Abstract
The recent demonstration and utilization of fluorescent proteins whose fluorescence can be switched on and off has greatly expanded the toolkit of molecular and cell biology. These photoswitchable proteins have facilitated the characterization of specifically tagged molecular species in the cell and have enabled fluorescence imaging of intracellular structures with a resolution far below the classical diffraction limit of light. Applications are limited, however, by the fast photobleaching, slow photoswitching, and oligomerization typical for photoswitchable proteins currently available. Here, we report the molecular cloning and spectroscopic characterization of mKikGR, a monomeric version of the previously reported KikGR that displays high photostability and switching rates. Furthermore, we present single-molecule imaging experiments that demonstrate that individual mKikGR proteins can be localized with a precision of better than 10 nanometers, suggesting their suitability for super-resolution imaging.
Highlights
Green fluorescent proteins (GFPs) and GFP-like fluorescent proteins have found extensive use in molecular and cellular biology [1,2,3,4]
To gain access to molecular dynamics on short timescales, two important requirements are an efficient and rapid photoswitching into the ‘on’ state, and a bright fluorescence after switching. Another promising application of photoswitchable proteins is their use in super-resolution microscopy
This technique relies on the stochastic photoactivation and localization of single molecules (PALM, STORM, FPALM), in which a fluorescence image is constructed from the high-accuracy localization of individual fluorescent molecules that are switched on and off optically [16,17,18,19,20]
Summary
Green fluorescent proteins (GFPs) and GFP-like fluorescent proteins have found extensive use in molecular and cellular biology [1,2,3,4]. Single-molecule photoswitching Figure 5A shows fluorescence images of individual red mKikGR
Sign-in/Register to view highlights & summary Sign-in/Register to access full text options 
Talk to us
Join us for a 30 min session where you can share your feedback and ask us any queries you have
Schedule a call
