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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
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