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Abstract
We present an implementation of fluorescence correlation spectroscopy with spectrally resolved detection based on a combined commercial confocal laser scanning/fluorescence correlation spectroscopy microscope. We have replaced the conventional detection scheme by a prism-based spectrometer and an electron-multiplying charge-coupled device camera used to record the photons. This allows us to read out more than 80,000 full spectra per second with a signal-to-noise ratio and a quantum efficiency high enough to allow single photon counting. We can identify up to four spectrally different quantum dots in vitro and demonstrate that spectrally resolved detection can be used to characterize photophysical properties of fluorophores by measuring the spectral dependence of quantum dot fluorescence emission intermittence. Moreover, we can confirm intracellular cross-correlation results as acquired with a conventional setup and show that spectral flexibility can help to optimize the choice of the detection windows.
Highlights
Fluorescence correlation spectroscopy (FCS) is a method originally developed in the 1970s [1,2,3,4] that allows to measure diffusion properties, concentrations and interaction properties of fluorescently labelled biomolecules as well as photophysical properties of fluorophores in vitro and in vivo [5,6,7,8]
We present an implementation of fluorescence correlation spectroscopy with spectrally resolved detection based on a combined commercial confocal laser scanning/fluorescence correlation spectroscopy microscope
Fluorescence correlation spectroscopy is a powerful tool widely employed in biochemistry and cell biology to measure diffusion and interaction properties of biomolecules
Summary
Fluorescence correlation spectroscopy (FCS) is a method originally developed in the 1970s [1,2,3,4] that allows to measure diffusion properties, concentrations and interaction properties of fluorescently labelled biomolecules as well as photophysical properties of fluorophores in vitro and in vivo [5,6,7,8]. The focus of a confocal laser illumination and fluorescence detection system such as that of a CLSM defines a small observation volume that is fixed at a position of interest Due to their diffusion, fluorescently labelled molecules can enter and leave the focus, resulting in signal fluctuations at the detector. From the crosscorrelation of the signals of corresponding detection channels, one can identify co-diffusion of the binding partners and extract quantitative interaction as well as diffusion parameters This concept was extended recently to more than two detection channels using conventional dichroic mirrors and filters, prisms or gratings as dispersive elements as well as combinations or arrays of avalanche photodiodes (APDs) or multichannel photomultiplier tube (PMT)/multichannel plate (MCP) combinations as detectors [12,13,14,15]. Received 13 Jul 2010; revised 29 Sep 2010; accepted 1 Oct 2010; published 27 Oct 2010 8 November 2010 / Vol 18, No 23 / OPTICS EXPRESS 23820 resolution and to measure molecular interactions in living cells with spectrally optimized settings
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