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The pH probe CypHer™5E is effectively quenched by FM dyes.
Oliver Welzel1, Kristina Loy, Carsten H Tischbirek
1Department of Psychiatry and Psychotherapy, Friedrich-Alexander-University of Erlangen-Nuremberg, Erlangen, Germany. oliver.welzel@uk-erlangen.de
Journal of Fluorescence
|February 12, 2013
Summary
FM dyes like FM®2-10 and FM®1-43 can quench the pH-dependent probe CypHer™5E in live-cell microscopy. Careful use of low FM dye concentrations is recommended for dual-color imaging to avoid signal interference.
Area of Science:
- Cell biology
- Microscopy techniques
- Biochemistry
Background:
- Concurrent imaging of spectrally distinct fluorescence probes is crucial for live-cell microscopy.
- Interactions between fluorescent dyes beyond spectral overlap are often overlooked.
- pH-dependent probes like CypHer™5E are valuable tools in live-cell studies.
Purpose of the Study:
- To investigate the interaction between FM®2-10/FM®1-43 dyes and the pH-dependent probe CypHer™5E.
- To determine if FM dyes affect CypHer™5E fluorescence in live-cell microscopy.
- To assess the suitability of dual-color experiments using these probes.
Main Methods:
- Spectrophotometry was used to analyze dye interactions.
- Live-cell fluorescence microscopy was employed to observe probe behavior.
- Control experiments were conducted to verify reversibility and rule out bleaching.
Main Results:
- FM®2-10 and FM®1-43 were found to be effective quenchers of CypHer™5E fluorescence.
- The quenching effect was reversible and not caused by photobleaching.
- Dual-color measurements are feasible at low FM dye concentrations.
Conclusions:
- FM dyes can significantly impact CypHer™5E signals in dual-color live-cell imaging.
- Researchers must exercise caution when combining these dyes, especially for dynamic analyses.
- Optimized concentrations are necessary to mitigate quenching effects and ensure data integrity.
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