Software for lattice light-sheet imaging of FRET biosensors, illustrated with a new Rap1 biosensor
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.
Document Details
- Document Type
- Pub Defense Publication
- Publication Date
- Aug 23, 2019
- Source ID
- 10.1083/jcb.201903019
Entities
People
- David Adalsteinsson
- Denis Tsygankov
- Ellen C. O’shaughnessy
- Eric Betzig
- Erika S Wittchen
- John M Heddleston
- Keith Burridge
- Klaus M Hahn
- Mihai L. Azoitei
- Orrin J. Stone
- Paul K LaFosse
- Teng-leong Chew
- Timothy C Elston
- Wesley R. Legant
Organizations
- Georgia Tech
- Gordon and Betty Moore Foundation
- Howard Hughes Medical Institute
- National Institutes of Health
- United States Department of Defense
- University of North Carolina at Chapel Hill