Total internal reflection fluorescence correlation spectroscopy (TIR-FCS) with low background and high count-rate per molecule
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. (c) 2005 Optical Society of America.
Keywords: (170.6280) Medical optics and biotechnology ; Spectroscopy ; fluorescence and luminescence ; (240.6490) Optics at surfaces ; Spectroscopy ; surface ; (300.2530) Spectroscopy ; Fluorescence ; laser-induced ; Spectroscopy
Record created on 2006-10-03, modified on 2016-08-08