title: The pH probe CypHer™5E is effectively quenched by FM dyes.
abstract: Concurrent imaging of spectrally distinct fluorescence probes has become an important method for live-cell microscopy experiments in many biological disciplines. The technique enables the identification of a multitude of causal relationships. However, interactions between fluorescent dyes beyond an obvious overlap of their fluorescent spectra are often neglected. Here we present the effects of the well-established fluorescent dyes FM®2-10 or FM®1-43 on the recently introduced pH-dependent probe CypHer™5E. Spectrophotometry as well as live-cell fluorescence microscopy revealed that both FM dyes are effective quenchers of CypHer™5E. Control experiments indicated that this effect is reversible and not due to bleaching. We conclude that, in general, parallel measurements of both dyes are possible, with low FM dye concentrations. Nevertheless, our results implicate that special care has to be taken in such dual colour experiments especially when analysing dynamic CypHer™5E signals in live-cell microscopy.
authors: Department of Psychiatry and Psychotherapy, Friedrich-Alexander-University of Erlangen-Nuremberg, Erlangen, Germany. oliver.welzel@uk-erlangen.de
authors: Oliver
authors: O
authors: Welzel
authors: Kristina
authors: K
authors: Loy
authors: Carsten H
authors: CH
authors: Tischbirek
authors: Alina
authors: A
authors: Tabor
authors: Peter
authors: P
authors: Gmeiner
authors: Johannes
authors: J
authors: Kornhuber
authors: Teja W
authors: TW
authors: Groemer
mesh_terms: D000818:Animals; D002232:Carbocyanines / Q000737:chemistry*; D005456:Fluorescent Dyes / Q000737:chemistry*; D006624:Hippocampus / Q000166:cytology; D006863:Hydrogen-Ion Concentration; D008856:Microscopy, Fluorescence; D057054:Molecular Imaging; D009474:Neurons / Q000166:cytology; D011726:Pyridinium Compounds / Q000737:chemistry*; D000644:Quaternary Ammonium Compounds / Q000737:chemistry*; D051381:Rats; D017208:Rats, Wistar; D013050:Spectrometry, Fluorescence; D013572:Synaptic Vesicles / Q000378:metabolism
chemical_list: D002232:Carbocyanines; C474471:CypHer 5 NHS ester; C073804:FM1 43; C086686:FM2 10; D005456:Fluorescent Dyes; D011726:Pyridinium Compounds; D000644:Quaternary Ammonium Compounds
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