Forster Resonance Energy Transfer (FRET) has enabled the visualization of dynamic protein interactions, studies of protease activity, alterations in membrane voltage potentials and calcium metabolism within living cells. It has also allowed the conduction of high-throughput screening assays such as for quantification of gene expression. However, FRET is confined to microscopy and it is becoming increasingly crucial to translate FRET assays 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.
The long term research goal is to establish optical fluorescence tomography, in particular FRET tomography, as a robust 3D quantitative imaging modality for pre-clinical and clinical oncology applications. As a step towards this goal, the research objective of this CAREER proposal is to test the hypothesis that quantitative whole-body FRET optical tomography of live small animals is achievable when patterned wide-field time-resolved Fluorescence Molecular Tomography (FMT) is employed. To accomplish this objective, the proposed research approaches are: (a) Implement and optimize patterned light strategies for multispectral time-resolved optical tomography; (b) Create new strategies for fast and robust FRET optical reconstructions based on time gates, Monte Carlo forward model reconstruction algorithms and graphical processor unit (GPU) computational architecture; (c) Establish FRET tomography in live animals by imaging NIR-Transferin labeled FRET pairs co-internalized in cells, implanted in live animals and quantify the donor fraction experiencing FRET (i.e. cell internalization).
