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. 2018 Nov 14;7:e36248. doi: 10.7554/eLife.36248

Figure 2. Comprehensive, fate-specific tracking and analysis of individual cells.

Figure 2.

(A–B) ‘Fate sensor’ midguts enable the live identification of cell types. (A) Stack projection of a single time point from a 10 hr movie (Video 7). Nuclei are distinguishable for four midgut cell types: stem cells (red pseudocolor), enteroblasts (yellow-green pseudocolor), enterocytes (gray, polyploid), and enteroendocrine cells (gray, diploid). Inset shows the zoom region depicted in (B). (B) Genetic design of the fate sensor line (esg >his2b::CFP, GBE-Su(H)-GFP:nls; ubi-his2av::mRFP). Cell types are distinguished by the combinatorial expression of three fluorescent, nuclear-localized markers: enterocytes/enteroendocrine cells (His2ab::mRFP only), stem cells (His2ab::mRFP, His2b::CFP), and enteroblasts (His2ab::mRFP, His2b::CFP, GFP:nls). All scale bars, 10 μm. (C–D) Workflow to identify, track and analyze cells in volumetric movies. (C) Nuclei from raw, multi-channel z-stacks are digitally separated into stem cell, enteroblast, and enterocyte/entero-endocrine populations using channel masks in ImageJ. (D) The three population sets are rendered in 4D in Imaris. Segmentation is performed on each population to identify individual nuclei. Enteroendocrine nuclei are separated from enterocyte nuclei by a size filter. The positions of individual nuclei are correlated between time points to track single cells over time.