Simplifying Ratiometric C-SNARF-1 pH Calibration Procedures with a Simple Post-Processing
International Journal of Photochemistry and Photobiology
Volume 1, Issue 2, December 2017, Pages: 36-43
Received: Feb. 23, 2017; Accepted: Mar. 21, 2017; Published: Apr. 14, 2017
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Rutjapan Kateklum, FEMTO-ST Institute, UMR CNRS 6174, Université Bourgogne Franche-Comté, Besançon, France
Bernard Gauthier-Manuel, FEMTO-ST Institute, UMR CNRS 6174, Université Bourgogne Franche-Comté, Besançon, France
Christian Pieralli, FEMTO-ST Institute, UMR CNRS 6174, Université Bourgogne Franche-Comté, Besançon, France
Samlee Mankhetkorn, Center of Excellence in Molecular Imaging, Chiang Mai University, Chiang Mai, Thailand
Bruno Wacogne, FEMTO-ST Institute, UMR CNRS 6174, Université Bourgogne Franche-Comté, Besançon, France; INSERM CIC 1431, Besançon University Hospital, Besançon, France
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A simple and easy to implement numerical method is proposed in order to considerably simplify the experimental calibration procedure of C-SNARF-1 indicator used for ratiometric pH sensing. Usually, calibration is based on the measurement of fluorescence spectra using perfectly calibrated equipment at extreme pH values. The calibration solutions must be extremely well controlled in terms of indicator concentration and path length. Also, the optical equipment used must be well controlled and excitation energy as well as fluorescence collection efficiency must be perfectly constant over the whole calibration procedure. The method we propose is based on the fact that the emission fluorescence energy does not only depend on pH but also on the excitation wavelength. In this paper, we propose a model describing the evolution of the emitted energy as a function of pH and excitation wavelength. We show that the emitted energy evolves linearly with pH and we express this linear evolution as a function of the excitation wavelength. We also show the evolution of the isosbestic (or isoemitting) point as a function of the excitation wavelength. Knowing the linear dependence of the emitted energy as a function of excitation wavelength allows post-processing calibration spectra obtained with basic optical equipment where the excitation energy, fluorescence collection efficiency, indicator concentration and path length can vary over the calibration session. Because the calibration procedure becomes independent of the above mentioned parameters, the post-processing we propose considerably simplify indicators calibration. This method can easily be transposed, not only to other ratiometric pH indicators, but also to ion sensing fluorescent indicators exhibiting dual emission peaks.
Fluorescence pH Sensing, Ratiometric Measurements, Calibration, C-SNARF-1
To cite this article
Rutjapan Kateklum, Bernard Gauthier-Manuel, Christian Pieralli, Samlee Mankhetkorn, Bruno Wacogne, Simplifying Ratiometric C-SNARF-1 pH Calibration Procedures with a Simple Post-Processing, International Journal of Photochemistry and Photobiology. Vol. 1, No. 2, 2017, pp. 36-43. doi: 10.11648/j.ijpp.20170102.11
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J. Han and K. Burgess, “Fluorescent Indicators for Intracellular pH”, Chem. Rev., Vol. 110, Issue 5, pp 2709–2728, 2010. doi: 10.1021/cr900249z.
J. Chao, Y. Liu, J. Sun, L. Fan, Y. Zhang, H. Tong and Z. Li, “A ratiometric pH probe for intracellular pH imaging”, Sensors and Actuators B: Chemical, Vol. 221, Issue 31, pp. 427-433, 2015.
W. J. Zhang, L. Fan, Z. B. Li, T. Ou, H. J. Zhai, J. Yang, C. Dong and S. M. Shuang, “Thiazole-based ratiometric fluorescence pH probe with large Stokes shift for intracellular imaging”, Sensors and Actuators B: Chemical, Vol. 233, pp. 566-573, 2016.
L. Ferrari, L. Rovati, P. Fabbri, and F. Pilati, “Disposable Fluorescence Optical pH Sensor for Near Neutral Solutions”, Sensors, Vol. 13, pp 484-499, 2013. doi: 10.3390/s130100484.
K. P. Dobmeier, G. W. Charvilleand M. H. Schoenfisch, “Nitric Oxide-Releasing Xerogel-Based Fiber-Optic pH Sensors”. Analytical chemistry. Vol. 78, Issue 21, pp. 7461-7466, 2006. doi: 10.1021/ac060995p.
M.-R. S. Fuh, L. W. Burgess, T. Hirschfeld, G. D. Christian and F. Wang, “Single fibre optic fluorescence pH probe”, Analyst, Vol. 112, pp 1159-1163, 1987. doi: 10.1039/AN9871201159.
