Large retrograde cargoes produce fluorescence voids within the axons of Lifeact-GFP-expressing neurons. (a–c) Cultured hippocampal neurons grown in a microfluidic device were transfected on DIV12 with Lifeact-GFP and cotransfected with either LC3-mRFP (autophagosome; a) Rab7-mRFP (late endosome; b), or Mito-TagRFP (mitochondria; c) and subjected to time-lapse imaging on DIV14. Representative dual-color 3D SIM projections of neurons expressing Lifeact-GFP and with different subcellular markers are magnified in the right panels, and overlapping regions are annotated. Scale bars as indicated. (d) Quantification of axon diameters with (+) and without (−) annotated markers. Data represent mean ± SEM (n = 10 axons for each marker from three independent cultures; **, P < 0.01; ***, P < 0.001, two-tailed unpaired t test). (e and f) Representative dual-color 3D SIM projections of axons expressing Lifeact-GFP, which were colabeled with the retrograde cargo marker CTB (e) or DIC^1B (f). Boxed regions are magnified in the right panels, and overlapping regions are annotated. Scale bars as indicated. Arrowheads indicate the locations of areas devoid of fluorescence (fluorescent voids).