Similar protocols

Pipeline publication

[…] 0 M with a plan-fluar ×100 1.45NA objective lens and a Roper Cascade EMCCD 512B camera. Illumination was achieved using a 300W Xenon system, (Sutter) which presented the system as an even field of illumination in addition to the appropriate neutral density and Schott filters to modulate the light source. Wavelength selection was achieved using external filter wheels, (Applied Scientific Instrumentation) and the ET-Sedat set, (Chroma). Data sets were captured with an axial resolution of 100 nm using the MS-2000 stage (Applied Scientific Instrumentation) and a lateral resolution of 0.1645 microns per pixel. The system was full controlled and automated via the FRAP-AI software, (MAG Biosystems/Metamorph). All of the data sets were deconvolved using Huygens (Scientific Volume Imaging) after which visualization and analysis was carried out using Imaris, (Bitplane). Deconvolved images were assessed utilizing the ImarisColoc software (Bitplane) in manual mode. A 2D scatter plot showing intensity pairs in the image was thresholded to include only co-localized points in the three dimensional volume. This data was then extracted to a separate channel containing three dimensional co-localized points only., For each in situ immunofluorescence investigation, a minimum of 50 cells per experimental condition were examined in at least duplicate. In addition a more detailed quantitative analysis (as above) of the patterns of expression (e.g. intracellular versus membrane) was performed on between 10–25 cells and representative images are presen […]

Pipeline specifications

Software tools MetaMorph, Huygens, Imaris