Computational protocol: TSSC1 is novel component of the endosomal retrieval machinery

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Protocol publication

[…] Live-cell imaging was performed on a Nikon Eclipse Ti Microscope System (Nikon Instruments, Melville, NY) at 37°C and 5% CO2. Images were captured using an electron-multiplying charge-coupled device camera (Photometrics Evolve 512; Photometrics, Tucson, AZ) and a Plan Apo VC 60× H objective (numerical aperture [NA] 1.40). Diffraction-limited fixed-cell imaging was performed with a Zeiss 780 (Carl Zeiss). Superresolution imaging was performed using a Zeiss 880 Airyscan microscope and a 63×/1.40 NA Plan-Apochromat Oil DIC M27 objective lens (Carl Zeiss) and processed at setting 6. Image processing was performed in ImageJ (http://imagej.nih.gov/ij/), including cropping and average/maximum intensity projections. Photobleaching experiments were performed at 37°C and 5% CO2 on a Nikon Eclipse Ti Microscope System equipped with a Galvo miniscanner to allow photobleaching and subsequent spinning-disk confocal imaging. TIRF images were acquired with an Apo TIRF 100× objective. [...] Quantification of Spearman’s r values were performed in ImageJ using the PSC colocalization plug-in (). Organelle distance mapping was performed using the Imaris imaging platform (Bitplane, Belfast, United Kingdom). A pseudonucleus was added to the observable nuclear center, with a diameter of 10 μm. Shiga toxin–containing organelles in z-stacked time series were mapped using an estimated xy-diameter of 1 μm and threshold of 600. Distance of at least 75 detectable organelles was measured. Using a distance transformation from the pseudonucleus, the 3D distance of each detectable object was calculated. Data were processed and plotted in Python 2.7. […]

Pipeline specifications

Software tools ImageJ, Imaris
Applications SPIM, Microscopic phenotype analysis