Journal
NATURE METHODS
Volume 5, Issue 6, Pages 545-551Publisher
NATURE PUBLISHING GROUP
DOI: 10.1038/nmeth.1209
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Funding
- NATIONAL INSTITUTE OF GENERAL MEDICAL SCIENCES [P20GM072033] Funding Source: NIH RePORTER
- NATIONAL INSTITUTE OF NEUROLOGICAL DISORDERS AND STROKE [R37NS027177, R01NS027177] Funding Source: NIH RePORTER
- Howard Hughes Medical Institute Funding Source: Medline
- NIGMS NIH HHS [P20 GM072033, P20 GM072033-04, GM72033] Funding Source: Medline
- NINDS NIH HHS [R01 NS027177, NS27177, R37 NS027177, R01 NS027177-18] Funding Source: Medline
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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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