Fluorescence analysis of single mitochondria with nanofluidic channels

Single mitochondrial assays are uncovering a new level of biological heterogeneity, holding promises for a better understanding of molecular respiration and mitochondria-related diseases. Here, we present a nanoscale approach to trapping single mitochondria in fluidic channels for fluorescence microscopy. We fabricate the nanofluidic channels in polydimethylsiloxane and bond them onto a glass slide, creating a highly reproducible device that can be connected to external pumps and mounted to a microscope. Having a unique nanoscale cross section, our channels can trap single mitochondria from a purified mitochondrial preparation flown across. Compared with the traditional fluorescence method to monitor single mitochondrial membrane potential with glass slides and open fluidic chambers, our nanofluidic channels reduce background fluorescence, enhance focus, and allow ease in experimental buffer exchanges. Hence, our channels offer researchers a new effective platform to test their hypotheses on single mitochondria.