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.
Original language | English (US) |
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Pages (from-to) | 7392-7404 |
Number of pages | 13 |
Journal | ACS Nano |
Volume | 8 |
Issue number | 7 |
DOIs | |
State | Published - Jul 22 2014 |
Keywords
- fluorescence correlation spectroscopy (FCS)
- fluorescence cross-correlation spectroscopy (FCCS)
- membrane cross-linking
- membrane dynamics
- nanofabrication
- near-field
- subdiffraction-limited
ASJC Scopus subject areas
- Engineering(all)
- Materials Science(all)
- Physics and Astronomy(all)