(689a) Cytometric Evaluation of the Fluorescence Lifetime for Measurements of FRET and Protein-Expression Applications

Many recent advances in high-throughput technologies for single cell analysis focus on compactness, portability, disposability, and sterility.  Many of these devices are flow cytometry-based in that they measure visible light scattering and fluorescence as cells flow past a finely focused laser beam. Yet among the many rapidly advancing cytometric technologies no instrument has been developed with time-resolved capabilities.  Time-resolved cytometry systems can provide quantitative information for cellular assays; some examples include time-dependent discrimination of spectrally overlapping species, separation of autofluorescence from dim exogenous signals, and quantification of Forster resonance energy transfer.  Therefore this contribution presents the latest outcomes from applications we are now studying that require high throughput multi-exponential decay detection using flow cytometry.  The approaches we present include (1) development of a new FRET pair using green fluorescent protein and a unique fluorophore with an affinity to estrogen receptor alpha, (2) measurement of energy transfer between fluorescent proteins and commercial fluorophores to detect cellular apoptosis, (3) measurement of the loss of energy transfer between two exogenous fluorophores for the detection of integrin conformational changes, and (3) measurement of the fluorescence lifetime of near-infrared fluorescent proteins.  Each application will be presented briefly highlighting the utility of the fluorescence lifetime and the instrumentation used to detect the lifetime values.  All measurements involve culturing of mammalian cells or E. coli cultures.  As a whole, a number of different fluorescence lifetime results can be obtained including time shifts as small as 0.5 ns, multiple fluorescence decays from single events, and progress with lifetime-dependent cell sorting.  One major conclusion from this work is the ability to translate a new, and biologically important, time-dependent parameter onto any cytometry system.