Measurement of drug distribution in vascular tissue using quantitative fluorescence microscopy

Quantitative tools to assess vascular macromolecular distributions have bee n limited by low signal-to-noise ratios, reduced spatial resolution, postex perimental motion artifact, and the inability to provide multidimensional d rug distribution profiles. Fluorescence microscopy offers the potential of identifying exogenous compounds within intact tissue by reducing autofluore scence, the process by which endogenous compounds emit energy at the same w avelength as fluorescent labels. A new technique combining fluorescence mic roscopy with digital postprocessing has been developed to address these lim itations and is now described in detail. As a demonstration, histologic cro ss-sections of calf carotid arteries that had been loaded endovascularly wi th FITC-Dextran (20 kD) ex vivo were imaged at two different locations of t he electromagnetic spectrum, one exciting only autofluorescent structures a nd the other exciting both autofluorescent elements and exogenous fluoresce nt labels. The former image was used to estimate the autofluorescence in th e latter. Subtraction of the estimated autofluorescence resulted in an auto fluorescence-corrected image. A standard curve, constructed from arteries t hat were incubated until equilibrium in different bulk phase concentrations of FITC-Dextran, was used to convert fluorescent intensities to tissue con centrations. This resulted in a concentration map with spatial resolution s uperior to many of the precious methods used to quantify macromolecular dis tributions. The transvascular concentration profiles measured by quantitati ve fluorescence microscopy compared favorably with those generated from the proven en face serial sectioning technique, validating the former. In addi tion, the fluorescence method demonstrated markedly increased spatial resol ution. This new technique may well prove to be a valuable tool for elucidat ing the mechanisms of macromolecular transport, and far the rational design of drug delivery systems.