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b
excitation conditions. This demonstrates that probe 1 can indeed be
used as a biphotonic pH probe with pKa close to 7.
Institut des Sciences Chimiques de Rennes (CNRS, UMR 6226), Université
de Rennes 1, F-35042 Rennes Cedex, France. Phone: +33 (2) 2323 5954; E-
DOI: 10.1039/C5CC04573H
In order to demonstrate the ability of these bolaamphiphilic probes to
label lipid membranes, we first tested their incorporation into
phospholipidic giant unilamellar vesicles (GUVs) which were grown
in presence of compound 1 under basic (8.1) and neutral (7.2) pH
buffer conditions (see SI). Confocal fluorescence microscopy reveals
a strong blue emission (415-500 nm) delimited to the membrane
(Figure 5) at both pH, while emission at higher wavelengths (550-
700) corresponding to the acidic form of 1 is also observed at neutral
pH. The confinement of the fluorescence at membranes location
shows that chromophore 1 is able to stain phospholipidic membranes
and is stable enough for imaging purposes. We thus tested the use of
this probe in two-photon imaging of living cells. COS cells were
mail: olivier.mongin@univ-rennes1.fr.
c Chimie et Photonique Moléculaires (UMR 6510), F-35042 Rennes, France.
Electronic Supplementary Information (ESI) available: synthetic
procedures, photophysical methods, absorption and emission spectra of 2,
determination of pKa values, two-photon absorption experimental details,
preparation of GUVs, confocal and two-photon cell imaging. See
DOI: 10.1039/c000000x/
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,
incubated with compound
1 then imaged under two-photon
excitation at 700 nm (Figure 5). Probe 1 was found to stain specific
part of cells membranes as well as intracellular organelles. Thanks to
the large two-photon brightness of probe 1 and to its ratiometic
behaviour, we were able to estimate an overall pH for these specific
environments of 6.8.
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Fig. 5 Left: Incorporation of 1 in GUVs at basic pH, images are the overlap of
bright field and fluorescence (ex
= 405 nm) detections to compare the
membrane and chromophore localisations. Middle and right: Two-photon
imaging of COS7 cells (middle) incubated with 1µM of for 1 (ex = 700 nm) and
overlap of bright field (right).
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In conclusion, we reported the synthesis of two bolaamphiphilic
quadrupoles built from a lipophilic fluorene core, which exhibit large
2PA response in NIR region. By integration of pyridine or quinoline
end-groups linked to the -system, we tuned their pKa close to
physiological pH. We further demonstrated that these probes are able
to stain efficiently lipidic membranes and have suitable
photostability to allow monitoring of pH in living COS cells. The
large shift of emission between protonated and basic forms and their
high 2PA cross-sections make these bolaamphiphilic chromophores
promising as ratiometric two-photon pH probes for bioimaging and
monitoring of slight pH variations close to physiological pH. As
such they hold promise for detection of acidic pH in specific cellular
compartiments.
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M.B.D. gratefully acknowledges Conseil Régional d’Aquitaine for a
Chaire d’Accueil grant and fellowship to J.D. This work was
partially supported by the European Commission and received
funding from the People Programme (Marie Curie Actions) of the
European Union's Seventh Framework Programme FP7/2007-2013/
under REA grant agreement n°607721. We thank Région Bretagne
and CNRS for a fellowship to A.S and MENESR for a fellowship to
J.M.V. We also wish to thank Y. Nguyen for assistance in the
synthesis and M. Palayret for providing the COS 7 cells.
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,
17969-17977.
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47, 2231-2234.
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B. R. Cho, Angew Chem Int Ed Engl, 2012, 51, 2673-2676.
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Yu, Biosens. Bioelectron., 2013, 50, 42-49.
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,
Notes and references
30. M. H. V. Werts, S. Gmouh, O. Mongin, T. Pons and M. Blanchard-
Desce, J. Am. Chem. Soc., 2004, 126, 16294-16295.
31. O. Mongin, L. Porrès, M. Charlot, C. Katan and M. Blanchard-Desce,
Chem. Eur. J., 2007, 13, 1481-1498.
a Université de Bordeaux, ISM, UMR 5255 CNRS, F-33400 Talence, France.
Phone: +33 (5) 4000 6732; E-mail: mireille.blanchard-desce@u-bordeaux.fr.
32. C. Rouxel, M. Charlot, Y. Mir, C. Frochot, O. Mongin and M.
Blanchard-Desce, New J. Chem., 2011, 35, 1771-1780.
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