Quantitative laser scanning confocal microscopy on single capillaries: permeability measurement

Abstract

Objective: We tested the hypothesis that there is significant solute loss to the superfusate during microvessel solute permeability measurement in frog mesentery which leads to underestimation of permeability coefficient. This is the first application of laser-scanning confocal microscopy to measure microvessel permeability.

Methods: Microvessel permeability to sodium fluorescein (0.5 mg/ml, MW 376) was measured in the presence of bovine serum albumin (10 mg/ml) in Ringer's solution. Every 11-12s an optical section transverse to the vessel axis (x-z section) was collected. The images were corrected for fluorescence attenuation in the z direction and nonuniformity of light collection (shading) in the x direction by comparison to a fluorescent standard. Extravascular fluorescence, which increased linearly with time, and the step increase in fluorescence intensity of the vessel lumen were used to calculate an apparent permeability. Measurements were made first with the tissue superfused with Ringer and second after changing the superfusate to mineral oil in order to retain solute potentially lost to Ringer superfusate.

Results: The permeability to fluorescein measured with Ringer superfusate (5.0 +/- SD 2.6 x 10(-5) cm/s; n = 3) was not different from that measured with mineral oil covering the upper mesothelial surface (4.5 +/- 1.9).

Conclusions: Therefore, the loss of sodium fluorescein through the upper mesothelium does not significantly alter permeability under these conditions. These techniques enable detailed analysis of solute concentration gradients surrounding microvessels.