I. Kasik, J. Mrazek, T. Martan, M. Pospisilova, O. Podrazky, V. Matejec, K. Hoyerova and M. Kaminek, “Fiber-optic pH detection in small volumes of biosamples”, Anal Bioanal Chem Vol. 398, pp 1883–1889, 2010. doi 10.1007/s00216-010-4130-9.
H. Diehl and R. Markuszewski, “Studies on fluorescein—VII”, Talanta, Vol. 36, Issue 3, pp. 416-418, 1989. doi. org/10.1016/0039-9140 (89)80213-9.
M. Yassine, J. M. Salmon, J. Vigoand P. Viallet, “C-SNARF-1 as a pHi fluoroprobe: discrepancies between conventional and intracellular data do not result from protein interactions”, Journal of Photochemistry and Photobiology B: Biology, Vol. 37, Issue 1, pp 18-25, 1997. doi. org/10.1016/S1011-1344 (96)07339-3.
B. Valeur, “Molecular Fluorescence: Principles and Applications”. 2001 Wiley-VCH Verlag GmbH. ISBNs: 3-527-29919-X (Hardcover); 3-527-60024-8 (Electronic).
J. E. Whitaker, R. P. Haugland and F. G. Prendergast, “Spectral and photophysical studies of benzo[c]xanthene dyes: Dual emission pH sensors”, Analytical Biochemistry, Volume 194, Issue 2, pp. 330-3441991. doi. org/10.1016/0003-2697 (91)90237-N.
T. M. Żurawik, A. Pomorski, A. BelczykCiesielska, G. Goch, K. Niedźwiedzka, R. Kucharczyk A. Krezel and W. Bal, “ Revisiting Mitochondrial pH with an Improved Algorithm for Calibration of the Ratiometric 5 (6)-carboxy-SNARF-1 Probe Reveals Anticooperative Reaction with H+ Ions and Warrants Further Studies of Organellar pH”, PLoS ONE 11 (8): e0161353, 2016. doi: 10.1371/journal. pone.0161353.
K. J. Buckler and R. D. Vaughan-Jones, Application of a new pH-sensitive fluoroprobe (earboxy-SNARF-1) for intracellular pH measurement in small, isolated cells, Plügers Archiv, Vol. 417, pp. 234-239, 1990. doi: 10.1007/BF00370705.
J. Bond, J. Varley, “Use of flow cytometry and SNARF to calibrate and measure intracellular pH in NS0 cells”, Cytometry, 64A: 43–50. doi: 10.1002/cyto.a.20066.
A. C. Ribou, J. Vigo and J. M. Salmon, “C-SNARF-1 as a fluorescent probe for pH measurements in living cells: two-wavelength-ratio method versus whole-spectral-resolution method”, J. of Chem. Educ., Vol. 79, Issue 12, pp. 1471-1474, 2002.
M. L. Graber, D. C. DiLillo, B. L. Friedman and E. Pastoriza-Munoz, “Characteristics of fluoroprobes for measuring intracellular pH”, Analytical Biochemistry, Vol. 156, pp. 202-212, 1986.
F. Bancel, J. Vigo, J. M. Salmon and P. Viallet, “Acid—base and calcium-binding properties of the fluorescent calcium indicator indo-1”, Journal of Photochemistry and Photobiology A: Chemistry, Vol. 53, Issue 3, pp. 397-409, 1990.
A. K. Mahapatra, S. K. Manna, C. D. Mukhopadhyay and D. Mandal, “Pyrophosphate-selective fluorescent chemosensor based on ratiometric tripodal-Zn (II) complex: Application in logic gates and living cells”, Sensors and Actuators B: Chemical, Vol. 200, pp. 123-131, 2014.
D. H. Kim, J. Seong, H. Lee and K. H. Lee, “Ratiometric fluorescence detection of Hg (II) in aqueous solutions at physiological pH and live cells with a chemosensor based on tyrosine”, Sensors and Actuators B: Chemical, Vol. 196, pp. 421-428,
G. Grynkiewicz, M. Poenie and R. Y. Tsien, “A new generation of Ca2+ indicators with greatly improved fluorescence properties”, The journal of biological chemistry, Vol. 260, Issue 6, pp. 3440-3450, 1985.
P. A. Negulescu and T. E. Machen, “Intracellular ion activities and membrane transport in parietal cells measured with fluorescent dyes”, Methods Enzymol., Vol. 192, pp. 38-81, 1990. doi: 10.1016/s1046-2023(05)80145-8. PMID: 2074799.
D. Bottenus, Y. J. Oh, Sang M. Han, Cornelius and F. Ivory, “Experimentally and theoretically observed native pH shifts in a nanochannel array”, Lab Chip, Vol. 9, pp. 219-231, 2009. doi: 10.1039/b803278e.
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