METHOD TO CREATE SMALL PHOTO-BLEACHED VOLUMES TO MONITOR BLOOD-PLASMAFLOW IN CAPILLARIES

A method has been developed to examine the movement of plasma in capil laries using intravital microscopy. Spatial transients in fluorescence properties are instantaneously induced by laser photo-bleach pulses a fter which the convective recovery can be monitored. The plasma is tag ged with fluorescent dyes coupled to bovine serum albumin, which is in jected well before the measurements and circulates with the blood stre am. A laser beam from an argon laser source, set to emit light with a wavelength of 488 nm, is focused on the illumination field diaphragm a nd creates a spot in the object plane of the microscope. At low laser power, the laser spot is aimed at a blood plasma gap between red blood cells in a capillary segment, using a steerable mirror. Light sensors , coupled to photo-multipliers in the secondary image plane of the mic roscope, record the light intensity of the moving plasma/dye while the preparation is continuously illuminated with a xenon epi-illuminating set-up. The laser photo-bleach spot is then used to bleach the dye co mplex within a 5.4 mu m segment of the capillary for less than 20 ms. The movement of the bleached plasma bolus is tracked by the photo-sens ors, placed sequentially along the capillary. Both dye and red blood c ell passage can be detected in the photo-multiplier signals, and the r elative velocities of the two blood components can be measured. Measur ements reveal that the ratio of transit times between blood plasma and red blood cells is 1.23 (SD = 0.22, N = 18), which is in good agreeme nt with measurements by other techniques.