Natalia B. Savchenko

Laser Doppler microscopy of biological objects with different optical properties

Quite a number of experimental techniques are used in biomedical research involving the registration of flow velocities of biological fluids. These arc: particle image velocimetry, ultrasonic Doppler velocimetry, speckle microscopy, transmission grating microscopy, etc. Each of these methods has its advantages but none of them provides means to study all the variety of biological objects and dynamic phenomena, via performing noninvasive measurements. An essential alternative method is laser Doppler (LD) spectroscopy and microscopy, based on the registration of Doppler frequency shifts of laser radiation scattered from moving particles. The method yields high spatial and temporal resolution and hence can be used in different fields of biophysics and biomedicine [1,2,3]. The amount of the integral information, averaged over all the particles traversing the probe volume, the real-time mode of measurements, the possibility of registration of the flow velocity profiles -all this makes LD microscopy an efficient method enabling to study biological objects of different levels of complexity with broad range of optical properties. The possibility of fluid flow measurements in live objects with the LD technique was first shown in 1972 [4] and since then the potentialities of this technique have been studied extensively [5, 6, 7J. Nonetheless, though there arc no apparent technical reasons which would prevent from designing an LD microscope (LDM) as a commercial device, LD microscopy has not become yet a conventional technique. There still exist problems of Doppler spectra interpretation and evaluation of data experimentally obtained from biological objects with different opticai properties. These will be discussed below. We describe here an LDM designed in Moscow State University specifically for biophysical and biomedical applications on the basis of our earlier experience in application of LD spectroscopy to the study of intracellular hydrodynamics [8] and of haemodynamics [9). The potentialities of the LDM are illustrated by the results of real time measurements of oscillating flow velocities in relation to two different phenomena: 1 -protoplasmicstreaming in plasmodium of myxomycete Physarum, and 2 -bloodflow in aquarium Danio rerio fish embryo. To carry out measurements in biological objects with different optical properties and a broad range of values of measured parameters the following innovations have been introduced: - inl.roduction of controlled high stability frequency shift in the probing beams; - two-steps formation of optimal probe volume; - generation of the output signal with high signal-to-noise ratio both in analog and in photon ounting regimes; - arrangement of computer controlled fast scanning;- elaboration of the software for signal processing and calculation of parameters under study

Decrease of hydrodynamic resistance in rat mesenteric arterioles in response to injection of polyethylene oxide polyox WSR-301

Adequate blood supply of tissues and organs is essential to the normal functioning of the organism. From the hemodynamic point of view, this is achieved, in particular, through variation of the ratio between local microcirculatory resistances. Since the classical studies of Poiseuille [6], the role of the diameter of resistive vessels arterioles in the total blood flow resistance and in the redistribution of the blood flow in the microcirculatory bed has been well documented. However, the particular contribution of the flow pattern to these processes, for example, microdisturbances caused by the pulsatile flow, the movement of formed elements in a shear flow, at bifurcations of the yessets has been less studied, primarily due to methodological complications of both a physiological and a mathematical nature. High-molecular linear polymers are widely used in hydrodynamics, as they reduce the hydrodynamic resistance of the liquid by acting on the time structure of the flow [3,9]. When injected into the circulation, these substances reduce to a greater or lesser extent the total vascular resistance due to a drop of the systemic arterial pressure and/or increase of the cardiac out-

Laser Doppler monitoring of alterations of blood-flow parameters in fish embryos in response to light irradiation: study of long-term and short-term reactions

In this paper new experimental results on monitoring of alterations of blood flow parameters in growing fish embryos under laser and non-laser light irradiation are discussed. The measurements were performed by means of laser Doppler technique with high temporal and spatial resolution. Two parameters of blood flows were mostly measured: average velocity and frequency of velocity pulsations. These parameters were shown to be an adequate characteristics of nonstationary blood flows in fishes. The problem of noninvasivity of such experiments is discussed. For this purpose absorption spectra of fish embryos were measured. The quantitative response of blood flows to irradiation at different light wavelengths was recorded. Different species of fishes were used to compare the responses. Different effects were recorded which depend upon the doze and the wavelength of irradiation, and upon the stage of the embryo development at which the irradiation had taken place. Among those effects long-term and short-term reactions can be distinguished.

Assessment of whole blood structure and dynamics in vitro and in vivo by laser light scattering

Laser backscattering nephelometry is used to retrieve information on the kinetics of structural changes of erythrocytes in whole blood due to aggregation and deformation in shear flow in vitro. Laser Doppler microscopy is used to monitor nonstationary blood flows in single vessels in vivo. Monte-Carlo numerical analysis of Doppler signal is given for the case of highly scattering tissue surrounding of low. This case is related to in vivo blood perfusion measurements.

Laser Doppler monitoring of alterations in fish embryo blood flow in response to external stimuli

Noninvasive registration and adequate analysis of response of living systems to low energy physical stimuli play an important role in contemporary biological and biomedical research. In this paper we discuss the possibilities of laser Doppler microscopy (LDM) application for the study of light irradiation and thermal effects on animal organisms. Fish embryos are shown to fit many requirements related to the performance of experiments in clearly defined conditions. LDM yielding quantitative data on the dynamic response of the embryos to temperature variations and light irradiation is proved to be an adequate diagnostic technique. A fringe- mode sign-sensitive LD microscope was used to monitor the alterations of blood flow in fish embryos. Measurements were performed with spatial resolution as small as 10 micrometers in real time scale. Experimental data on the biological effects of thermal and nonthermal light stimuli on different fish species are presented.

Biological effects of low-energy laser and nonlaser light irradiation on fish embryos

The problems of choice of live models for the study of laser and nonlaser light irradiation effects on animal organisms are discussed. Fish embryos are shown to fit many requirements related to the performance of experiments in clearly defined conditions. Laser Doppler microscopy yielding quantitative data on the dynamic response of the embryos irradiation have proved to be an adequate technique. Preliminary experimental data on the biological effects of light on different types of embryos are presented.

LASER DOPPLER MICROSCOPY OF BLOOD FLOWS IN FISH EMBRYOS - AT DIFFERENT STAGES OF ONTOGENESIS

Laser Doppler microscopy is an efficient method of in vivo measurements of flow velocities in different biological objects. It is based on the registration of frequency shifts in light quasielastically scattered from particles moving in the flows. To study the embryonic development of the cardiac-vascular system in embryos of warm water fishes, embryos of Macropodus opercularis have been used. Doppler spectra from pulsatile blood flows in selected vessels and their changes in the process of ontogenesis have been registered. The recording of the successive spectra and their computer processing yield the varying dynamics of blood flows. Typical age dependencies of velocity patterns in the embryos are presented.

Light-scattering diagnostics of blood dynamics and structure

Laser backscattering nephelometry is used to retrieve information on the kinetics of structural changes of erythrocytes in whole blood due to aggregation and deformation in shear flow in vitro. Laser Doppler microscopy is used to monitor nonstationary blood flows in single vessels in vivo. Monte-Carlo numerical analysis of Doppler signal is given for the case of highly scattering tissue surrounding the flow. This case is related to in vivo blood perfusion measurements.