Images were captured on a Leica inverted TCS SP8 X confocal or DM2500 epifluorescence microscope. For time-lapse image capture, embryos were imaged as they developed in sea water-filled chambers on coverslip-bottom petri dishes (MatTek). Confocal image stacks were processed in Leica Application Suite or ImageJ. Video annotations were made using Camtasia software (TechSmith). 3D slices and projections were generated using Imaris (Bitplane) or Volocity (PerkinElmer) software. Kaede∷nls47 (link) was photoconverted as previously described48 (link). Neurite lengths and Golgi apparatus positioning were measured using ImageJ. Not all cells, neurites, and/or Golgi were visible in every embryo. Golgi positioning relative to BTN nuclei was measured in degrees of angle formed between a line traced anteriorly from the nucleus and another line traced through the middle of the Golgi complex. Thus, when the Golgi complex is perfectly aligned anterior to the nucleus, the angle is 0°, whereas if the Golgi complex is perfectly posterior to the nucleus, the angle is 180°. Rose plots (angle histograms) were generated in Matlab (http://www.mathworks.com/help/matlab/ref/rose.html ).
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Quantifying Neurodevelopmental Changes via Confocal Imaging
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Other organizations : New York University, Dalhousie University
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Variable analysis
independent variables
- Photoconversion of Kaede::nls protein
- Neurite lengths
- Golgi apparatus positioning
- Embryos imaged as they developed in sea water-filled chambers on coverslip-bottom petri dishes (MatTek)
- Confocal image stacks processed in Leica Application Suite or ImageJ
- Video annotations made using Camtasia software (TechSmith)
- 3D slices and projections generated using Imaris (Bitplane) or Volocity (PerkinElmer) software
- Kaede::nls photoconversion performed as previously described
- Not explicitly mentioned
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