Date of Graduation


Document Type


Degree Name

Bachelor of Science in Biomedical Engineering

Degree Level



Biomedical Engineering


Muldoon, Timothy


Macrophages are a type of immune cell that are characterized by their ability to differentiate into various active phenotypes based on environmental stimuli. They can generally be classified as one of two extremes: inflammatory/M1 or wound-healing/M2. Macrophage populations in vivo, however, are rarely homogenous, and since macrophages play vital roles in the immune response, particularly in the context of the tumor microenvironment (TME), it’s important to be able to investigate and understand heterogenous macrophage populations to develop more effective treatments. Recently, autofluorescence imaging such as multiphoton microscopy and fluorescence lifetime imaging microscopy (FLIM) of metabolic cofactors NADH and FAD have been used to determine the phenotypes of induvial macrophages based on their characteristic metabolic profiles. M1s show preferential utilization of glycolysis, whereas M2s more heavily utilize oxidative phosphorylation (OXPHOS). However, concentrations of these cofactors within the cells, while a potential indicator of metabolic shifts, can also be affected by environmental factors, such as temperature, pH, and oxygen availability, to name a few. The goal of this research was to verify that the changes observed using autofluorescence imaging were due to changes in metabolism rather than in environmental factors. This was done by comparing the imaging data to gold-standard extracellular flux assays (Seahorse assays) and characteristic metabolic intermediates (lactate and succinate) for the M1 phenotype. Correlations were then done between each of the 3 imaging methods, the 7 parameters from the Seahorse assays, and the concentrations of the 2 intermediates – 41 correlations total. Of the 41 correlations, 10 were statistically significant (p < 0.05), and they linked changes in FLIM to changes in metabolite concentrations and Seahorse parameters. These correlations indicate that autofluorescence imaging is a reliable method of monitoring macrophage metabolism during activation.


macrophage, metabolism, Seahorse, NADH, FLIM, autofluorescence imaging

Available for download on Friday, October 18, 2024