Figure 1

Schematic Overview of Multicolor Cell Labeling and Multispectral Imaging Approach. (A) Four different cell labeling strategies were compared in chick embryos, including lipophilic dye (DiI), membrane label (Gap43-GFP), 1-color nuclear label (H2B-RFP) or 3-color (H2B-YFP, -RFP, -CFP or GFP), and membrane/nuclear labels (Gap43-YFP, Gap43-CFP or Gap43-GFP, and H2B-mCherry). (B) For each of the different experiments, one of the four different labeling options were microinjected into the lumen of the chick neural tube and the embryo was electroporated. (C or D) After re-incubation, the embryos were either sectioned in the hindbrain region and mounted on a glass slide with a coverslip or mounted as whole embryos for live imaging. (D-E) Each slice was imaged using either channel or lambda scanning in confocal multispectral imaging to obtain a 3D or 4D data set. (F) Images were processed to ensure that background was reduced for accurate cell identification in spot detection. (G) NC cells were tracked throughout time with spectral identification using a combination of AIM line intensity profile and Imaris spot detection. (H) Alternatively, the 3D static data were analyzed in Imaris with spot detection for automated cell identification and manual cell count. (I) Filament tracing (Imaris) was used to measure cellular processes on individual cells and provide a picture of cell-cell interactions.