Open AccessTotal internal reflection fluorescence correlation spectroscopy (TIR-FCS) with low background and high count-rate per molecule
Author(s) -
Kaï Hassler,
Marcel Leutenegger,
Per Rigler,
Ramachandra Rao,
Rudolf Rigler,
Michael Gösch,
Theo Lasser
Publication year - 2005
Publication title -
optics express
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.394
H-Index - 271
ISSN - 1094-4087
DOI - 10.1364/opex.13.007415
Subject(s) - fluorescence correlation spectroscopy , total internal reflection fluorescence microscope , total internal reflection , optics , fluorescence , materials science , spectroscopy , fluorescence spectroscopy , microscopy , oil immersion , analytical chemistry (journal) , chemistry , physics , chromatography , quantum mechanics
We designed a fluorescence correlation spectroscopy (FCS) system for measurements on surfaces. The system consists of an objective-type total internal reflection fluorescence (TIRF) microscopy setup, adapted to measure FCS. Here, the fluorescence exciting evanescent wave is generated by epi-illumination through the periphery of a high NA oil-immersion objective. The main advantages with respect to conventional FCS systems are an improvement in terms of counts per molecule (cpm) and a high signal to background ratio. This is demonstrated by investigating diffusion as well as binding and release of single molecules on a glass surface. Furthermore, the size and shape of the molecule detection efficiency (MDE) function was calculated, using a wave-vectorial approach and taking into account the influence of the dielectric interface on the emission properties of fluorophores.