Related Experiment Video
Updated: Aug 8, 2026
Fluorescence-mediated Tomography for the Detection and Quantification of Macrophage-related Murine Intestinal Inflammation
Published on: December 15, 2017
Two-photon fluorescence lifetime imaging microscopy of macrophage-mediated antigen processing
1Department of Physics, University of Illinois at Urbana-Champaign 61801, USA.
Insights
This study used two-photon fluorescence lifetime imaging microscopy to track antigen processing in macrophages. It revealed time-dependent changes in fluorescence reflecting protease activity during endocytosis and degradation.
Area of Science:
- Cell Biology
- Immunology
- Biophysics
Background:
- Macrophage-mediated endocytosis is crucial for immune responses.
- Intracellular antigen processing involves complex biochemical events.
- Noninvasive monitoring of these processes is essential for understanding cellular mechanisms.
Purpose of the Study:
- To noninvasively monitor antigen processing within macrophages using advanced microscopy.
- To investigate the role of proteases in antigen degradation.
- To correlate fluorescence changes with specific intracellular events.
Main Methods:
- Utilized two-photon fluorescence lifetime imaging microscopy (2p-FLIM).
- Employed fluorescein-conjugated bovine serum albumin (FITC-BSA) as a model antigen.
- Conducted control experiments with fluorescein-conjugated poly-L-lysine and poly-D-lysine.
Main Results:
- Observed a significant increase in fluorescein fluorescence lifetime from 0.5 ns to 3.0 ns during antigen processing.
- Demonstrated that proteolysis is responsible for the observed fluorescence changes, as shown by inert D-isomer controls.
- Indicated active macrophage localization of FITC-dextran within vacuoles.
- Validated 2p-FLIM findings against flow cytometry data for antigen degradation kinetics.
Conclusions:
- Two-photon fluorescence lifetime imaging microscopy is a powerful tool for real-time monitoring of intracellular antigen processing.
- Protease activity is a key determinant of antigen processing and fluorescence changes within macrophages.
- The study validates flow cytometry as a reliable method for future antigen processing studies.
Abstract:
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 approximately 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.
