Journal of Fluorescence

, Volume 23, Issue 3, pp 487–494 | Cite as

The pH Probe CypHer™5E is Effectively Quenched by FM Dyes

  • Oliver Welzel
  • Kristina Loy
  • Carsten H. Tischbirek
  • Alina Tabor
  • Peter Gmeiner
  • Johannes Kornhuber
  • Teja W. Groemer
ORIGINAL PAPER

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.

Keywords

FM dye CypHer™5E Fluorescence microscopy Synaptic vesicles Hippocampal neurons 

Abbreviations

FM® 2-10

N-(3-Triethylammoniumpropyl)-4-(4-(Diethylamino) Styryl) Pyridinium Dibromide

FM® 1-43

N-(3-Triethylammoniumpropyl)-4-(4-(Dibutylamino) Styryl) Pyridinium Dibromide

GFP

Green fluorescent protein

CHAPS

3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonate

CMC

Critical micelle concentration

Supplementary material

10895_2013_1164_MOESM1_ESM.docx (117 kb)
Fig. S1Dye structure and corresponding spectra of the styryl dye FM®1-43. (A) Dye structure of FM®1-43 [14]. (B) Excitation (dashed) and emission spectra of the dye FM®1-43 for two different pH values. Typical excitation and emission filters are depicted. (DOCX 116 kb)
10895_2013_1164_MOESM2_ESM.docx (111 kb)
Fig. S2Fluorescence emission spectra of CypHer™5E at pH = 5.3 for multiple measurements. Emission spectra of CypHer™5E at pH = 5.3 for 12 consecutive measurements (A) and corresponding mean and standard deviation (B). (DOCX 110 kb)
10895_2013_1164_MOESM3_ESM.docx (117 kb)
Fig. S3Stern–Volmer plots. The term \( \frac{{{F_0}}}{F}-1 \) is plotted versus the respective FM dye concentration, where F0 is CypHer™5E fluorescence is absence of the quencher (FM®2-10 or FM®1-43) and F in presence of the quencher. Data was taken from the spectrophotometric and in vitro experiments, respectively. (A) In FM®2-10 experiments the Stern-Volmer constant for the spectrophotometric data is 0.0058 μM−1 with a coefficient of determination R2 of 0.94 and in the in vitro experiments 0.0032 μM−1 (R2 = 0.99). (B) In FM®1-43 experiments the Stern–Volmer constant for the spectrophotometric data is 0.0196 μM−1 (R2 = 0.99) and in the in vitro experiments 0.0111 μM−1 (R2 = 0.97). (DOCX 117 kb)

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Copyright information

© Springer Science+Business Media New York 2013

Authors and Affiliations

  • Oliver Welzel
    • 1
  • Kristina Loy
    • 1
  • Carsten H. Tischbirek
    • 1
  • Alina Tabor
    • 2
  • Peter Gmeiner
    • 2
  • Johannes Kornhuber
    • 1
  • Teja W. Groemer
    • 1
  1. 1.Department of Psychiatry and PsychotherapyFriedrich-Alexander-University of Erlangen-NurembergErlangenGermany
  2. 2.Department of Chemistry and Pharmacy, Emil Fischer CenterFriedrich-Alexander-University of Erlangen-NurembergErlangenGermany

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