in a single electrodynamically levitated microdroplet and to measure their motion within it. The approach was to develop a method for holding saturated salt water droplets in an approximate thermodynamic equilibrium in a quadrupole balance. We then captured droplets containing subparticles or guests approximating the size of typical micro-organisms. The presence and motion of these guests in the host droplet is indicated by both photon correlation spectroscopy and by fluctuations in the emission of fluorescence. Finally we show that the system may be used with real micro-organisms by capturing a microdroplet of the growth medium (minus nutrients) for the micro-organism, Halobacterium halobium with those micro-organisms contained in it. Halobacteria halobium were chosen for the model system because they thrive in a growth medium containing a nearly saturated solution of NaCl which we have found is particularly amenable to handling in our present apparatus.

In the present paper we report on initial attempts to correlate changes in polarized light scattering signals from bacteria and bacterial spores with changes in physical parameters as a result of heat and UV exposure. Clear reproducible changes can be observed, but quantitative correlation such as measuring the degree of germination or survivability needs further study. Average cell size changes can be measured quickly in bacteria such as after UV exposure or during the growth cycle, making the used technique very valuable to monitor changes. Computer modelling is needed to distinguish effects caused by size changes and by changes in index of refraction.

Laser Doppler (LD) microscopy is a technique, providing high-resolution noninvasive measurements of microstructures dynamics. It can be used in different fields of biophysics and biomedicine. This technique yields quantitative information on diffusion coefficients, velocities, and velocity profiles of dynamic microstructures in vivo and in vitro. LD microscopy is an alternative method of velocity measurement to such methods as computer- aided microphotography and imaging, diffraction grating microscopy, FRAP, etc. In this paper we describe the results of our LDM measurements of one of the main hemodynamic parameters -- the blood-flow velocities in the microvessels of Salmo salar and Danio rerio fish embryos, as well as of the rat mesentery.

Taking into account the current research results of the probabilistic model describing the laser Doppler flowmetry signal, the probability-density function is derived and the potential attainable in the measurements of the unknown parameters in the laser Doppler flowmetry accuracy is analyzed.

The description of special features of laser light interaction with biotissues, such as the skin, eye tissues, etc., with respect to laser diagnostics and therapy methods development is given. Optical models of transparent and turbid biotissues are analyzed. The role of static and dynamic light scattering in the light dosimetry, tissue heating, and receiving information of biotissue optical parameters, its structure, movements, and vibrations are considered.

At present, the available procedures to determine optical parameters of tissue suffer from several limitations, either from a practical or a theoretical point of view, which restrict their use. The aim of the present study was to verify whether a single steady-state diffuse reflectance measurement could be exploited to estimate the optical characteristics of in vivo skin. Using integrating spheres, reflectance spectra of in vivo skin in different anatomical sites were measured. Further, transmittance of relatively thin structures (e.g., lobe of the ear) was measured at 633 and 800 nm. Results were compared with data obtained from a Monte Carlo model. As input parameters we used values of absorption and reduced scattering coefficients either estimated from previously determined in vitro data or calculated from reflectance and transmittance measurements. Analysis of results shows several peculiar features: (1) based upon the estimated set of absorption and reduced scattering coefficients, calculated diffuse reflectance spectrum matches well the experimental data, whereas intensity of light fluence in depth is underestimated; (2) the backward photon flux is strictly albedo dependent; (3) the in vivo scattering coefficient is lowered, with respect to the in vitro datum, by the presence of blood.

The kinetics of aggregation and disaggregation of red blood cells in whole blood subjected to shear stress is different with normal and pathological blood. To measure the kinetics we have applied a backscattering nephelometric technique, and quantitatively registered the alternation of the scattered intensity due to appearance or disappearance of different types of cell aggregates under controlled shear stresses. The latter were obtained in a Couette flow in a thin gap between two concentrical cylinders, the inner one rotating at controlled velocities. The peculiarities of backscattered light signal formation and obtained results were discussed.

