Two‐photon fluorescence lifetime imaging microscopy of macrophage‐mediated antigen processing

Abstract

SummaryTwo-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 Ϸ 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.

Fluorescence lifetime imaging in an optically sectioning programmable array microscope (PAM)

“…However, as with many spectroscopic techniques, lifetimes measured in a conventional widefield microscope are subject to distortions arising from contributions of out-of-focus light originating above and below the plane of best focus. This realization has led to the development of optically sectioned lifetime imaging techniques implemented in single (3,4, this publication) and multiphoton configurations (1,(5)(6)(7)(8). Image reconstruction procedures applied to widefield and structured illumination data have also been used to generate sectioned fluorescence lifetime images.…”

Metabolic Mapping of Breast Cancer with Multiphoton Spectral and Lifetime Imaging

“…FLIM is a recently developed imaging mode where the excited state lifetimes of the fluorophores within a specimen are revealed (usually in false color) (28)(29)(30)(31)44). The lifetime of the excited state, which gives rise to the emitted fluorescence photons, provides another dimension of information that is often independent of color.…”

Fluorescence-Lifetime Imaging Techniques for Microscopy

“…The sensitivity of fluorescence lifetime to the microenvironment has been used to measure pH (Sanders et al, 1995a), metal ion concentration (Piston et al, 1994;Lakowicz et al, 1994a), fluorescence resonance energy transfer (Oida et al, 1993;Gadella and Jovin, 1995), cellular photostress (Kö nig et al, 1996), and antigen processing (French et al, 1997). Other quantities such as molecular oxygen concentration, environmental polarity, and local order also may be measured using lifetime imaging techniques.…”