Confocal Microscopy and Image Analysis
All slides were imaged on a Nikon A1R confocal microscope using either a
40X or 60X objective. To analyze the percent area occupied by a specific
organelle, 10 fields per slide were imaged using the 40X objective.
These frames were processed using an in-house plugin for FIJI (National
Institutes of Health), which contained components inspired by the ImageJ
Mitochondrial Morphology Macro (Ruben K. Dagda Ph.D.; University of
Nevada School of Medicine, Stefan Strack Ph.D.; University of Iowa, and
Charleen Chu; University of Pittsburgh). Briefly, cell perimeters were
manually outlined, the channels were split, thresholded, and analyzed
for percent area of the appropriate channel.