title: Near-field fluorescence cross-correlation spectroscopy on planar membranes.
abstract: The organization and dynamics of plasma membrane components at the nanometer scale are essential for biological functions such as transmembrane signaling and endocytosis. Planarized nanoscale apertures in a metallic film are demonstrated as a means of confining the excitation light for multicolor fluorescence spectroscopy to a 55 ± 10 nm beam waist. This technique provides simultaneous two-color, subdiffraction-limited fluorescence correlation spectroscopy and fluorescence cross-correlation spectroscopy on planar membranes. The fabrication and implementation of this technique are demonstrated for both model membranes and live cells. Membrane-bound proteins were observed to cluster upon the addition of a multivalent cross-linker: On supported lipid bilayers, clusters of cholera toxin subunit B were formed upon cross-linking by an antibody specific for this protein; on living cells, immunoglobulin E bound to its receptor (FcεRI) on the plasma membranes of RBL mast cells was observed to form clusters upon exposure to a trivalent antigen. The formation of membrane clusters was quantified via fluorescence intensity vs time and changes in the temporal auto- and cross-correlations above a single nanoscale aperture. The illumination profile from a single aperture is analyzed experimentally and computationally with a rim-dominated illumination profile, yielding no change in the autocorrelation dwell time with changes in aperture diameter from 60 to 250 nm. This near-field fluorescence cross-correlation methodology provides access to nanoscale details of dynamic membrane interactions and motivates further development of near-field optical methods.
authors.affiliation: Department of Chemistry and Chemical Biology, Cornell University , Ithaca, New York 14853, United States.
authors.forename: Christopher V
authors.initials: CV
authors.lastname: Kelly
authors.forename: Devin L
authors.initials: DL
authors.lastname: Wakefield
authors.forename: David A
authors.initials: DA
authors.lastname: Holowka
authors.forename: Harold G
authors.initials: HG
authors.lastname: Craighead
authors.forename: Barbara A
authors.initials: BA
authors.lastname: Baird
mesh_terms: D000818:Animals; D045744:Cell Line, Tumor; D002462:Cell Membrane / Q000737:chemistry*; D002470:Cell Survival; D020342:Finite Element Analysis; D008051:Lipid Bilayers / Q000737:chemistry; D008407:Mast Cells / Q000166:cytology; D051381:Rats; D013050:Spectrometry, Fluorescence / Q000379:methods*
chemical_list: D008051:Lipid Bilayers