Abstract
Sodium ions (Na+) play an important role in a plethora of cellular processes, which are complex and partly still unexplored. For the investigation of these processes and quantification of intracellular Na+ concentrations ([Na+]i), two-photon coupled fluorescence lifetime imaging microscopy (2P-FLIM) was performed in the salivary glands of the cockroach Periplaneta americana. For this, the novel Na+-sensitive fluorescent dye Asante NaTRIUM Green-2 (ANG-2) was evaluated, both in vitro and in situ. In this context, absorption coefficients, fluorescence quantum yields and 2P action cross-sections were determined for the first time. ANG-2 was 2P-excitable over a broad spectral range and displayed fluorescence in the visible spectral range. Although the fluorescence decay behaviour of ANG-2 was triexponential in vitro, its analysis indicates a Na+-sensitivity appropriate for recordings in living cells. The Na+-sensitivity was reduced in situ, but the biexponential fluorescence decay behaviour could be successfully analysed in terms of quantitative [Na+]i recordings. Thus, physiological 2P-FLIM measurements revealed a dopamine-induced [Na+]i rise in cockroach salivary gland cells, which was dependent on a Na+-K+-2Cl− cotransporter (NKCC) activity. It was concluded that ANG-2 is a promising new sodium indicator applicable for diverse biological systems.
Article PDF
Similar content being viewed by others
References
N. Sperelakis, Cell Physiology Sourcebook: Essentials of Membrane Biophysics, Academic Press, London, 4th edn, 2011.
V. V. Martin, A. Rothe, Z. Diwu and K. R. Gee, Fluorescent sodium ion indicators based on the 1,7-diaza-15-crown-5 system, Bioorg. Med. Chem. Lett., 2004, 14, 5313–5316.
B. Valeur and I. Leray, Design principles of fluorescent molecular sensors for cation recognition, Coord. Chem. Rev., 2000, 205, 3–40.
A. Minta and R. Y. Tsien, Fluorescent indicators for cytosolic sodium, J. Biol. Chem., 1989, 264, 19449–19457.
J. R. Lakowicz, Principles of Fluorescence Spectroscopy, Springer, New York, 3rd edn, 2006.
A. E. Schreiner and C. R. Rose, Quantitative Imaging of Intracellular Sodium, Curr. Microsc. Contrib. Adv. Sci. Technol., 2012, 119–129.
S. D. Meier, Y. Kovalchuk and C. R. Rose, Properties of the new fluorescent Na+ indicator CoroNa Green: comparison with SBFI and confocal Na+ imaging, J. Neurosci. Methods, 2006, 155, 251–259.
S. Dietrich, S. E. Stanca, C. G. Cranfield, B. Hoffmann, K. Benndorf and C. Biskup, New strategies to measure intracellular sodium concentrations, Multiphot. Microsc. Biomed. Sci. X, 2010, 7569, 756914.
H. Szmacinski and J. R. Lakowicz, Sodium Green as a potential probe for intracellular sodium imaging based on fluorescence lifetime, Anal. Biochem., 1997, 250, 131–138.
S. Despa, P. Steels and M. Ameloot, Fluorescence lifetime microscopy of the sodium indicator sodium-binding benzofuran isophthalate in HeLa cells, Anal. Biochem., 2000, 280, 227–241.
H. Szmacinski and J. R. Lakowicz, Potassium and sodium measurements at clinical concentrations using phase-modulation fluorometry, Sens. Actuators, B, 1999, 60, 8–18.
C. M. Lamy, J.-Y. Chatton, Optical probing of sodium dynamics in neurons and astrocytes, Neuroimage, 2011, 58, 572–578.
A. Diaspro, P. Bianchini, G. Vicidomini, M. Faretta, P. Ramoino and C. Usai, Multi-photon excitation microscopy, Biomed. Eng. Online, 2006, 5, 36.
C. Hille, M. Lahn, H.-G. Löhmannsröben and C. Dosche, Two-photon fluorescence lifetime imaging of intracellular chloride in cockroach salivary glands, Photochem. Photobiol. Sci., 2009, 8, 319–327.
F. Just and B. Walz, Salivary glands of the cockroach, Periplaneta americana: new data from light and electron microscopy, J. Morphol., 1994, 220, 35–46.
M. Rumi, J. E. Ehrlich, A. A. Heikal, J. W. Perry, S. Barlow, Z. Hu, D. McCord-Maughon, T. C. Parker, S. Thayumanavan, S. R. Marder, D. Beljonne and J. Bredas, Structure - Property Relationships for Two-Photon Absorbing Chromophores: Bis-Donor Diphenylpolyene and Bis(styryl)benzene Derivatives, J. Am. Chem. Soc., 2000, 122, 9500–9510.
M. Lahn, C. Dosche and C. Hille, Two-photon microscopy and fluorescence lifetime imaging reveal stimulus-induced intracellular Na+ and Cl− changes in cockroach salivary acinar cells, Am. J. Physiol., 2011, 300, C1323–C1336.
N. S. Makarov, M. Drobizhev and A. Rebane, Two-photon absorption standards in the 550-1600 nm excitation wavelength range, Opt. Express, 2008, 16, 4029–4047.
R. Sjöback, J. Nygren and M. Kubista, Absorption and fluorescence properties of fluorescein, Spectrochim. Acta, Part A, 1995, 51, L7–L21.
P. R. Escamilla and A. Minta, Cytosolic fluorescent ion indicators, patent WO2012040204 A2, 2012.
K. Sagolla, H.-G. Löhmannsröben and C. Hille, Time-resolved fluorescence microscopy for quantitative Ca2+ imaging in living cells, Anal. Bioanal. Chem., 2013, 405, 8525–8537.
