Imager d1m

For imaging, animals were mounted by placing them in a drop of cold, liquid 36% Pluronic F-127 with 1 mM tetramisole solution and pressed between two coverslips. The slides were brought to room temperature, solidifying the Pluronic F-127 gel and immobilizing the animals. Co-localization images were made using iVision software. Images were taken using a Zeiss Imager D1m upright compound microscope with a 40x dry objective. For confocal imaging, animals were immobilized by using 7.5% M9 agarose pads with 2.5 μl PolySciences 0.05 μm polystyrene microspheres. A Zeiss spinning disk confocal upright microscope with 100x oil immersion objective was used for select images to show additional details, including lysosomal imaging and connection imaging.

MitoTimer encodes a dsRed derivative that fluoresces green when reduced (first synthesized), but irreversibly shifts tored fluorescence as it oxidizes ^{20 (link)}. Adult day 2 rbw2834Si[P_mec-3::mitotimer::T54, CB-unc-119 + II ttTi5605] in unc-119 (ed3)^{20 (link)} animals were mounted as above on a Zeiss Imager D1m upright compound microscope with a 63x oil immersion lens. Samples were alternately measured under GFP and dsRed channels, keeping light intensity and exposure times constant between images. Images were quantified using ImageJ by selecting the ROI, subtracting the background, measuring red and green intensities, and calculating the red/green ratio.