Autofluorescence intensity imaging for studying mitochondrial dynamics

Mitochondria are highly dynamic organelles that continuously go through fission and fusion, a process that characterizes mitochondrial dynamics. These dynamics are important for the maintenance of the cell and are factors in aging, metabolic-related diseases, and cancer. Autofluorescence imaging (AFI) is used to study metabolic changes in the cell using the endogenous fluorophores reduced nicotinamide adenine dinucleotide (phosphate) NAD(P)H and flavin adenine dinucleotive (FAD). AFI can be used to study the dynamics of mitochondria, but requires high imaging speeds to capture the mitochondria movement. Here, we describe a multiphoton imaging technique that simultaneously captures NAD(P)H and FAD with a single excitation wavelength of 790 nm, reducing imaging times. A cyanide experiment was performed to verify that AFI at this optimal wavelength captures metabolic changes in cells. The optical redox ratios were computed from NAD(P)H and FAD images obtained both simultaneously using a single excitation wavelength and sequentially using the absorption-matched excitation wavelengths. The AFI results at 790 nm support the simultaneous acquisition of NAD(P)H and FAD for further research of mitochondrial dynamics and the metabolic state of the cell.