Abstract
Förster resonance energy transfer (FRET) is a powerful biological tool for reading out cell signaling processes. In vivo use of FRET is challenging because of the scattering properties of bulk tissue. By combining diffuse fluorescence tomography with fluorescence lifetime imaging (FLIM), implemented using wide-field time-gated detection of fluorescence excited by ultrashort laser pulses in a tomographic imaging system and applying inverse scattering algorithms, we can reconstruct the three dimensional spatial localization of fluorescence quantum efficiency and lifetime. We demonstrate in vivo spatial mapping of FRET between genetically expressed fluorescent proteins in live mice read out using FLIM. Following transfection by electroporation, mouse hind leg muscles were imaged in vivo and the emission of free donor (eGFP) in the presence of free acceptor (mCherry) could be clearly distinguished from the fluorescence of the donor when directly linked to the acceptor in a tandem (eGFP-mCherry) FRET construct.
Highlights
The combination of genetically encoded fluorescent proteins and Förster resonance energy transfer (FRET) has become an important tool for reading out cell signaling processes [1,2] and a significant arsenal of FRET probes has been developed
We are developing a tomographic imaging capability for small animals that utilizes fluorescence lifetime imaging (FLIM) to read out and localize FRET, which we have demonstrated by applying it to live mice transfected with genetically expressed fluorophores
The donor eGFP of the GCLink FRET construct exhibits a consistently lower mean fluorescence lifetime compared to the free eGFP co-expressed with mCherry, showing that the lifetime reduction is specific to the GCLink FRET construct and is not due to intermolecular
Summary
The combination of genetically encoded fluorescent proteins and Förster resonance energy transfer (FRET) has become an important tool for reading out cell signaling processes [1,2] and a significant arsenal of FRET probes has been developed. To date, such readouts have been largely confined to fluorescence microscopy of cells in culture. We are developing a tomographic imaging capability for small animals that utilizes FLIM to read out and localize FRET, which we have demonstrated by applying it to live mice transfected with genetically expressed fluorophores. The instrumentation for time-resolved detection that is required to determine fluorescence lifetimes provides a means to characterize diffuse light transport and, by employing a time-resolved model for diffuse optical tomography, we are able to reconstruct three dimensional maps of fluorescence lifetime and quantum yield, as well as the optical properties of the sample [6]
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