Abstract
The calcium-ion indicator dye, Calcium Green 1 (CG-1), has been characterized using a combination of ensemble and single-molecule optical spectroscopy measurements. In terms of ensemble measurements, CG-1 demonstrated a strong increase in fluorescence emission as a function of increasing [Ca2+]. This was accompanied by a change in the relative proportions of two chemical forms of the dye, each with a different fluorescence lifetime, which were found to co-exist in solution. From single-molecule fluorescence measurements, it was found that the fluorescence intensity and photobleaching time (on-time) of each CG-1 molecule was invariant with [Ca2+] and that changes in ensemble fluorescence intensity simply correlates with the number of fluorescent molecules in solution. These results are compared with that of the related system, Calcium Green 2 (CG-2), and the mechanisms of operation of these two indicator dyes are discussed.
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References
Weiss S (1999) Fluorescence spectroscopy of single biomolecules. Science 283(5408):1676–1683
Moerner WE, Orrit M (1999) Illuminating single molecules in condensed matter. Science 283(5408):1670–1676
Moerner WE (2002) A dozen years of single-molecule spectroscopy in physics, chemistry, and biophysics. J Phys Chem B 106(5):910–927
Ambrose WP, Goodwin PM, Jett JH, Van Orden A, Werner JH, Keller RA (1999) Single molecule fluorescence spectroscopy at ambient temperature. Chem Rev 99(10):2929–2956
Prodi L (2005) Luminescent chemosensors: from molecules to nanoparticles. New J Chem 29(1):20–31
Prodi L, Bolletta F, Montalti M, Zaccheroni N (2000) Luminescent chemosensors for transition metal ions. Coord Chem Rev 205:59–83
Czarnik AW (1994) Chemical communication in water using fluorescent chemosensors. Acc Chem Res 27(10):302–308
de Silva A, Gunnlaugsson T, Rice T (1996) Recent evolution of luminescent photoinduced electron transfer sensors. Analyst 121:1759–1762
Callan JF, de Silva AP, Magri DC (2005) Luminescent sensors and switches in the early 21st century. Tetrahedron 61(36):8551–8588
Zang L, Liu RC, Holman MW, Nguyen KT, Adams DM (2002) A single-molecule probe based on intramolecular electron transfer. J Am Chem Soc 124(36):10640–10641
Brasselet S, Moerner WE (2000) Fluorescence behavior of single-molecule pH-sensors. Single Mol 1:17–23
Brasselet S, Peterman EJG, Miyawaki A, Moerner WE (2000) Single-molecule fluorescence resonant energy transfer in calcium concentration dependent cameleon. J Phys Chem B 104(15):3676–3682
Ha T, Zhuang XW, Kim HD, Orr JW, Williamson JR, Chu S (1999) Ligand-induced conformational changes observed in single RNA molecules. Proc Natl Acad Sci U S A 96(16):9077–9082
Cossart R, Lkegaya Y, Yuste R (2005) Calcium imaging of cortical networks dynamics. Cell Calcium 37(5):451–457
Knot HJ, Laher I, Sobie EA, Guatimosim S, Gomez-Viquez L, Hartmann H, Song LS, Lederer WJ, Graier WF, Malli R, Frieden M, Petersen OH (2005) Twenty years of calcium imaging: cell physiology to dye for. Mol Interv 5(2):112–127
Bagh S, Paige MF (2006) Ensemble and single-molecule fluorescence spectroscopy of a calcium-ion indicator dye. J Phys Chem A 110(22):7057–7066
Haugland RP (2002) Handbook of fluorescent probes and research products, 9th edn. Molecular Probes, Eugene, OR
Tsien R, Pozzan T (1989) Measurement of cytosolic free Ca-2+ with Quin2. Methods Enzymol 172:230–262
Tsien RY (1980) New calcium indicators and buffers with high selectivity against magnesium and protons—design, synthesis, and properties of prototype structures. Biochemistry 19(11):2396–2404
Grynkiewicz G, Poenie M, Tsien RY (1985) A new generation of Ca-2+ indicators with greatly improved fluorescence properties. J Biol Chem 260(6):3440–3450
Minta A, Kao JPY, Tsien RY (1989) Fluorescent indicators for cytosolic calcium based on rhodamine and fluorescein chromophores. J Biol Chem 264(14):8171–8178
de Silva AP, Gunaratne HQN (1990) Fluorescent pet (photoinduced electron-transfer) sensors selective for submicromolar calcium with quantitatively predictable spectral and ion-binding properties. J Chem Soc. Chem Commun 2:186–188
Bagh S, Paige MF (2005) Construction and application of a single-molecule fluorescence microscope. Can J Chem 83(5):435–442
Dickson RM, Norris DJ, Tzeng YL, Moerner WE (1996) Three-dimensional imaging of single molecules solvated in pores of poly(acrylamide) gels. Science 274(5289):966–969
Dickson RM, Cubitt AB, Tsien RY, Moerner WE (1997) On/off blinking and switching behaviour of single molecules of green fluorescent protein. Nature 388(6640):355–358
Sanders R, Gerritsen HC, Draaijer A, Houpt PM, Levine YK (1994) Fluoresence lifetime imaging of free calcium in single cells. Bioimaging 2:131–138
Tessier A, Meallet-Renault R, Denjean P, Miller D, Pansu RB (1999) Conformational dynamics of Calcium Green 1 by fluorescence correlation spectroscopy. PCCP 1:5767–5769
Basche T, Ambrose WP, Moerner WE (1992) Optical-spectra and kinetics of single impurity molecules in a polymer–spectral diffusion and persistent spectral hole burning. J Opt Soc Am B 9(5):829–836
Acknowledgements
Funding for this work was provided by the Natural Sciences and Engineering Research Council of Canada and by the University of Saskatchewan. Professor Ron Steer, Dr. Sophie Brunet and the Saskatchewan Structural Sciences Centre are acknowledged for providing access and technical assistance with fluorescence lifetime measurements.
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Lu, Y., Paige, M.F. An Ensemble and Single-molecule Fluorescence Spectroscopy Investigation of Calcium Green 1, a Calcium-ion Sensor. J Fluoresc 17, 739–748 (2007). https://doi.org/10.1007/s10895-007-0185-1
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DOI: https://doi.org/10.1007/s10895-007-0185-1