Quantification of the distribution of macromolecules in vascular tissue

Quantitative tools to assess vascular macromolecular distributions have so far been limited by post-experiment artifact, low signal-to-noise ratios, limited spatial resolution, and the inability to provide multi-dimensional drug distribution profiles. This suggests the need to develop new techniques that overcome these limitations. A new technique combining fluorescence microscopy with digital post-processing has been developed. Quantitative fluorescence microscopy studies have been limited by autofluorescence, the process where endogenous compounds emit energy at the same wavelength as fluorescent labels, and the proper conversion of fluorescent intensities to physical concentrations. Specimens were imaged with two different filter sets with one image consisting of signal from both autofluorescent molecules and fluorescent labels and the other image containing signal only from autofluorescence. The latter image is then used to estimate the autofluorescence in the former. Subtraction of the estimated autofluorescence results in an autofluorescence-free image. A standard curve to convert fluorescent intensities to macromolecular concentrations was constructed by examining tissue specimens that were incubated until equilibrium in different concentrations. The end result is a two-dimensional concentration profile with spatial resolution superior to many of the previous methods to quantify macromolecular distributions. To illustrate the utility of this technique, the transvascular transport of fluorescently labeled 20 kD dextrans in the calf carotid artery is examined using an in vitro perfusion apparatus. A mathematical model of transport is evaluated using measured concentration profiles yielding estimates of important transport parameters such as the effective diffusivity, and endovascular and perivascular permeabilities. Future experiments incorporating this new technique will be able to elucidate details of macromolecular transport which can then be used to improve the efficacy and efficiency of drug delivery systems. Thesis Supervisor: Elazer R. Edelman Title: Thomas D. and Virginia W. Cabot Associate Professor, Division of Health Sciences and Technology