Abstract

The ability to observe the behavior of living cells and tissues provides unparalleled access to information regarding the organization and dynamics of complex cellular structures. While great strides have been made over the past thirty to forty years in the design and application of a variety of novel optical microscopic techniques, until recently, it has not been possible to image biological phenomena which occur over very short time periods (nanosecond to millisecond) or over short distances (10–100 Angstroms). However, the recent combination of: 1) very rapidly gated and sensitive image intensifiers; and 2) the ability to deliver fluorescence excitation energy to intact living biological specimens in a pulsed or sinusoidally modulated fashion, has allowed such measurements to become a reality through the imaging of the lifetimes of fluorescent molecules. This capability has resulted in the ability to observe the dynamic organization and interaction of cellular components on a spatial and temporal scale previously not possible using other microscopic techniques. This paper discusses: 1) the implementation of a fluorescence lifetime imaging microscope (FLIM); 2) a comparison, between time domain vs. frequency domain FLIM; 3) a review of some of the applications of such an instrument, including measurements of receptor topography and subunit interactions using fluorescence resonance energy transfer (FRET), fluorescence anisotropy of phospholipids in cell membranes, cytosolic free calcium (Ca²⁺_i) and the detection of Human Papillomavirus (HPV) infection in clinical cervicovaginal smears; and 4) future improvements in technology which would lead to enhanced performance of FLIM.

© 1995 Optical Society of America