A newly developed system to determine red blood cell (RBC) flexibility is presented. It combines ektacytometry (laser diffraction) with image analysis. Laser light is passed through erythrocyte (RBC) suspensions which are sheared in a Searle like viscosimeter. The laser diffraction patterns are photographed by a linear CCD camera, and further analyzed by a computer. Flexibility is characterized by the quotient of minor and major axes of isointensity lines of the elliptically transformed diffraction patterns. The variation coefficient of the measurement is less than 1%. Exposing RBC to mechanical stress within the viscosimeter (shear rates from 130 to 2620/s, viscosity of the suspending medium 22 - 24 cpoise) is followed by a decrease of RBC flexibility. This effect is only detectable if elongation is measured at low shear rates (e.g., 260/s) and a threshold of stress (1100/s) is exceeded. It is not reversible and not accompanied with detectable hemolysis. Exposing whole blood to ozone (2 - 77 (mu) g/ml) does not alter RBC flexibility, but induces significant hemolysis at ozone > 7 (mu) g/ml. Thus the action of ozone can be regarded as an all or nothing effect either leaving the cell unaffected (flexibility) or destroying it (hemolysis).

When a narrow collimated light beam is incident on biological tissue with inhomogeneity, the characteristics of light backscattered from the sample carry information about the inhomogeneity location. A model is developed, based on the diffusion approximation to radiative transfer, which yields analytical expression for backscattered light beam intensity from tissue with an inhomogeneity. The inhomogeneity may be an object with given reflection coefficient or a variation of scattering and absorption parameters of tissue. The relative contribution (delta) I/I of spatially limited inhomogeneity to backscattered light intensity is expressed in terms of inhomogeneity diffuse scattering amplitude and the probability density distribution of photon paths in depth. This contribution is a function of the positional inhomogeneity coordinates and the separation between the incident and receiving points on medium surface. It is shown that the function (delta) I/I has three conditional maximums and one minimum. The effect of medium scattering and absorption coefficients on conditional extremums of function (delta) I/I is studied. The discovered extremums of analytical solution to searchlight problem on backscattering of narrow pencil light from turbid medium with inhomogeneity permit us to formulate a simple theoretical method for solution of the location problem of the inhomogeneity in tissue. The model predictions make it possible to give correct quantitative interpretation of the experiment results on the change of the backscattered light beam intensity from tissue phantom due to the existence of small absorber.

Unified approach is suggested to determine the real (n) and imaginary (x) parts of refractive index and average size r of biological particle suspensions, biopolymers, and so on for a wide range of sizes (r equals 10^-1 - 10² micrometers , n equals 1.02 - 1.15 and x equals 10 ^-6 - 10^-4). The values to be measured are the diffuse reflection and transmission coefficients and the temporal spectra scattering intensity fluctuations of optically thick layers in the case of weak absorption (this condition is determined experimentally). The sensitivity of the proposed approach for the determination of n and x (as well as the average cosine of the phase function of light scattering <cos(phi) > and radiation pressure cross section (beta) for finding the force acting on the particle) is very high and exceeds the known methods. The methods of determining n, x, r, <cos(phi) >, (beta) have been evaluated for suspensions of erythrocytes, blood substitute (emulsions of PFC), and latexes.

New technology for the definition of the skin epidermis optical parameters, which includes upper layers of epidermis stripping, diffuse reflection, transmission, and angular measurement of scattered light by stripping samples, calculations using 4-flux Kubelka-Munk approximation, have been obtained. This technology was successfully used for depth dependence monitoring of epidermis optical parameters, and for laser light dosimetry in percutaneous irradiation of blood and laser PUVA therapy.

Normal cornea transmits greater than 90% of visible light, but its transmission would be less than 30% if the stroma's collagen fibrils scattered independently of one another. Thus modern transparency theories are based on there being sufficient order in fibril positions for destructive interference to cause cancellation among the scattered fields. Two types of structure have been proposed: long-range crystalline order as used in the earliest theory, and short-range liquid-like order such as that depicted by electron microscopy. Of course structures depicted in electron micrographs may be distorted and other tests are required to determine the nature of the order. Light scattering measurements can afford such a test. Specifically, the two types of order produce different dependencies on wavelength for the scattering cross-section (angular or total) in the long-wavelength limit. Measurements must be analyzed appropriately to obtain the long-wavelength limit. The results reported in this paper demonstrate that measurements of both angular and total scattering cross-sections support short-range order of fibril positions.

