<title>Fluorescence lifetime imaging microscopy (FLIM) and its applications</title>

Abstract

Recent technological advances have provided an opportunity for the development of "Fluorescence Lifetime Imaging Microscopy" (FLIM). FLIM is an extremely important advance, as it allows for the first time, the sensitivity of the fluorescence lifetime to environmental parameters to be monitored in a spatial manner in single living cells. FLIM can be developed both on conventional and confocal fluorescence microscopes. Fluorescence lifetime detection can be performed using either time-or frequency-domain methods. In this paper, we report on the development of conventional and confocal FLIM systems currently underway in our laboratory. We also present three examples of current biological research projects in which we employ FLIM.

Fluorescence Lifetime Imaging with Near-Field Scanning Optical Microscopy

“…Alternatively, the system can be used to determine the ratio of the amplitude of two fluorescence decay components. Similar measurements have been made with confocal microscopy with reduced resolution and no topographic information. − …”

Probing Single Molecule Dynamics

“…The fluorescence lifetime measures an intrinsic property of the molecular species and its particular environment, such as pH, the presence of quenchers, or binding to a protein or DNA. In recent years, fluorescence lifetime imaging with the use of confocal microscopy has undergone rapid advancements (26,27). Below we demonstrate the feasibilitv of picosecond lifetime imaging with sub-diffraction-limit spatial resolution and single molecule sensitivity for two-dimensional samples.…”

Quantitative FRET imaging of leptin receptor oligomerization kinetics in single cells

“…Another manifestation of the FRET event is a decrease in the donor lifetime (Gadella and Jovin, 1995; Lakowicz, 1999; Krishnan et al, 2003). Experimentally, this can be calculated by measuring the fluorescence lifetime of the donor in the presence and absence of the acceptor (Wang et al, 1996; Bastiaens and Squire, 1999; Pepperkok and Squire, 1999) using the following expression: E =1−(τ DA /τ D ), where τ DA and τ D are the donor lifetimes in the presence and absence of acceptor. We carried out FLIM measurements using a newly developed multiphoton FLIM system using a streak camera (Krishnan et al, 2003).…”