title: Refractive index sensing of green fluorescent proteins in living cells using fluorescence lifetime imaging microscopy.
abstract: We show that fluorescence lifetime imaging microscopy (FLIM) of green fluorescent protein (GFP) molecules in cells can be used to report on the local refractive index of intracellular GFP. We expressed GFP fusion constructs of Rac2 and gp91(phox), which are both subunits of the phagocyte NADPH oxidase enzyme, in human myeloid PLB-985 cells and showed by high-resolution confocal fluorescence microscopy that GFP-Rac2 and GFP-gp91(phox) are targeted to the cytosol and to membranes, respectively. Frequency-domain FLIM experiments on these PLB-985 cells resulted in average fluorescence lifetimes of 2.70 ns for cytosolic GFP-Rac2 and 2.31 ns for membrane-bound GFP-gp91(phox). By comparing these lifetimes with a calibration curve obtained by measuring GFP lifetimes in PBS/glycerol mixtures of known refractive index, we found that the local refractive indices of cytosolic GFP-Rac2 and membrane-targeted GFP-gp91(phox) are approximately 1.38 and approximately 1.46, respectively, which is in good correspondence with reported values for the cytosol and plasma membrane measured by other techniques. The ability to measure the local refractive index of proteins in living cells by FLIM may be important in revealing intracellular spatial heterogeneities within organelles such as the plasma and phagosomal membrane.
authors.forename: Henk-Jan
authors.initials: HJ
authors.lastname: van Manen
authors.forename: Paul
authors.initials: P
authors.lastname: Verkuijlen
authors.forename: Paul
authors.initials: P
authors.lastname: Wittendorp
authors.forename: Vinod
authors.initials: V
authors.lastname: Subramaniam
authors.forename: Timo K
authors.initials: TK
authors.lastname: van den Berg
authors.forename: Dirk
authors.initials: D
authors.lastname: Roos
authors.forename: Cees
authors.initials: C
authors.lastname: Otto
mesh_terms: D002468:Cell Physiological Phenomena; D049452:Green Fluorescent Proteins / Q000502:physiology*; D007090:Image Interpretation, Computer-Assisted / Q000379:methods*; D008856:Microscopy, Fluorescence / Q000379:methods*; D012031:Refractometry / Q000379:methods*
chemical_list: D049452:Green Fluorescent Proteins