Theoretical and experimental results are presented on the diagnostics of erythrocyte mechanical properties by two recently emerged techniques: Flicker Spectroscopy (FS) and Dielectro-Deformation (DD). FS allows us to monitor the bending modulus of a cell membrane and the viscosity of a cell interior, while DD allows monitoring of the shear modulus and the surface viscosity of a cell membrane. All four moduli are very sensitive to physiological state and pathological processes in erythrocytes, as well as to various environmental changes. The correct evaluation of these moduli or their changes from the measured curves requires elaborate theory. This theory should adequately take into account rather complex geometrical, mechanical, and electric characteristics of erythrocyte, as well as instrumental function of the measuring optical set-up. The approximate models, which give analytical solutions are formulated, namely, the modified planar model of erythrocyte flickering, and the general dynamic equation of erythrocyte dielectro-deformations. The instrumental function is introduced and its role is discussed. The flicker spectra are measured in a broad frequency range 0.05 divided by 500 Hz using different optical set-ups. The results are compared with the theory. The amplitude-frequency characteristics of forced dielectro- deformational oscillations of erythrocyte are measured also, giving the characteristic time of viscoelastic relaxation of its shape. Some problems of diagnostic applications of FS and DD are discussed.

General parameters of polydispersive suspensions of homogeneous aspherical particles of different orientation structure have been found, and their integral optical characteristics are accorded with such characteristics of sphere in general coordinates. On the basis of exact theory new optical effects absent in conclusions of known approximation have been found, particularly appearance of unreal polydispersive function. It has been proved that in first approximation the coefficient of spectrum absorption (scattering) transformation of suspension is invariable. On this basis the method of determination of spectrum indices of absorption and scattering of matter of `soft' dispersive particles has been proposed. Analytical expression for integral light scattering phase function has been obtained. Using this expression in approximation of RGD (Rayleigh-Gans-Debye) approach, express-method and apparatus for the estimate of average sizes of suspension particles are worked out.

The flow velocity profiles and particles concentration profiles in a flow are often required for design and optimal operation of various flow-exploiting biomedical instruments. A new optical probing technique named Integral Doppler Anemometry (IDA) allows the fast and precise measurements of these profiles in narrow channels with the optically transparent walls and characteristic width from tens of microns up to several millimeters. The shape of IDA spectrum is determined by the flow velocity profile and the concentrational profile of light scattering particles in a flow. These profiles can be reconstructed from the measured spectrum by means of special measuring and data processing procedures. The basic principles of IDA technique and the main requirements to IDA measurements are formulated. The analytical expressions giving the shape of IDA spectrum for the practically used optical arrangement of anemometer are presented. The algorithms of reconstruction of flow velocity and particles concentration profiles from IDA spectra are discussed. Measurements showed an essentially nonuniform lateral distribution of erythrocytes in a flow in such channels, namely, the transverse hydrodynamic focusing of cells.

The diffuse reflection and transmission coefficients, other optical parameters of normal and cancer tissues have been investigated in visible and infrared spectra. The optimal spectral range for distinguishing the cancer is found. The spectral absorption coefficients and size of cells parameter determined using our approach are analyzed to be different for normal and pathological tissues. The method is proposed for calculating the diffuse reflectance and transmittance of multiple tissue layers. The investigations have shown that cancer may be distinguished under the layers of skin and normal tissue.

To develop methods of some skin diseases diagnostics and of these diseases treatment monitoring the experimental investigations of the laser beam scattered by epidermis thin layers have been carried out. For the analysis of the specific structures with typical sizes about 10¹ - 10³ micrometers in the human upper skin layers the coherent optical analyzer (COA) has been developed. By means of COA the spatial distributions of epidermis strippings amplitude-phase transmission functions were investigated. The spatial power spectrum (SPS) instantaneous values registration for various spatial frequencies at the mechanical scanning of a pattern in direction, normal to the system's optical axis, was carried out. The registration was fulfilled by means of the specialized pulse-counting registrator of photo-electric signals. Well marked differences have been discovered between level crossing statistics for normal and psoriatic skin.

