CONFOCAL LASER-SCANNING FLUORESCENCE MICROSCOPY OF INTACT UNFIXED RATLUNGS

Fluorescence microscopy is a potentially powerful tool for assessing t he pulmonary disposition of drugs. Difficulties maintaining lung tissu e in its native hydrated state, sectioning, and other disruptions of t he internal milieu create artifacts and complicate data interpretation . Confocal laser scanning fluorescence microscopy, which obviates many of the liabilities of conventional microscopy, was used to image inta ct, fully-hydrated, unfixed rat lung at depths up to 25 mu m beneath t he outer pleural surface. Rhodamine B, a relatively lipophilic fluores cent marker introduced ex vivo either intratracheally or by vascular p erfusion, was used to help define endothelial and epithelial barriers to drug transport, macrophages, interstitial spaces, and Type II alveo lar epithelial cells. The technique was used to characterize the dispo sition of the fluorescent 'model drug' 6-carboxyfluorescein at a singl e time point following its intratracheal instillation into anesthetize d rats. Dual wavelength excitation of both rhodamine B and 6-carboxyfl uorescein resulted in 'multi-colored' images showing the latter within pulmonary interstitial spaces and localized within punctate regions a long alveolar epithelial surfaces. (C) 1998 Elsevier Science B.V. All rights reserved.