Representative fluorescence traces of the internalized pH sensor in endolysosomes within iNs and SVG-A cells (associated with Fig. 4 ). (A) Sequential Z-maximum projections from a 3D time series of an iN cell, captured with 0.25 µm z-spacing and 2.6-s intervals using a ZEISS Lattice Lightsheet 7. The images are from a 10-min time series that began 4 h after a 2-h incubation with a Dextran mixture tagged with pHrodo Green (pH-sensitive) and Alexa Fluor 560 (pH-insensitive). For visualization, red and green channels were shifted horizontally by 10 pixels. Regions showing a red signal without green indicate neutral endolysosomes; the white arrow highlights a neutral endolysosome. The image represents two biological replicates. Scale bar: 5 µm. Data include 10-min time series from seven fields in a single differentiated iN sample. (B–D) Ratiometric fluorescence intensity plots of neutral (B) and acidic (C) endolysosomes in the somas of seven iNs, and acidic endolysosomes (D) from four SVG-A cells. iN data were acquired from 3D, 10-min time series captured every 2.6 s with 0.25 µm z-spacing, using a ZEISS Lattice Lightsheet 7. SVG-A data were acquired from 3D, 7-min time series captured every 2 s, with 0.25 µm z-spacing, using a lattice light sheet microscope modified with adaptive optics (MOSAIC). Vertical scale is logarithmic, and traces are arbitrarily aligned at t = 0 s. Shorter traces reflect incomplete tracking by the CME program.