title: Fluorescence recovery after photobleaching and photoconversion in multiple arbitrary regions of interest using a programmable array microscope.
abstract: Photomanipulation (photobleaching, photoactivation, or photoconversion) is an essential tool in fluorescence microscopy. Fluorescence recovery after photobleaching (FRAP) is commonly used for the determination of lateral diffusion constants of membrane proteins, and can be conveniently implemented in confocal laser scanning microscopy (CLSM). Such determinations provide important information on molecular dynamics in live cells. However, the CLSM platform is inherently limited for FRAP because of its inflexible raster (spot) scanning format. We have implemented FRAP and photoactivation protocols using structured illumination and detection in a programmable array microscope (PAM). The patterns are arbitrary in number and shape, dynamic and adjustable to and by the sample characteristics. We have used multispot PAM-FRAP to measure the lateral diffusion of the erbB3 (HER3) receptor tyrosine kinase labeled by fusion with mCitrine on untreated cells and after treatment with reagents that perturb the cytoskeleton or plasma membrane or activate coexpressed erbB1 (HER1, the EGF receptor EGFR). We also show the versatility of the PAM for photoactivation in arbitrary regions of interest, in cells expressing erbB3 fused with the photoconvertible fluorescent protein dronpa.
authors.affiliation: Laboratory of Cellular Dynamics, Max Planck Institute for Biophysical Chemistry, Göttingen, Germany. guy.hagen@lf1.cuni.cz
authors.forename: Guy M
authors.initials: GM
authors.lastname: Hagen
authors.forename: Wouter
authors.initials: W
authors.lastname: Caarls
authors.forename: Keith A
authors.initials: KA
authors.lastname: Lidke
authors.forename: Anthony H B
authors.initials: AH
authors.lastname: De Vries
authors.forename: Cornelia
authors.initials: C
authors.lastname: Fritsch
authors.forename: B George
authors.initials: BG
authors.lastname: Barisas
authors.forename: Donna J
authors.initials: DJ
authors.lastname: Arndt-Jovin
authors.forename: Thomas M
authors.initials: TM
authors.lastname: Jovin
mesh_terms: D002460:Cell Line; D004847:Epithelial Cells / Q000737:chemistry / Q000648:ultrastructure; D036681:Fluorescence Recovery After Photobleaching; D017930:Genes, Reporter; D006801:Humans; D008856:Microscopy, Fluorescence / Q000379:methods*; D020893:Receptor, ErbB-3 / Q000378:metabolism; D011993:Recombinant Fusion Proteins / Q000378:metabolism
chemical_list: D011993:Recombinant Fusion Proteins; D020893:Receptor, ErbB-3