Enhanced Yellow Fluorescent Protein (eYFP) is one of the most widely used fluorescent proteins in cell biology. However, its applicability in localization based super-resolution imaging is affected by limited photostability and the need of UV-reactivation of dark states. For a detailed analysis of the intrinsic photophysical properties of YFP molecules the group of Prof. Tinnefeld synthesized semisynthetic conjugates of the fluorescent protein with single stranded DNA to enable targeted placement of single protein molecules on DNA origami nanostructures at defined positions. The analysis revealed a 6fold enhanced photostability under oxygen depletion and presence of aliphatic thiols in the measuring buffer. The beneficial effect for super-resolution imaging was demonstrated by resolving the distances between immobilized proteins on DNA-nanostructures with intermark distances of 160/100 nm at high quality without UV-reactivation and by imaging of fixated mammalian Vero-cells expressing YFP-Tubulin protein-fusions. Additionally, the compatibility with common imaging of organic fluorescent dyes (dSTORM method) was validated by two-color super-resolution imaging combining the yellow fluorescent protein with Alexa647 as red organic dye.
Jusuk I., Vietz, C., Raab M., Dammeyer T., Tinnefeld P.
Super-Resolution Imaging Conditions for Yellow Fluorescent Protein (eYFP) Demonstrated on DNA Origami Nanorulers
Scientific Reports 5, Article number: 14075 (2015)
DOI:10.1038/srep14075
Prof. Philip Tinnefeld
Institute for Physikal and Theoretical Chemistry
Technische Universität Braunschweig
Hans Sommer Str. 10
D-38106 Braunschweig