Fluorescence lifetime imaging: Multi-point calibration, minimum resolvabledifferences, and artifact suppression

Background: Frequency-domain fluorescence lifetime imaging microscopy (FLIM ) is finding increasing use in the analysis of biological systems. However, the calibration. determination of resolvable lifetime differences, and eva luation of artifacts have not been extensively treated. We describe a multi -point method for calibrating a frequency-domain FLIM system, characterize the minimum detectable heterogeneity and intra- and inter-image lifetime di fferences, discuss the statistical treatment of FLIM data. and suggest meth ods for minimizing artifacts. Methods: A set of solutions exhibiting single-component lifetimes suffice f or accurately calibrating a reference material with a single-component life time, even in the absence of accurate data on the lifetimes of the individu al solutions or the reference material. We used a set of rhodamine 6G solut ions quenched with varying concentrations of iodide, leading to lifetimes o f 0.5-4.0 ns, to calibrate a 1 muM reference solution of rhodamine 6G in wa ter. Results: We measured a value of 4.11 ns with an estimated absolute error of +/-0.05 ns for the rhodamine 6G reference solution. With 57.7 MHz modulati on, the minimum detectable inter-image lifetime difference was 0.1-0.15 ns and the minimum detectable intra-image lifetime difference was 1-5 ps, allo wing solutions differing in lifetime by 40 and 70 ps to be easily distingui shed. The minimum detectable lifetime heterogeneity was 50-80 ps. Evaluatio n of replicate measurements of single solutions demonstrated that inter-ima ge instrument errors exceeded those predicted from intra-image statistics b y more than an order of magnitude. We also measured lifetimes and heterogen eity in 4 GFP variants (WTGFP, EGFP, S65T, and EYFP) with the technique. Conclusion: The multi-point calibration method is applicable to any system consisting of single-component lifetimes. Applying the method in our FLIM m icroscope allowed us to demonstrate a previously unreported degree of lifet ime resolution in a FLIM microscope. Cytometry 13:248-260, 2001. (C) 2001 W iley-Liss, Inc.