3D and timelapse autofluorescence imaging of mitochondrial networking and morphology using 2-photon microscopy

The regulation of mitochondrial dynamics, involving both fission and fusion, is vital for maintaining a healthy population of mitochondria within the cell. Alterations in mitochondrial dynamics have been associated with various disease states, such as metabolic and neurodegenerative diseases and cancer. We describe a protocol for imaging NAD(P)H intensity to visualize the movement of mitochondria over time and in 3D to visualize the distribution of the mitochondrial network within the cell using multiphoton microscopy. This protocol motivates using label-free imaging techniques to study mitochondrial dynamics in a non- destructive manner, suitable for drug screening and understanding the effects of mitochondrial dynamic alterations in disease.