Date of Graduation


Document Type


Degree Name

Master of Science in Biomedical Engineering (MSBME)

Degree Level



Biomedical Engineering


Timothy J. Muldoon

Committee Member

Jeffrey C. Wolchok

Second Committee Member

Kyle P. Quinn


Fluorescence Lifetime, Glycolysis, Metabolism, Muscle Physiology, NADH, Oxidative Phosphorylation


Nicotinamide adenine dinucleotide (NAD) is a cofactor that serves to shuttle electrons during metabolic processes such as glycolysis, the tricarboxylic acid cycle, and oxidative phosphorylation (OXPHOS) [1-5]. The reduced form of NAD, NADH, is autofluorescent, making it a measurable characteristic in the investigation of metabolic changes. The fluorescence lifetime of NADH is dependent on the state of protein binding, specifically free or bound NADH, allowing for detection of metabolic shifts between glycolysis (free NADH) and OXPHOS (bound NADH) [6-16]. Fiber-coupled time-correlated single photon counting (TCSPC) equipped with an implantable needle probe can be used to measure NADH lifetime in vivo [17]. This method is minimally invasive, making it suitable for use in living tissue as opposed to current terminal methods [18-19]. Here, we use TCSPC to investigate the changes in energy metabolism during tetanic contraction of healthy and injured muscle. NADH lifetime was measured in the tibialis anterior (TA) muscles of male Sprague Dawley rats. On day zero, volumetric muscle loss injuries were surgically created in the left TA muscle of each rat. Following injury creation, NADH lifetime measurements were taken from the uninjured leg every other day and in the injured leg at the endpoints (days six and fourteen). A Harvard apparatus electrophysiology system was used for electrical stimulation of muscular contraction for serial measurements. The needle optrode was inserted into the center of the TA muscle and lifetime measurements were taken every 0.2 s for 90 s beginning 30 s prior to contraction and finishing 30 s following muscle relaxation. TCSPC measurements were then fit with biexponential decay functions to extract the ratio of free to protein-bound NADH (A1/A2 ratio), which also underwent fractal dimension (FD) analysis. Overall, there were no significant differences between the A1/A2 ratios and FD between the phases of contraction or between injured and healthy muscle tissue, but a few trends emerged. On average, the A1/A2 ratio was higher during and after contraction in healthy muscle compared to muscle at rest. This indicates that there is more free NADH in contracting and recovering muscle, suggesting that the rate of glycolysis increases at the onset of contraction. In injured muscle, the A1/A2 ratio was higher than healthy muscle across all phases of contraction, suggesting higher rates of glycolysis overall, which is consistent with current knowledge of inflammatory response to injury [20-21]. The A1/A2 ratio values decreased from day six to day fourteen post-injury, suggesting that as the tissue is regenerated, the A1/A2 ratio becomes more comparable to healthy muscle. The FD for all groups remained above 1.5, indicating meaningful frequency content above the noise floor. This data suggests that fiber-coupled TCSPC has the potential to measure changes in NADH binding in vivo in a minimally invasive manner. The small differences measured throughout this study indicate the need for large datasets and further investigation of system specificity. In future studies, this technique could be used to measure metabolic changes associated with NADH binding, such as metabolic diseases, cancer, or injury [7, 9-16].

Available for download on Monday, February 17, 2025