Abstract
Calcium is a ubiquitous intracellular messenger that has important functions in normal neuronal function. The pathology of Alzheimer’s disease has been shown to alter calcium homeostasis in neurons and astrocytes. Several calcium dye indicators are available to measure intracellular calcium within cells, including Oregon Green BAPTA-1 (OGB-1). Using fluorescence lifetime imaging microscopy, we adapted this single wavelength calcium dye into a ratiometric dye to allow quantitative imaging of cellular calcium. We used this approach for in vitro calibrations, single-cell microscopy, high-throughput imaging in automated plate readers, and in single cells in the intact living brain. While OGB is a commonly used fluorescent dye for imaging calcium qualitatively, there are distinct advantages to using a ratiometric approach, which allows quantitative determinations of calcium that are independent of dye concentration. Taking advantage of the distinct lifetime contrast of the calcium-free and calcium-bound forms of OGB, we used time-domain lifetime measurements to generate calibration curves for OGB lifetime ratios as a function of calcium concentration. In summary, we demonstrate approaches using commercially available tools to measure calcium concentrations in live cells at multiple scales using lifetime contrast. These approaches are broadly applicable to other fluorescent readouts that exhibit lifetime contrast and serve as powerful alternatives to spectral or intensity readouts in multiplexing experiments.
An erratum to this chapter is available at http://dx.doi.org/10.1007/978-1-61779-328-8_33
An erratum to this chapter can be found at http://dx.doi.org/10.1007/978-1-61779-328-8_33
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References
Wen Z, Guirland C, Ming GL, Zheng LQ (2004) A CaMKII/calcineurin switch controls the direction of Ca(2+)-dependent growth cone guidance. Neuron 43, 835–846.
Malenka RC, Bear MF (2004) LTP and LTD: an embarrassment of riches. Neuron 44, 5–21.
Demuro A, Mina E, Kayed R, Milton SC, Parker I, Glabe CG (2005) Calcium dysregulation and membrane disruption as a ubiquitous neurotoxic mechanism of soluble amyloid oligomers. J Biol Chem 280, 17294–17300.
Guo Q, Sebastian L, Sopher BL, Miller MW, Ware CB, Martin GM, Mattson MP (1999) Increased vulnerability of hippocampal neurons from presenilin-1 mutant knock-in mice to amyloid beta-peptide toxicity: central roles of superoxide production and caspase activation. J. Neurochem 72, 1019–1029.
Mattson MP, Cheng B, Davis D, Bryant K, Lieberburg I, Rydel RE (1992) Beta-Amyloid peptides destabilize calcium homeostasis and render human cortical neurons vulnerable to excitotoxicity. J Neurosci 12, 376–389.
Mattson MP, Barger SW, Cheng B, Lieberburg I, Smith-Swintosky VL, Rydel RE (1993) Beta-Amyloid precursor protein metabolites and loss of neuronal Ca2+ homeostasis in Alzheimer’s disease. Trends Neurosci 451, 720–724.
Tu H, Nelson O, Bezprozvanny A, Wang Z, Lee SF, Hao, YH, Serneels L, deStrooper B, Yu G, Bezprozvanny I (2006) Presenilins form ER Ca2+ leak channels, a function disrupted by familial Alzheimer’s disease-linked mutations. Cell 126, 981–993.
Nelson O, Tu H, Lei T, Bentahir M, deStrooper B, Bezprozvanny I (2007) Familial Alzheimer disease-linked mutations specifically disrupt Ca2+ leak function of presenilin 1. J Clin Invest, 1230–1239.
Cheung KH, Shineman D, Müller M, Cárdenas C, Mei L, Yang J, Tomita T, Iwatsubo T, Lee VM, Foskett JK (2008) Mechanism of Ca2+ disruption in AD by presenilin regulation of InsP3 receptor channel gating. Neuron 58, 871–883.
Stutzmann GE, Caccamo A, LaFerla FM, Parker I (2004) Dysregulated IP3 signaling in cortical neurons of knock-in mice expressing an Alzheimer’s-linked mutation in presenilin1 results in exaggerated Ca2+ signals and altered membrane excitability. J Neurosci 24, 508–513.
Stutzmann GE, Smith I, Caccamo A, Oddo S, LaFerla FM, Parker I (2006) Enhanced ryanodine receptor recruitment contributes to Ca2+ disruptions in young, adult, and aged Alzheimer’s disease mice. J Neurosci 26, 5180–5189.
Kuchibhotla KV, Goldman ST, Lattarulo CR, Wu H-Y, Hyman BT, Bacskai BJ (2008) Amyloid-beta plaques lead to aberrant regulation of calcium homeostasis in vivo resulting in structural and functional disruption of neuronal networks. Neuron 29, 214–225.
Kuchibhotla KV, Lattarulo CR, Hyman BT, Bacskai BJ (2009) Synchronous hyperactivity and intercellular calcium waves in astrocytes in Alzheimer mice. Science , 1211–1215.
Hendel T, Mank M, Schnell B, Griesbeck O, Borst A, Reiff DF (2008) Fluorescence changes of genetic calcium indicators and OGB-1 correlated with neural activity and calcium in vivo and in vitro. J Neurosci 28, 7399–7411.
Spires TL, et al. (2005) J NeuroSci 25, 7278.
Skoch J, Hickey GA, Kajdasz ST, Hyman BT, Bacskai BJ (2005) In vivo imaging of amyloid-beta deposits in mouse brain with multiphoton microscopy. Methods Mol Biol 299: 349–363.
Stosiek C, Garaschuk O, Holthoff K, Konnerth A (2003) In vivo two-photon calcium imaging of neuronal networks. Proc Natl Acad Sci USA 100, 7319–7324.
Nimmerjahn A, Kirchhoff F, Kerr JN, Helmchen F (2004) Sulforhodamine 101 as a specific marker of astroglia in the neocortex in vivo. Nat Methods 1, 31–37.
Bacskai BJ, Klunk WE, Mathis CA, Hyman BT (2002) Imaging amyloid-beta deposits in vivo. J Cereb Blood Flow Metab 22, 1035–41.
Grynkiewicz G, Poenie M, Tsien RY (1985) A new generation of Ca2+ indicators with greatly improved fluorescence properties. J Biol Chem 25, 3440–3450.
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Lattarulo, C., Thyssen, D., Kuchibholta, K.V., Hyman, B.T., Bacskaiq, B.J. (2011). Microscopic Imaging of Intracellular Calcium in Live Cells Using Lifetime-Based Ratiometric Measurements of Oregon Green BAPTA-1. In: Manfredi, G., Kawamata, H. (eds) Neurodegeneration. Methods in Molecular Biology, vol 793. Humana Press, Totowa, NJ. https://doi.org/10.1007/978-1-61779-328-8_25
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DOI: https://doi.org/10.1007/978-1-61779-328-8_25
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