title: Z-scan fluorescence profile deconvolution of cytosolic and membrane-associated protein populations.
abstract: This study introduces a technique that characterizes the spatial distribution of peripheral membrane proteins that associate reversibly with the plasma membrane. An axial scan through the cell generates a z-scan intensity profile of a fluorescently labeled peripheral membrane protein. This profile is analytically separated into membrane and cytoplasmic components by accounting for both the cell geometry and the point spread function. We experimentally validated the technique and characterized both the resolvability and stability of z-scan measurements. Furthermore, using the cellular brightness of green fluorescent protein, we were able to convert the fluorescence intensities into concentrations at the membrane and in the cytoplasm. We applied the technique to study the translocation of the pleckstrin homology domain of phospholipase C delta 1 labeled with green fluorescent protein on ionomycin treatment. Analysis of the z-scan fluorescence profiles revealed protein-specific cell height changes and allowed for comparison between the observed fluorescence changes and predictions based on the cellular surface area-to-volume ratio. The quantitative capability of z-scan fluorescence profile deconvolution offers opportunities for investigating peripheral membrane proteins in the living cell that were previously not accessible.
authors: School of Physics and Astronomy, University of Minnesota, Minneapolis, MN 55455, USA.
authors: Elizabeth M
authors: EM
authors: Smith
authors: School of Physics and Astronomy, University of Minnesota, Minneapolis, MN 55455, USA.
authors: Jared
authors: J
authors: Hennen
authors: School of Physics and Astronomy, University of Minnesota, Minneapolis, MN 55455, USA; Institute for Molecular Virology, University of Minnesota, Minneapolis, MN 55455, USA.
authors: Yan
authors: Y
authors: Chen
authors: School of Physics and Astronomy, University of Minnesota, Minneapolis, MN 55455, USA; Institute for Molecular Virology, University of Minnesota, Minneapolis, MN 55455, USA; Department of Biomedical Engineering, University of Minnesota, Minneapolis, MN 55455, USA. Electronic address: mueller@physics.umn.edu.
authors: Joachim D
authors: JD
authors: Mueller
mesh_terms: D002460:Cell Line; D002462:Cell Membrane / Q000737:chemistry*; D003600:Cytosol / Q000737:chemistry*; D005453:Fluorescence; D049452:Green Fluorescent Proteins / Q000737:chemistry*; D006801:Humans; D008565:Membrane Proteins / Q000737:chemistry*; D054803:Phospholipase C delta / Q000737:chemistry*
chemical_list: D008565:Membrane Proteins; D049452:Green Fluorescent Proteins; C518024:PLCD1 protein, human; D054803:Phospholipase C delta
keywords: Brightness; Peripheral membrane protein; Phospholipase C; Pleckstrin homology domain; Point spread function; Translocation