The role of photon statistics in fluorescence anisotropy imaging.
Anisotropy imaging can be used to image resonance energy transfer between pairs of identical fluorophores and, thus, constitutes a powerful tool for monitoring protein homo-association in living single cells. The requirement for only a single fluorophore significantly simplifies biological preparation and interpretation. We use quantitative methods for the acquisition and image processing of anisotropy data that return the expected error of the anisotropy per pixel based on photon statistics. The analysis methods include calibration procedures and allow for a balance in spatial, anisotropy, and temporal resolution. They are featured here with anisotropy images of fluorescent calibration beads and enhanced green fluorescent protein complexes in live cells.
Lidke
Keith A
KA
Department of Molecular Biology, Max Planck Institute for Biophysical Chemistry, 37077 Göttingen, Germany. dlidke@gwdg.de
Rieger
Bernd
B
Lidke
Diane S
DS
Jovin
Thomas M
TM
D000465:Algorithms; D016880:Anisotropy; D001185:Artificial Intelligence; D003198:Computer Simulation; D031541:Fluorescence Resonance Energy Transfer / Q000379:methods*; D049452:Green Fluorescent Proteins; D006367:HeLa Cells; D006801:Humans; D007089:Image Enhancement / Q000379:methods*; D007090:Image Interpretation, Computer-Assisted / Q000379:methods*; D018613:Microscopy, Confocal / Q000379:methods*; D008856:Microscopy, Fluorescence / Q000379:methods*; D008859:Microscopy, Polarization / Q000379:methods*; D008954:Models, Biological; D015233:Models, Statistical; D017785:Photons
D049452:Green Fluorescent Proteins