2-PHOTON FLUORESCENCE LIFETIME IMAGING MICROSCOPY OF MACROPHAGE-MEDIATED ANTIGEN-PROCESSING

Two-photon fluorescence lifetime imaging microscopy was used noninvasi vely to monitor a fluorescent antigen during macrophage-mediated endoc ytosis, intracellular vacuolar encapsulation, and protease-dependent p rocessing, Fluorescein-conjugated bovine serum albumin (FITC-BSA) serv ed as the soluble exogenous antigen. As a relatively nonfluorescent pr obe in the native state, the antigen was designed to reflect sequentia l intracellular antigen processing events through time-dependent chang es in fluorescence properties. Using two-photon lifetime imaging micro scopy, antigen processing events were monitored continuously for sever al hours. During this time, the initial fluorescein fluorescence lifet ime of 0.5 ns increased to approximate to 3.0 ns. Control experiments using fluorescein conjugated polya-lysine and poly-D-lysine demonstrat ed that the increase in fluorescence parameters observed with FITC-BSA were due to intracellular proteolysis since addition of the inert D-i somer did not promote an increase in fluorescence lifetime or intensit y, Comparisons of intravacuolar and extracellular FITC-dextran concent ration suggested active localization of dextran in the vacuoles by the macrophage. In addition, the kinetics of degradation observed using t wo-photon microscopy were similar to results obtained on the flow cyto meter, thus validating the use of now cytometry for future studies.