Simultaneous near-field and far-field fluorescence microscopy of single molecules

A new microscope objective is presented for the parallel fluorescence detection below and above the critical angle of total internal reflection with single molecule sensitivity. The collection of supercritical angle fluorescence (SAF) leads to a strongly surface confined detection volume whereas the...

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Bibliographic Details
Published in:Optics express 2011-03, Vol.19 (7), p.6836-6844
Main Authors: Ruckstuhl, Thomas, Verdes, Dorinel, Winterflood, Christian M, Seeger, Stefan
Format: Article
Language:English
Subjects:
Equipment Design
Equipment Failure Analysis
Image Enhancement - instrumentation
Lenses
Microscopy, Fluorescence - instrumentation
Molecular Probe Techniques - instrumentation
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Summary:A new microscope objective is presented for the parallel fluorescence detection below and above the critical angle of total internal reflection with single molecule sensitivity. The collection of supercritical angle fluorescence (SAF) leads to a strongly surface confined detection volume whereas the collection of undercritical angle fluorescence (UAF) allows for the observation of deeper axial sections of the specimen. By simultaneous detection of the near-field-mediated SAF and the far-field UAF emission modes the z-position of emitters can be obtained on the nanometer scale. We investigate the point spread function of the optics and demonstrate nanoscopic z-localization of single molecules. The oil immersion objective, developed for use on common microscope bodies, opens up new possibilities for the study of topographies and dynamics at surfaces and on membranes.
ISSN:1094-4087
1094-4087
DOI:10.1364/OE.19.006836