S. Despa, J. Vecer, P. Steels and M. Ameloot, Fluorescence lifetime microscopy of the Na+ indicator Sodium Green in HeLa cells, Anal. Biochem., 2000, 281, 159–175.
M. A. Albota, C. Xu and W. W. Webb, Two-Photon Fluorescence Excitation Cross Sections of Biomolecular Probes from 690 to 960 nm, Appl. Opt., 1998, 37, 7352–7356.
A. Baartscheer, C. A. Schumacher and J. W. Fiolet, Small changes of cytosolic sodium in rat ventricular myocytes measured with SBFI in emission ratio mode, J. Mol. Cell. Cardiol., 1997, 29, 3375–3383.
A. Sillen and Y. Engelborghs, The Correct Use of “Average” Fluorescence Parameters, Photochem. Photobiol., 1998, 67, 475–486.
G. Grynkiewicz, M. Poenie and R. Y. Tsien, A new generation of Ca2+ indicators with greatly improved fluorescence properties, J. Biol. Chem., 1985, 260, 3440–3450.
J.-Y. Chatton and K. R. Spring, The Sodium Concentration of the Lateral Intercellular Spaces of MDCK Cells: A Microspectrofluorimetric Study, J. Membr. Biol., 1995, 19, 11–19.
S. Despa, M. A. Islam, S. M. Pogwizd and D. M. Bers, Intracellular [Na+] and Na+ pump rate in rat and rabbit ventricular myocytes, J. Physiol., 2002, 539, 133–143.
G. P. Amorino and M. H. Fox, Intracellular Na+ measurements using sodium green tetraacetate with flow cytometry, Cytometry, 1995, 21, 248–256.
B. A. Wallace, Gramicidin channels and pores, Annu. Rev. Biophys. Biophys. Chem., 1990, 19, 127–157.
T. Jimenez, G. Sánchez, E. Wertheimer and G. Blanco, Activity of the Na,K-ATPase α4 isoform is important for membrane potential, intracellular Ca2+, and pH to maintain motility in rat spermatozoa, Reproduction, 2010, 139, 835–845.
H. Ishiguro, M. C. Steward, A. R. Lindsay and R. M. Case, Accumulation of intracellular HCO3, − by Na+-HCO3, − cotransport in interlobular ducts from guinea-pig pancreas, J. Physiol., 1996, 495, 169–178.
A. Takahashi, P. Camacho, J. D. Lechleiter and B. Herman, Measurement of intracellular calcium, Physiol. Rev., 1999, 79, 1089–1125.
A. Malgaroli, D. Milani, J. Meldolesi and T. Pozzan, Fura-2 Measurement of Cytosolic Free Ca2+ in Monolayers and Suspensions of Various Types of Animal Cells Cell Loading with Fluorescent Probes, J. Cell Biol., 1987, 105, 2145–2155.
K. M. Hirshfield, D. Toptygin, B. S. Packard and L. Brand, Dynamic fluorescence measurements of two-state systems - applications to calcium-chelating probes, Anal. Biochem., 1993, 209, 209–218.
H. Szmacinski and J. R. Lakowicz, Fluorescence lifetime-based sensing and imaging, Sens. Actuators, B, 1995, 29, 16–24.
C. D. Wilms, H. Schmidt and J. Eilers, Quantitative two-photon Ca2+ imaging via fluorescence lifetime analysis, Cell Calcium, 2006, 40, 73–79.
B. Walz, O. Baumann, W. Blenau and C. Krach, The Aminergic Control of Cockroach Salivary Glands, Arch. Insect Biochem. Physiol., 2006, 62, 141–152.
I. Lang and B. Walz, Dopamine-induced epithelial K+ and Na+ movements in the salivary ducts of Periplaneta americana, J. Insect Physiol., 2001, 47, 465–474.
J. M. Russell, Sodium-potassium-chloride cotransport, Physiol. Rev., 2000, 80, 211–276.
B. L. Gupta and T. A. Hall, Ionic distribution in dopamine-stimulated NaCl fluid-secreting cockroach salivary glands, Am. J. Physiol., 1983, 244, R176–R186.
S. Dissing and B. Nauntofte, Na+ transport properties of isolated rat parotid acini, Am. J. Physiol., 1990, 259, G1044–G1055.
J. E. Lee, J. H. Nam and S. J. Kim, Muscarinic activation of Na+-dependent ion transporters and modulation by bicarbonate in rat submandibular gland acinus, Am. J. Physiol., 2005, 288, G822–G831.
M. A. Robertson and J. K. Foskett, Na+ transport pathways in secretory acinar cells: membrane cross talk mediated by [Cl−]i, Am. J. Physiol., 1994, 267, C146–C156.
Author information
Authors and Affiliations
Corresponding author
Rights and permissions
This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons licence, and indicate if changes were made. The images or other third party material in this article are included in the article's Creative Commons licence, unless indicated otherwise in a credit line to the material. If material is not included in the article's Creative Commons licence and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this licence, visit http://creativecommons.org/licenses/by/4.0/.
About this article
Cite this article
Roder, P., Hille, C. ANG-2 for quantitative Na+ determination in living cells by time-resolved fluorescence microscopy. Photochem Photobiol Sci 13, 1699–1710 (2014). https://doi.org/10.1039/c4pp00061g
Received:
Accepted:
Published:
Issue Date:
DOI: https://doi.org/10.1039/c4pp00061g