What is behind all those lifetimes anyway, and where do we go from here?

Abstract

Fluorescence lifetime-resolved imaging microscopy (FLIM) has made tremendous strides in the last two decades. Exciting applications are being presented weekly, an extensive diversity of instrumentation and commercial devices have appeared and have improved dramatically, sophisticated algorithms for analysis and interpretation are now available, and FLIM is being coupled to other imaging modalities, such as spectral dispersion and anisotropy. In other words, FLIM has matured considerably, and is approaching a point where it can be routinely applied by researchers who are not involved with the instrumentation and analysis side of things. The number of interested users in FLIM has almost certainly surpassed that of the audience that previously employed single channel fluorescence lifetime measurements (in a cuvette). The reason is, of course, the imaging capability of FLIM and its exciting possibilities for biological applications, especially FRET. In this lecture I will attempt to give an overview of where we have come from together with a personal judgment on where we stand, propose possible areas of growth that will be of importance for the future of FLIM, and discuss some specific challenges that remain, especially considering the unique nature of the FLIM measurement and the complex biological and medical systems that require innovative and novel solutions from the FLIM community. © 2009 SPIE.