Nature Methods
- 5, 545 - 551 (2008)
Published online: 4 May 2008; | doi:10.1038/nmeth.1209
Improving the photostability of bright monomeric orange and red fluorescent proteinsNathan C Shaner1, 5, Michael Z Lin1, 2, Michael R McKeown1, 2, Paul A Steinbach1, 2, Kristin L Hazelwood4, Michael W Davidson4 & Roger Y Tsien1, 2, 31
Department of Pharmacology, University of California at San Diego, 9500 Gilman Drive, La Jolla, California 92093, USA. 2
Howard Hughes Medical Institute, University of California at San Diego, 9500 Gilman Drive, La Jolla, California 92093, USA. 3
Department of Chemistry and Biochemistry, University of California at San Diego, 9500 Gilman Drive, La Jolla, California 92093, USA. 4
National High Magnetic Field Laboratory and Department of Biological Science, The Florida State University, 1800 East Paul Dirac Drive, Tallahassee, Florida 32310, USA. 5
Present address: The Salk Institute for Biological Studies, 10010 N. Torrey Pines Rd., La Jolla, California 92037, USA.
Correspondence should be addressed to Roger Y Tsien rtsien@ucsd.edu All organic fluorophores undergo irreversible photobleaching during prolonged illumination. Although fluorescent proteins typically bleach at a substantially slower rate than many small-molecule dyes, in many cases the lack of sufficient photostability remains an important limiting factor for experiments requiring large numbers of images of single cells. Screening methods focusing solely on brightness or wavelength are highly effective in optimizing both properties, but the absence of selective pressure for photostability in such screens leads to unpredictable photobleaching behavior in the resulting fluorescent proteins. Here we describe an assay for screening libraries of fluorescent proteins for enhanced photostability. With this assay, we developed highly photostable variants of mOrange (a wavelength-shifted monomeric derivative of DsRed from Discosoma sp.) and TagRFP (a monomeric derivative of eqFP578 from Entacmaea quadricolor) that maintain most of the beneficial qualities of the original proteins and perform as reliably as Aequorea victoria GFP derivatives in fusion constructs.
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