Hardware and software for single-molecule fluorescence analysis

Over the last three decades, fluorescence spectroscopy has developed into a unique tool for probing biologically relevant systems. In particular, fluorescence correlation spectroscopy (FCS) is now a routine measurement enabling biologists access to reaction kinetics (Wu et al. 2006; Heyduk and Niedziela-Majka 2002; Allen and Thompson 2006) and sizes of biomolecular assemblies (Pitschke et al. 1998). Likewise, single molecule Förster resonance energy transfer (smFRET) has proved to be useful for a variety of biologically relevant measurements ranging from DNA/RNA mechanics (Iqbal et al. 2008; Vafabakhsh and Ha 2012; Laurence et al. 2005; Wo ́zniak et al. 2008) to protein conformation (Wo ́zniak et al. 2005) and protein function (Gopich et al. 2009; Sevenich et al. 1998). When used in tandem these two techniques can provide unique perspective onto dynamics on molecular length-scales and over a wide range of time-scales (Nettels, Hoffmann, and Schuler 2008).