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 (V_mean) and red cell velocity using the two-slit method under epifluorescence (V_epi) and transillumination (V_trans) microscopy. The velocities V_epi and V_trans obtained in vivo for 47 measurements in arterioles and venules (12- to 51-μm internal diameter) were linearly related by V_epi = 0.83 V_trans + 0.074, and the ratio V_epi V_trans decreased gradually with increasing vessel diameter (P ≤ 0.05). In vitro studies in tapered glass tubes (diameter 30-70 μm) were conducted for feed hematocrits (H_F) from 10 to 40%. Under transillumination, V_trans V_mean was nearly constant with an average of 1.56 ± 0.16 (SD) for all hematocrits and diameters. The velocity ratio, V_epi V_mean, however, decreased with H_F from 1.8 to 0.8 as H_F was increased from 10 to 40%. Theoretical considerations suggest that the variations of V_epi V_mean 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 microscope objective.