1. Software for lattice light-sheet imaging of FRET biosensors, illustrated with a new Rap1 biosensor.
- Author
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O'Shaughnessy EC, Stone OJ, LaFosse PK, Azoitei ML, Tsygankov D, Heddleston JM, Legant WR, Wittchen ES, Burridge K, Elston TC, Betzig E, Chew TL, Adalsteinsson D, and Hahn KM
- Subjects
- Human Umbilical Vein Endothelial Cells cytology, Humans, Microscopy, Fluorescence, Shelterin Complex, Telomere-Binding Proteins genetics, Biosensing Techniques, Fluorescence Resonance Energy Transfer, Human Umbilical Vein Endothelial Cells metabolism, Image Processing, Computer-Assisted, Signal Transduction, Software, Telomere-Binding Proteins metabolism
- Abstract
Lattice light-sheet microscopy (LLSM) is valuable for its combination of reduced photobleaching and outstanding spatiotemporal resolution in 3D. Using LLSM to image biosensors in living cells could provide unprecedented visualization of rapid, localized changes in protein conformation or posttranslational modification. However, computational manipulations required for biosensor imaging with LLSM are challenging for many software packages. The calculations require processing large amounts of data even for simple changes such as reorientation of cell renderings or testing the effects of user-selectable settings, and lattice imaging poses unique challenges in thresholding and ratio imaging. We describe here a new software package, named ImageTank, that is specifically designed for practical imaging of biosensors using LLSM. To demonstrate its capabilities, we use a new biosensor to study the rapid 3D dynamics of the small GTPase Rap1 in vesicles and cell protrusions., (© 2019 O'Shaughnessy et al.)
- Published
- 2019
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