Towards Deep Tissue Imaging of Fluorescence Resonance Energy Transfer Parameters

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0854249WebbFluorescence resonance energy transfer (FRET) has proved to be of immense value in the study of chemical transport into cells and the underlying cause of disease due to, for example, protein misfolding (which may lead to Alzheimer's disease). The quenching of donor fluorescence by FRET transfer has been measured, and the intramolecular FRET parameters (lifetime and yield, and in the case of a flexible linker, the mean and variance of the linker distance) have been widely used. However, FRET imaging has been limited to in vitro or surface microscopy because of the deleterious effects of substantial scatter. The goal of this project is to demonstrate a method for imaging FRET parameters in highly scattering media, thereby providing the opportunity for deep tissue in vivo FRET studies. The new work planned is as follows:- Development on an Approach to Image FRET Parameters with ODT: A fluorescence resonance energy transfer optical diffusion tomography (FRET-ODT) imaging approach will be developed. This will involve a solution for the intramolecular FRET parameters with both rigid and flexible linkers that are incorporated as unknown sources in a diffusion equation representation for the donor fluorescence. The sensitivity to the parameter space (modulation frequencies, source-detector arrangement, FRET parameters, signal to noise ratio) will be studied and software suitable for use with experimental data for FRET sources in a scattering medium will be developed. The merits of incorporating acceptor kinetics will also be investigated. In a similar way, intermolecular FRET sources will be implemented in an ODT framework.- FRET-ODT Experimental Studies: A model tissue experiment will be constructed to obtain data to evaluate the FRET-ODT imaging approach to be developed. Data allowing the influence of varying degrees of scatter, varying FRET chemical concentration, varying donor-acceptor distance and distance distribution, and multiple modulation frequencies, as well as the effect of an inhomogeneous background, will be obtained. As an in vivo FRET-ODT application, a mouse model for targeted drug delivery to cancer cells, with the subsequent drug entry and release, will be studied using a FRET model that incorporates information about the transport kinetics and which emulates the drug delivery process. Information from this study will aid in the development of targeted drugs that are selectively delivered to cancer cells, thereby preventing damage to healthy cells that usually occurs during chemotherapy (for example, leucopenia - a deficiency in white blood cells, alopecia - hair loss, constipation, and hypertension). This work will be done with the Low group in the Dept. of Chemistry at Purdue.