Abstract
Forster resonance energy transfer (FRET) is a nonradiative transfer of energy between two fluorescent molecules (a donor and an acceptor) in nanometer range proximity. FRET imaging methods have been applied to proteomic studies and drug discovery applications based on intermolecular FRET efficiency measurements and stoichiometric measurements of FRET interaction as quantitative parameters of interest. Importantly, FRET provides information about biomolecular interactions at a molecular level, well beyond the diffraction limits of standard microscopy techniques. The application of FRET to small animal imaging will allow biomedical researchers to investigate physiological processes occurring at nanometer range in vivo as well as in situ. In this work a new method for the quantitative reconstruction of FRET measurements in small animals, incorporating a full-field tomographic acquisition system with a Monte Carlo based hierarchical reconstruction scheme, is described and validated in murine models. Our main objective is to estimate the relative concentration of two forms of donor species, i.e., a donor molecule involved in FRETing to an acceptor close by and a nonFRETing donor molecule.
Highlights
Fluorescence is a widely used readout of molecular localization that has enabled the elucidation of many biological processes
While Forster Resonance energy transfer (FRET) has been extensively applied in the nondestructive assessment of molecular phenomena in cell-based assays, it is becoming increasingly important to translate FRET assays to detect intra- as well as intermolecular interactions to small animal imaging where the in vivo physiological context is critical for drug development, the study of diseases, and fundamental cellular and molecular biology [12]
Quantitative imaging of FRET activity in preclinical models promises to be a very powerful tool which will allow the study of intermolecular specific protein-protein interactions in the nanometer range in situ
Summary
Fluorescence is a widely used readout of molecular localization that has enabled the elucidation of many biological processes. In cell-based assays employing intermolecular FRET interaction, the fluorophore pairs are typically conjugated to soluble ligands, e.g., EGF, transferrin and others, that can come into nanometer-range FRET proximity upon binding to their dimerized/oligomerized respective receptors The translation of such techniques to preclinical models holds tremendous potential for investigation of cancer biology and drug development. The principle of time resolved FMT is based on the measurement of photons exiting the animal model over time (usually over a few ns) upon the Received 2 Aug 2012; revised 15 Oct 2012; accepted 15 Oct 2012; published 8 Nov 2012 1 December 2012 / Vol 3, No 12 / BIOMEDICAL OPTICS EXPRESS 3164 injection of an ultra-short laser pulse into the animal model This measurement data type referred to as the temporal point spread function (TPSF) provides a wealth of information for tomographic reconstruction of fluorescent markers in thick tissue. The fluorophore characterization and tomographic studies presented demonstrate an accurate quantification of intermolecular FRET activity in vivo in small animal murine models
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