Computational protocol: Spinning-Spot Shadowless TIRF Microscopy

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Protocol publication

[…] Electron-multiplied CCCD (emCCD) cameras (Cascade, Roper Scientific) were used for acquisition of dynamic fluorescence signals; a Cascade 128 for fast (5 ms exposure time) imaging of Ca2+ signals, and a Cascade 650 to provide greater pixel resolution for slower (500 ms exposure time) imaging of STIM/Orai dynamics. By utilizing a TTL pulse generated by the scanner software to initiate acquisition by the camera, each frame represents an average across all 360° azimuthal incidence angles, producing the effect of a spatially and temporally uniform field of illumination. High resolution and color images (Figs and ) were obtained using a 35mm digital camera (Canon EOS M) with long (2s) exposures to summate over hundreds of scan cycles.Images from the Cascade emCCD cameras were acquired using MetaMorph (Universal Imaging/ Molecular Dynamics), but other software, including open-source packages such as Micro˗Manager (www.micro-manager.org) would be equally applicable. Processing and analysis was facilitated by a custom software package written in Python (). This enables black level subtraction, de-interleaving into separate image stacks (e.g. stacks representing near-simultaneous shadowless TIRF and WF images) and processing of each stack to yield pseudo-ratio (ΔF/F0) image stacks where the value of each pixel is normalized relative to the average value calculated from a specified range of resting frames acquired before stimulation. Duplicate regions of interest can then be defined on multiple image stacks, which are linked so that movement of one region is mirrored by its paired region on the other stack. A window displays superimposed traces over time of average pixel intensities within the paired regions, and these traces can be saved as ASCII files for subsequent analysis and plotting.The open-source software routines for controlling the scan mirrors are available at https://github.com/kyleellefsen/shadowlessTIRF. Open-source software used for image processing is available at https://github.com/kyleellefsen/Flika. […]

Pipeline specifications

Software tools MetaMorph, μManager, Flika
Application Laser scanning microscopy