| The development of two different methods for fluorescence lifetime imaging in microscopy are presented in addition to an application to an important immunological problem. The first method uses a fast CCD camera and a gated image intensifier to collect lifetime data with the frequency domain heterodyning technique. The camera system is capable of generating lifetime images in a few seconds, has a frame rate of up to 200 Hz, and a time resolution of 200 ps. The instrument I built has several advantages over previously reported systems. Because of the data collection technique, this instrument can measure the correct lifetime even when the sample undergoes strong photobleaching which is the major problem in the other systems using a camera. The technique used to acquire lifetime images, demonstrations of the capabilities of a fast camera system, and sample images of biological specimens are presented.;The second fluorescence lifetime imaging method uses the relatively new technique of two photon excitation in microscopy. This instrument has a spatial point spread function of 0.3 ;Fluorescence lifetime resolved two photon microscopy was used to (non-invasively) monitor a fluorescent hapten-immunogen during intracellular vacuolar encapsulation and enzymatic processing. Fluorescein conjugated bovine serum albumin served as the soluble exogenous antigen. As a relatively non-fluorescent probe in the native state, the antigen was designed to reflect sequential intracellular antigen processing events through time dependent changes in the fluorescence properties. The fluorescence was limited to intracellular vacuoles. Three dimensionally resolved fluorescence lifetime images of the fluorescent probe in macrophages show that the initial lifetime of 0.5 ns increased to 2.2 ns after 24 hours of incubation indicating processing of the antigen. |
