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Two‐photon fluorescence lifetime imaging microscopy of macrophage‐mediated antigen processing
Author(s) -
French T.,
So P. T. C.,
Weaver, Jr D. J.,
CoelhoSampaio T.,
Gratton E.,
Voss, Jr E. W.,
Carrero J.
Publication year - 1997
Publication title -
journal of microscopy
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 0.569
H-Index - 111
eISSN - 1365-2818
pISSN - 0022-2720
DOI - 10.1046/j.1365-2818.1997.d01-632.x
Subject(s) - fluorescence microscope , fluorescein , fluorescence , intracellular , biophysics , confocal microscopy , two photon excitation microscopy , microscopy , alexa fluor , antigen , bovine serum albumin , flow cytometry , dextran , chemistry , endocytosis , fluorescence lifetime imaging microscopy , microbiology and biotechnology , biology , biochemistry , cell , pathology , immunology , optics , medicine , physics
Two‐photon fluorescence lifetime imaging microscopy was used noninvasively to monitor a fluorescent antigen during macrophage‐mediated endocytosis, intracellular vacuolar encapsulation, and protease‐dependent processing. Fluorescein‐conjugated bovine serum albumin (FITC–BSA) served as the soluble exogenous antigen. As a relatively nonfluorescent probe in the native state, the antigen was designed to reflect sequential intracellular antigen processing events through time‐dependent changes in fluorescence properties. Using two‐photon lifetime imaging microscopy, antigen processing events were monitored continuously for several hours. During this time, the initial fluorescein fluorescence lifetime of 0.5 ns increased to α 3.0 ns. Control experiments using fluorescein conjugated poly‐ l ‐lysine and poly‐ d ‐lysine demonstrated that the increase in fluorescence parameters observed with FITC–BSA were due to intracellular proteolysis since addition of the inert d ‐isomer did not promote an increase in fluorescence lifetime or intensity. Comparisons of intravacuolar and extracellular FITC–dextran concentration suggested active localization of dextran in the vacuoles by the macrophage. In addition, the kinetics of degradation observed using two‐photon microscopy were similar to results obtained on the flow cytometer, thus validating the use of flow cytometry for future studies.