The methods of the analysis of angular and shear cardiovibrations have been developed. The experimental results of vibration investigations using these methods are presented. The advantages and deficiencies of the methods are discussed.

We have analyzed the light scattering by whole bovine lens in cold cataract state. The intensity of unscattered component dependent on the temperature was measured. To describe the cold cataract we have determined parameters related to variations of the unscattered component. The relations between the parameters are discussed.

Angular dependence of light scattered by skull samples was investigated. Measurements were performed on full-thickness skull samples by the use of a goniometric system. The angular dependence of the scattered light intensity was measured. The wavelength of the incident radiation was 501.7 nm. The transmission of the light through the samples was estimated as a function of sample thickness. The contribution of the fluorescent light in the scattered radiation was estimated and discussed.

The problem of unique sensitivity of living cells to weak forces and fields remains to be solved. A generally accepted principle of investigations of the cell amplification mechanisms is that of detailing. As a result all the more structural and chemical details have been discovered in regard to biological processes. Unfortunately the living system has been broken down into artificial fragments and a prospect has been removed to reveal how the details are united in real time allowing a cell to accept low-energy signals and to respond adequately to them. According to our conception the native cell self must be used as a measuring instrument for resolution of this problem. Internal organization and operation of the living cell can be understood by way of variations of nature and parameters of external forces and recording the cell response by a suitable technique. In this report a simple and sensitive technique of the red blood cell (RBC) response includes the creation of circulating RBC flow, the RBC's hydrodynamic orienting and plane focusing in an optical cell with parabolic profile of velocities, and application of Fraunhofer diffraction analysis to detect changes of the plane position as a result of the cell exposure to low-level action. As examples of such action the low-intensity laser irradiation and temperature-induced changes are presented.

In solution the surface of protein macromolecules is always charged and so the electrostatic interaction between the polar solvent and charged groups on the biopolymer surface is strong enough. It is of interest to consider three types of interaction: protein - protein, protein - contrion, protein - solvent. All of these interactions can change as static parameters of macromolecules solution. This paper deals with the most suitable methods for measurement of these parameters. The methods are NMR (nuclear magnetic resonance) and laser light scattering.

Conformational changes of (lambda) -phage DNA macromolecules by dynamic light scattering (DLS) method were investigated. It was shown that the persistence length of B-form DNA could reduce to small value in some conditions, and could achieve approximately equals 12 nm. When studying the internal motions in the linear form of (lambda) -DNA, there was observed the deviation of the experimental angular dependence of the first cumulant (Gamma) from theoretical predictions for the (Gamma) in Rouse-Zimm model including hydrodynamic interactions. This deviation appears in region qR_g >> 1. The first cumulant deviation to the higher frequencies is observed only for B-form DNA. Experimental behavior of the first cumulant (Gamma) for the A-form DNA coincides with the theoretical one in the Rouse-Zimm model.

The dynamic light scattering technique of photon correlation spectroscopy (PCS) is a sensitive and noninvasive method of probing spontaneous fluctuations in macromolecular solutions. PCS was used to study the wavevector dependence (wavevector magnitude, q) for a wide range of q of intracoil relaxations for a very large, linear polystyrene in both good and theta solvents and a clear coupling between intracoil excluded volume behavior and hydrodynamic interactions was observed. Moreover, the marked concentration dependence of the overall intracoil relaxation for the same polystyrene in good solvent revealed the important role strong intercoil interactions, i.e. `collisions,' played in this system while the absence of this dependence and thus of collisions, in the corresponding theta system highlighted the important role played by thermodynamics in determining the magnitude of the effect of intercoil behavior on intracoil behavior. These results were also consistent with those of an earlier study of coil-coil interactions in dilute solution in which it was also found that thermodynamics played the dominant role.