Estimating leukocyte velocities from high-speed 1D line scans oriented orthogonal to blood flow

The fast and direct measurement of leukocyte behaviour in microvessels in situ is possible by labelling them with fluorescent compounds and subsequently imaging them by confocal laser scanning microscopy. Using a high-speed single line scanning technique a time dependent trace of leukocytes in microvessels can be recorded (x-t scan). These x-t traces, which are orientated orthogonal to the blood flow, are characterized by distinct, but often noise-affected elliptical streaks, which carry information on speed and frequency of leukocytes crossing the scan line. Here we describe the combination of a confocal laser scanning system and an image analysis software package that allows rapid and reproducible estimation of leukocyte velocities in microvessels.