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Multiple exposure speckle imaging (MESI) allows to map relative blood flows at the surface of biological tissues. MESI is an extension of laser speckle contrast imaging (LSCI). It relies on the computation of speckle contrast K for several exposure times T, allowing to discriminate the contribution of static scatters (bulk tissues) from that of moving scatterers (red blood cells). First, we have evaluated how a synthetic exposure acquisition scheme could strongly simplify the instrument for MESI, while remaining quantitative over a range of relevant flows. A microfluidic chip with controlled flows in channels with dimension representative of mice brain cerebral vasculature has been imaged using the classical modulated intensities approach and the synthetic exposure mode. This study allowed to propose guidelines in terms of readout dark noise and spatial response uniformity for the choice of a camera for MESI in the synthetic exposure mode. Second, we have evaluated how unwanted movements introduce bias in the speckle contrast calculation for a representative range of movement speeds. Mixed solutions of intralipid and glycerin in Brownian motion have been characterized to provide calibrated samples in terms of scatterers de-correlation times. High concentration of glycerin led to decorrelation times of several ms corresponding to actual values in small capillaries while low concentration of glycerin led to decorrelation times of 1ms or less corresponding to arterioles and arteries. The effects of the unwanted movement speed and direction have been measured for both lateral (x-y) and axial (z) movements. The bias introduced by unwanted movement in the (x-y) plane depends on the relative values of the time between frames and the scatterers decorrelation. In addition, for axial movements, parameters such as the numerical aperture (NA) and the magnification level (M) need to be considered due to their role in defining the depth of field.
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