Fluorescent proteins for single‐molecule fluorescence applications

Abstract

We present single-molecule fluorescence data of fluorescent proteins GFP, YFP, DsRed, and mCherry, a new derivative of DsRed. Ensemble and single-molecule fluorescence experiments proved mCherry as an ideally suited fluorophore for single-molecule applications, demonstrated by high photostability and rare fluorescence-intensity fluctuations. Although mCherry exhibits the lowest fluorescence quantum yield among the fluorescent proteins investigated, its superior photophysical characteristics suggest mCherry as an ideal alternative in single-molecule fluorescence experiments. Due to its spectral characteristics and short fluorescence lifetime of 1.46 ns, mCherry complements other existing fluorescent proteins and is recommended for tracking and localization of target molecules with high accuracy, fluorescence resonance energy transfer (FRET), fluorescence lifetime imaging microscopy (FLIM), or multicolor applications.

In vivo analysis of the 2-Cys peroxiredoxin oligomeric state by two-step FRET

“…E.g. mCherry was demonstrated to be more photostable than YFP and CFP, whereas its quantum yield is only a third of the YFP quantum yield (Shaner et al, 2004;Seefeldt et al, 2008). In a second approach, FRET-efficiency between CFP and mCherry was estimated in the absence of YFP.…”

Photoswitches: Key molecules for subdiffraction‐resolution fluorescence imaging and molecular quantification

“…Although, fluorescent proteins have the advantage of being genetically targeted, they exhibit a lower photostability than conventional organic fluorophores [105,106]. Bright organic fluorophores are therefore more attractive because the ultimate optical resolution is determined mainly by the number of photons detected from each individual molecule [38].…”

Single-molecule spectroscopy of fluorescent proteins

“…Monomeric red-emitting fluorescent proteins derived from the tetrameric DsRed are becoming increasingly available, and some have also been studied on the singlemolecule level [100,106,118]. mCherry was found to be the most suitable protein for single-molecule studies [118]; the pH-dependent blinking observed for these monomeric red fluorescent proteins was linked to conformational rearrangements that enable radiationless deactivation resulting in dark states [106].…”

“…mCherry was found to be the most suitable protein for single-molecule studies [118]; the pH-dependent blinking observed for these monomeric red fluorescent proteins was linked to conformational rearrangements that enable radiationless deactivation resulting in dark states [106].…”