PROJECT SUMMARY/ABSTRACT
This is an application for a Maximizing Investigator’s Research Award (MIRA) submitted by the Early Stage
Investigator (ESI), Dr. Alex Walsh. Dr. Walsh’s research program focuses on the characterization and
development of autofluorescence lifetime microscopy for the quantification of cell metabolism and mitochondria
function of living cells. Abnormal cell metabolism and mitochondria dysfunction is a hallmark of many diseases
and pathological states. Furthermore, many drugs and pharmacological agents, including anesthesia, either
have direct mechanisms of action through altered metabolism signaling or indirect toxicities due to impaired
mitochondria function. Current research assays to evaluate cellular metabolism include cell-based assays such
genomic, metabolomic, and oxygen-consumption assays and whole-body imaging assays such as
fluorodeoxyglucose-positron emission tomography and carbon-13 magnetic resonance imaging. However,
these existing tools have limited spatial and temporal resolutions and the label-dependent nature of the assays
prevents assessment of dynamic metabolic states and multiple longitudinal assessments of the same samples.
Autofluorescence lifetime imaging of reduced nicotinamide adenine dinucleotide (NADH) within the mitochondria
and cytosol and flavin adenine dinucleotide (FAD) within mitochondria presents a unique, label-free, high-
resolution technique to evaluate cell metabolism. Autofluorescence lifetime microscopy is not dependent on
chemical or antibody labels, is non-contact, broadly applicable to any cell or tissue, well-suited for longitudinal
studies, and compatible with secondary assays. However, adoption of autofluorescence lifetime is currently
hindered by the advanced expertise needed to design and perform fluorescence lifetime experiments, obscurity
in the correlation between fluorescence lifetime metrics and molecular specificity, and a lack of robust models to
determine metabolic phenotype and mitochondria function from autofluorescence features. Therefore, Dr.
Walsh’s goals for the next five years will address these limitations. Goal 1: Determine the sensitivity and
specificity of autofluorescence lifetime imaging to identify cellular metabolic states and pathway utilization. Goal
2: Quantify autofluorescence lifetime imaging features across common lab models to determine the robustness
of autofluorescence metrics to report cellular metabolism. Goal 3: Develop, test, and optimize autofluorescence
imaging protocols to quantify mitochondria dynamics and function. The outcomes of this proposal will enable
metabolic measurements with high temporal resolution and specificity to evaluate cellular metabolism in living
cells. Additionally, autofluorescence imaging will provide a platform to evaluate the impacts of drug and
pharmacological agents, including anesthetics, on cell metabolism and mitochondria function in living cells,
tissues, and in vivo.
