Zeiss ELYRA - Fluorescent Proteins for PALM; Recommended FP-Pairs for dual-color PALM

Fluorescent Proteins for PALM
Theadvantageofphotoswitchableuorescentproteins(PS-FPs)liesintheiroutstandingspecicityandtheirsmallsize,
whichisaround2nm.Thelatterfeaturepotentiallyallowsforhighlabelingdensities.Amongphoto-switchableproteins
photoconvertable ones are the easiest to use as they - and the structure they stain – can be visualized at a different spec-
tralrangebeforeconversion.Alsoonecaneasilychecktransfectionefcienciesandexpressionlevels.E.g.tdEOSor
mEOScanbecheckedinthegreenspectralrange,whilethePALMexperimentwillbecarriedoutdetectingphoto-
switchedEOSmoleculesinamoreredshiftedspectralband.ThereforetdEOSoritsmonomericvariantmEOShave
been in extensive use as they also yield reasonable photon numbers.
Figure 3 Examples of fluorescent proteins that can be used for PALM imaging
Mostoftheseproteinsareavailablethroughe.g.Addgene,Cambridge,MA02139,USA
Recommended FP-Pairs for dual-color PALM
Pair1:mEOS2+Dronpa
Pair2:NeonGreen+PA-mCherry1
Pair3:Padron+Dronpa
IftwodifferentlystainedmoleculesinthesamesamplearesubjectedtoPALM,itisadvisabletorstimagethehigher
wavelength dye as this does not cross-talk into the shorter wavelength channel. Hence, cross-excitation of the longer
wavelength dye by the shorter wavelength and cross-emission of the shorter wavelength dye into the longer wave-
lengthchannelareminimized.Underexperimentalconditionsmanymoleculesoftheshorterwavelengthdyeare
equallyactivatedwiththe405nmlaserline,thatisusedforconversion/PALMimagingofthelongerwavelengthdye.
Thereforereversibleswitchableuorophoresarethepreferredchoicefortheshorterwavelengthastheycanberecov-
ered and are not irretrievably lost.
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