0.05 μm polystyrene microspheres

Synchronized Is[P_mec-4mCh1] adult day 2 animals were immobilized on 7.5% M9 agarose pads with 2.5 μL PolySciences 0.05 μm polystyrene microspheres. Exopher centers were photo-bleached with 7 pulses of the MicroPoint pulsed nitrogen pumped dye laser (neutral density filter at position 9, Lumencor solid state light source) attached to a Zeiss Inverted Axio Observer microscope (100x 1.4 N.A. objective) on an anti-vibration table. 1 frame was recorded every 5 seconds using constant excitation intensity and exposure time with a Qimaging EXi Blue camera. Images were analyzed with ImageJ. Exopher fluorescence intensity was normalized to the intensity of the first data point in each series.

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.