A Quantitative In Vitro Fluorescence Imaging Method for Phospholipid Loss from Respirable Mineral Particles

Abstract

Respirable quartz and kaolin particles were treated with fluorescent-labeled phospholipids to model contact of fibrogenic and nonfibrogenic particles with pulmonary surfactant in the alveolar regions of the lung. Particles were used to challenge rat pulmonary macrophages in vitro at times from 1 d to 10 d. The objective was to develop a quantitative method to track surfactant components that adsorb to respirable particles in the lung or inside cells. Confocal laser scanning microscopy was used to image and quantify surfactant remaining on particles internalized by cells. Results indicate that the fluorescent label is removed from quartz particles quickly, with the fluorescence intensity less than 15% of initial value at 3 d, and about 5% at 10 d. In contrast, the kaolin particle-associated fluorescence was still ∼39% of initial intensity at 3 d, and 10–15% at 10 d. Unchallenged cells showed a background of ∼5%, and noninternalized particles did not exhibit any loss of fluorescence over the 10-d exposure. The results indicate the method may be useful in label-removal rate studies of respirable particles in vitro, with some cautions and limitations. Results are discussed and compared with similar studies using nonimaging techniques.