Resonant Energy Transfer (FRET) imaging has long been established as the best method for detecting protein interaction and sensing physiological state in living systems. However, 3D in vivo FRET imaging of whole organism remains highly challenging, because existing microscopy methods cannot perform deep- penetrating sub-cellular multi-channel FRET imaging. We propose to develop a deep tissue Fluorescence lifetime imaging (FLIM) method, named Lifetime Excitation-Emission Matrix Scanning Laser Optical Tomography (LEEM-SLOT) to: 1. Perform isotropic sub-cellular 3D imaging in deep tissue, 2. Perform multi- color FRET imaging in one scan, 3. Fully analyze multi-color FRET. LEEM-SLOT will permit fast FLIM tomography and reconstruction of lifetime images in 4x4 excitation emission matrices with 1-?m isotropic resolution over 500-?m depth. Lifetime analysis of spectrally unmixed LEEM images will allow parallel visualization of multiple FRET channels with CFP/GFP/YFP/mCherry and equivalents. We will apply the LEEM-SLOT method to two pilot studies of: 1. Map Kinase pathway activation in Ciona heart progenitor cells, and 2. Intracellular cAMP level during renal cystogenesis in live zebrafish. Through these studies we expect to establish the LEEM-SLOT method as a novel tool for studying the regional activation of signaling pathways in live embryos or tissues, and performing multi-modal FRET biosensing in live organisms.

Agency
National Institute of Health (NIH)
Type
Research Project (R01)
Project #
5R01EB015481-03
Application #
8662262
Study Section
Enabling Bioanalytical and Imaging Technologies Study Section (EBIT)
Program Officer
Conroy, Richard
Project Start
Project End
Budget Start
Budget End
Support Year
3
Fiscal Year
2014
Total Cost
Indirect Cost
Name
University of Arizona
Department
None
Type
Other Specialized Schools
DUNS #
City
Tucson
State
AZ
Country
United States
Zip Code
85721