Fluorescent Proteins (FP) have become essential reporter molecules for the elucidation of the function of proteins within cells, the bio-distribution of immune and stem cells and for evaluation of drug candidates in vivo. Volumetric detection and accurate quantification of Fluorescent Proteins in entire animals would greatly enhance our ability to monitor biological processes in vivo. Currently however, whole body fluorescent protein imaging is largely facilitated by photographic techniques that compromise the quantification and any volumetric imaging ability. The overall goal of this proposal is therefore to develop whole body, quantitative Fluorescence Protein Tomography (FPT) in entire animals. This development necessitates 1) the use of novel scanning technologies, 2) the advancement of theoretical models of photon propagation appropriate for imaging in the visible and far-red, 3) the design of computationally efficient inversion techniques with multispectral characteristics and 4) the interrogation of appropriate fluorescent proteins that can yield high detection sensitivity. Of particular importance for achieving high imaging performance is the utilization of freespace 360 degree projection tomographic principles and appropriate auto-fluorescent subtraction schemes that lead to unprecedented imaging performance compared to the current state of the art. Overall we hypothesize that the imaging accuracy imparted by FPT, combined with the high specificity and versatility achieved by fluorescent protein reporters and the high detection sensitivity afforded by novel red-shifted constructs can revolutionize biological imaging and propagate FPT as the method of choice in the biological laboratory for volumetric whole animal imaging.
