title: Imaging Cellular Dynamics with Spectral Relaxation Imaging Microscopy: Distinct Spectral Dynamics in Golgi Membranes of Living Cells.
abstract: Spectral relaxation from fluorescent probes is a useful technique for determining the dynamics of condensed phases. To this end, we have developed a method based on wide-field spectral fluorescence lifetime imaging microscopy to extract spectral relaxation correlation times of fluorescent probes in living cells. We show that measurement of the phase and modulation of fluorescence from two wavelengths permit the identification and determination of excited state lifetimes and spectral relaxation correlation times at a single modulation frequency. For NBD fluorescence in glycerol/water mixtures, the spectral relaxation correlation time determined by our approach exhibited good agreement with published dielectric relaxation measurements. We applied this method to determine the spectral relaxation dynamics in membranes of living cells. Measurements of the Golgi-specific C6-NBD-ceramide probe in living HeLa cells revealed sub-nanosecond spectral dynamics in the intracellular Golgi membrane and slower nanosecond spectral dynamics in the extracellular plasma membrane. We interpret the distinct spectral dynamics as a result of structural plasticity of the Golgi membrane relative to more rigid plasma membranes. To the best of our knowledge, these results constitute one of the first measurements of Golgi rotational dynamics.
authors.affiliation: Centre for Micro-Photonics, Faculty of Science, Engineering and Technology, Swinburne University of Technology, Hawthorn, Victoria, Australia.
authors.forename: Alireza
authors.initials: A
authors.lastname: Lajevardipour
authors.affiliation: Centre for Micro-Photonics, Faculty of Science, Engineering and Technology, Swinburne University of Technology, Hawthorn, Victoria, Australia.
authors.forename: James W M
authors.initials: JW
authors.lastname: Chon
authors.affiliation: CSIR-Centre for Cellular and Molecular Biology, Uppal Road, Hyderabad, 500 007, India.
authors.forename: Amitabha
authors.initials: A
authors.lastname: Chattopadhyay
authors.affiliation: Centre for Micro-Photonics, Faculty of Science, Engineering and Technology, Swinburne University of Technology, Hawthorn, Victoria, Australia.
authors.forename: Andrew H A
authors.initials: AH
authors.lastname: Clayton
mesh_terms: D009327:4-Chloro-7-nitrobenzofurazan / Q000031:analogs & derivatives* / Q000032:analysis / Q000528:radiation effects; D065638:Absorption, Radiation; D002462:Cell Membrane / Q000737:chemistry / Q000648:ultrastructure; D002518:Ceramides / Q000032:analysis / Q000528:radiation effects*; D005456:Fluorescent Dyes / Q000032:analysis / Q000528:radiation effects*; D005990:Glycerol; D006367:HeLa Cells; D006801:Humans; D007425:Intracellular Membranes / Q000737:chemistry / Q000648:ultrastructure*; D008563:Membrane Lipids / Q000032:analysis / Q000528:radiation effects*; D018613:Microscopy, Confocal; D008856:Microscopy, Fluorescence / Q000379:methods*; D059010:Single-Cell Analysis; D012997:Solvents; D013050:Spectrometry, Fluorescence; D014867:Water
chemical_list: D002518:Ceramides; D005456:Fluorescent Dyes; D008563:Membrane Lipids; D012997:Solvents; D014867:Water; C037974:N-(7-(4-nitrobenzo-2-oxa-1,3-diazole))-6-aminocaproyl sphingosine; D009327:4-Chloro-7-nitrobenzofurazan; D005990:Glycerol