Ti e eclipse inverted scope

The Dendra2 plasmid was generously provided by Dr. David Sherwood. Photoconversion was performed as previously described (Ihara et al., 2011 (link)). The ventral nerve cord of live transgenic animals was photoconverted and imaged using an UltraVIEW Vex (PerkinElmer) spinning disc confocal microscope (Nikon Ti-E Eclipse inverted scope; Hamamatsu C9100-50 camera) with a 40x objective and Volocity FRAP Plugin (Improvision). Selected ventral nerve cord and cell bodies were scanned with a 405 nm laser at 1% power. The photoconverted red-Mito-Dendra2 within axons was captured immediately after photoconversion as 0.5 μm z-stacks with a 60x objective. For experiments involving injured axons, GABA neurons were severed using a pulsed laser axotomy and then photoconverted within about 5 minutes. Imaging immediately after axotomy did not show red-Mito::Dendra2 signal within the injured axon commissures. To test mitochondrial trafficking from the ventral nerve cord to the axons, animals with or without axotomy were recovered from the agar pad after photoconversion, cultured at 20°C for 24 hour s, and then reimaged with identical settings using the 60x objective.

Transgenic animals expressing Mito::GFP or mCherry were analyzed at the young adult stage (see table S1). To assess mitochondria density in GABAergic neurons after axotomy, selected axons in transgenic animals expressing Mito::GFP or mCherry were cut using a Micropoint laser from Photonic Instruments (10 pulses, 20 Hz). The axotomized animals were recovered to NGM plates and cultured at 20°C. 6, 12 or 24 hours later, images were acquired as 0.3~0.5 μm z-stacks at room temperature on an UltraVIEW Vex (PerkinElmer) spinning disc confocal microscope (Nikon Ti-E Eclipse inverted scope; Hamamatsu C9100-50 camera) with a 60× CFI Plan Apo numerical aperture (NA) 1.4 oil-immersion objective using Volocity software (Improvision). To score mitochondria density, Mito::mCherry puncta in individual GABAergic axons were counted and then the axon length was measured. The number of mitochondria was normalized by the axon length and converted to density per 100 μm axon length. To compare the volume of Mito::mCherry puncta, Volocity software (Improvision) was used to measure the volume of individual puncta. All animals within each experiment were imaged on the same day with identical conditions including camera gain, exposure settings, and fluorescence filters.