ABSTRACT
Following a light-induced excitation, the uorescence lifetime is the average residence time that a uorophore will spend in its excited electronic state prior to returning to the ground state (Figure 4.1). Since the excited state is energetically unstable, this time (usually in the range of picoseconds to microseconds) is sensitive to a number of intramolecular interactions, dened by the uorophore’s structure and the surrounding microenvironment. Such sensitivity makes the uorescence lifetime an important observable for identifying a molecule and its conformational states (e.g., folding and ligand binding) and sensing its microenvironment (e.g., pH, temperature, and collisional encounters with other molecules or surfaces). While such interactions will also aect the spectral signature of the molecule, these changes are usually minor and oen dicult to eciently resolve due to their characteristically broad emission spectra. In contrast, changes in the uorescence lifetime can be very pronounced (as much as 20-fold in some cases). In addition, the uorescence lifetime does not depend on the concentration of the uorophore under most experimental conditions, which makes uorescence lifetime probing less sensitive to artifacts produced by photobleaching and scattering. ese advantages are utilized in uorescence lifetime imaging microscopy (FLIM) techniques-a number of methods complimentary to conventional uorescence intensity measurements, such as wide-eld and confocal microscopy (Bastiaens and Squire 1999; Gadella et al. 1993; Lakowicz 2006; Lakowicz and Berndt 1991; Levitt et al. 2009; Periasamy and Clegg 2009; Wang et al. 1989, 1992). While single-point uorescence lifetime measurements of molecules in dierent environments have been around for almost a century, the rst spatially resolved FLIM image was acquired 20 years ago (Berezin and Achilefu 2010; Wang et al. 1989). Since then, the applications of FLIM techniques have grown exponentially in both basic and applied research. In particular, FLIM of intrinsically
4.1 Introduction: Why FLIM? .................................................................77 4.2 eoretical Underpinnings ...............................................................78 4.3 Experimental Designs, Data Acquisition, and Analysis ...............80
