Fluorescence microscopy imaging of mitochondrial metabolism in cancer cells

Mitochondrial metabolism is an important contributor to cancer cell survival and proliferation that coexists with enhanced glycolytic activity. Measuring mitochondrial activity is useful to characterize cancer metabolism patterns, to identify metabolic vulnerabilities and to identify new drug targets. Optical imaging, especially fluorescent microscopy, is one of the most valuable tools for studying mitochondrial bioenergetics because it provides semiquantitative and quantitative readouts as well as spatiotemporal resolution of mitochondrial metabolism. This review aims to acquaint the reader with microscopy imaging techniques currently used to determine mitochondrial membrane potential (ΔΨm), nicotinamide adenine dinucleotide (NADH), ATP and reactive oxygen species (ROS) that are major readouts of mitochondrial metabolism. We describe features, advantages, and limitations of the most used fluorescence imaging modalities: widefield, confocal and multiphoton microscopy, and fluorescent lifetime imaging (FLIM). We also discus relevant aspects of image processing. We briefly describe the role and production of NADH, NADHP, flavins and various ROS including superoxide and hydrogen peroxide and discuss how these parameters can be analyzed by fluorescent microscopy. We also explain the importance, value, and limitations of label-free autofluorescence imaging of NAD(P)H and FAD. Practical hints for the use of fluorescent probes and newly developed sensors for imaging ΔΨm, ATP and ROS are described. Overall, we provide updated information about the use of microscopy to study cancer metabolism that will be of interest to all investigators regardless of their level of expertise in the field.