In vivo and in vitro measurements of red cell velocity under epifluorescence microscopy

Studies of blood flow in mesentery, cremaster muscle, and small bore glass tubes were performed to obtain a relationship between mean velocity (Vmean) and red cell velocity using the two-slit method under epifluorescence (Vepi) and transillumination (Vtrans) microscopy. The velocities Vepi and Vtrans obtained in vivo for 47 measurements in arterioles and venules (12- to 51-micron internal diameter) were linearly related by Vepi = 0.83 Vtrans + 0.074, and the ratio Vepi/Vtrans decreased gradually with increasing vessel diameter (P less than or equal to 0.05). In vitro studies in tapered glass tubes (diameter 30-70 micron) were conducted for feed hematocrits (HF) from 10 to 40%. Under transillumination, Vtrans/Vmean was nearly constant with an average of 1.56 +/- 0.16 (SD) for all hematocrits and diameters. The velocity ratio, Vepi/Vmean, however, decreased with HF from 1.8 to 0.8 as HF was increased from 10 to 40%. Theoretical considerations suggest that the variations of Vepi/Vmean with tube hematocrit and diameter might result from attenuation of the excitation light by absorption and scattering by red cells, and also due to a finite depth of field of the microscopic objective.