Nanoscale fluorescence correlation spectroscopy on intact living cell membranes with NSOM probes.
Characterization of molecular dynamics on living cell membranes at the nanoscale is fundamental to unravel the mechanisms of membrane organization and compartmentalization. Here we demonstrate the feasibility of fluorescence correlation spectroscopy (FCS) based on the nanometric illumination of near-field scanning optical microscopy (NSOM) probes on intact living cells. NSOM-FCS applied to fluorescent lipid analogs allowed us to reveal details of the diffusion hidden by larger illumination areas. Moreover, the technique offers the unique advantages of evanescent axial illumination and straightforward implementation of multiple color excitation. As such, NSOM-FCS represents a powerful tool to study a variety of dynamic processes occurring at the nanometer scale on cell membranes.
Carlo
C
Manzo
Thomas S
TS
van Zanten
Maria F
MF
Garcia-Parajo
D004058:Diffusion; D021962:Membrane Microdomains / Q000737:chemistry* / Q000648:ultrastructure*; D018613:Microscopy, Confocal / Q000379:methods; D020527:Microscopy, Scanning Probe / Q000295:instrumentation / Q000379:methods*; D056004:Molecular Dynamics Simulation; D049329:Nanostructures / Q000737:chemistry* / Q000648:ultrastructure*; D013050:Spectrometry, Fluorescence / Q000379:methods*
Biophys J. 2005 Dec;89(6):4029-42
16199500
Proc Natl Acad Sci U S A. 2006 Jan 10;103(2):329-34
16407160
Proc Natl Acad Sci U S A. 2010 Apr 13;107(15):6829-34
20351247
Biophys J. 2005 Jun;88(6):L43-5
15821167
Biophys J. 2006 May 1;90(9):3288-99
16461393
Nat Methods. 2007 Nov;4(11):963-73
17971781
Science. 2010 Jan 1;327(5961):46-50
20044567
Nano Lett. 2008 Feb;8(2):642-6
18229969
Appl Opt. 2001 Aug 20;40(24):4011-21
18360435
Annu Rev Biophys Biomol Struct. 2004;33:269-95
15139814
Opt Express. 2006 Jan 23;14(2):956-69
19503416
Cytometry. 1999 Jul 1;36(3):176-82
10404965
J Cell Biol. 2002 Jun 10;157(6):1071-81
12058021
Small. 2010 Jan;6(2):270-5
19943247
Nano Lett. 2009 Sep;9(9):3330-6
19591452
Nature. 2009 Feb 26;457(7233):1159-62
19098897
Biophys J. 2007 Feb 1;92(3):913-9
17085499