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Routine fluorescence microscopy of single untethered protein molecules confined to a planar zone
Author(s) -
GAI HONGWEI,
GRIESS GARY A.,
DEMELER BORRIES,
WEINTRAUB SUSAN T.,
SERWER PHILIP
Publication year - 2007
Publication title -
journal of microscopy
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 0.569
H-Index - 111
eISSN - 1365-2818
pISSN - 0022-2720
DOI - 10.1111/j.1365-2818.2007.01776.x
Subject(s) - microscopy , fluorescence microscope , agarose , aqueous solution , planar , molecule , materials science , microscope , fluorescence , single molecule experiment , chemistry , analytical chemistry (journal) , nanotechnology , chromatography , optics , physics , computer science , computer graphics (images) , organic chemistry
Summary To bypass limitations of ensemble averaging biochemical analysis, microscopy‐based detection and tracking are needed for single protein molecules that are diffusing in aqueous solution. Confining the molecules to a planar zone dramatically assists tracking. Procedures of microscopy should be routine enough so that effort is focused on the biochemistry. Fluorescence microscopy and partial planar confinement of single, untethered, aqueous protein molecules have been achieved here by use of a routine procedure. With this procedure, multiple thermally diffusing Alexa 488‐stained bovine serum albumin molecules were observed during partial confinement to a thin aqueous zone next to a cover slip. The procedure produces confinement by partial re‐swelling of a previously dried agarose gel on the microscope slide. Confinement was confirmed through analysis that revealed thermal motion lower in the third dimension than it was in the plane of observation.