Csu 21

An inverted fluorescence confocal microscope (Nikon Ti-E) equipped with a Yokogawa spinning disc (CSU-21), digital CMOS camera (Hamamatsu ORCA-Flash TuCam), and precision microscope stage (Piezo) was used for all microscopy work. An environmental control chamber was used for live-cell imaging experiments to maintain a 37° C and 5% CO₂ environment. Z-stacks were acquired with 0.2μm steps throughout the cell depth using a Nikon 100X Plan Apo objective for analysis of peroxisome morphology and colocalization and a Nikon 60X Plan Apo objective for all other experiments. For long-term peroxisome imaging, Z-stacks were acquired at 50ms exposure per step to limit laser exposure to <10 seconds per cell. Image acquisition was automated for sequential imaging of 20 individual cells over an 8-hour period. Time points in which cells moved out of the field of view were discarded.