Abstract
Fluorescence spectroscopy and fluorescence microscopy carried out on the single molecule level are elegant methods to decipher complex biological systems; it can provide a wealth of information that frequently is obscured in the averaging of ensemble measurements. Fluorescence can be used to localise a molecule, study its binding with interaction partners and ligands, or to follow conformational changes in large multicomponent systems. Efficient labelling of proteins and nucleic acids is very important for any fluorescence method, and equally the development of novel fluorophores has been crucial in making biomolecules amenable to single molecule fluorescence methods. In this paper we review novel coupling strategies that permit site-specific and efficient labelling of proteins. Furthermore, we will discuss progressive single molecule approaches that allow the detection of individual molecules and biomolecular complexes even directly isolated from cellular extracts at much higher and much lower concentrations than has been possible so far.
Highlights
Biophysical techniques have been invaluable to gain a detailed understanding of biological systems often providing quantitative and time-resolved data that complement data obtained by traditional biochemical experimental setups
Because single molecule techniques avoid the averaging effect seen in bulk experiments, subpopulations, competing reaction pathways and transient intermediates can be identified
Among the measurable parameters are the spectral properties of the fluorophore, the fluorescence intensity (‘brightness’), the quantum yield, the fluorescence lifetime and anisotropy
Summary
Biophysical techniques have been invaluable to gain a detailed understanding of biological systems often providing quantitative and time-resolved data that complement data obtained by traditional biochemical experimental setups. Circular holes of 50– 200 nm diameter in a metal cladding film of 100 nm thickness deposited on a transparent substrate (so called zeromode waveguides), for example, reduce the observation volumes and enable monitoring of enzymatic reactions at high substrate concentration (Figure 3c) [41] This technique led to the visualization of DNA polymerization and translation at the single-molecule level [36,42]. Since the created hot-spots are ultra-small the enhancement is restricted to the molecules in the hotspot and additional labelled species (even present at elevated concentrations) in the surrounding solution vanish compared to the increased signal in the hot-spot This opens the possibility to solve the concentration issue and allow single molecule assays at elevated concentrations. Fluorescence enhancement in the hot-spot might increase the signal-to-noise ratio to an extent that single molecules can even be detected in much bigger volumes opening the route to single molecule diagnostics at ultra-low concentrations
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