Fluorescent Probes Used for Measuring Intracellular Calcium


Calcium (Ca2+) is a ubiquitous messenger in many intracellular signalling cascades. With resting Ca2+ concentrations around 100 nM and peak amplitudes of Ca2+ transients of the order of 10–100 μM, the specificity of the diverse Ca2+ signals comes to a lesser degree from the affinities of the different biological Ca2+ sensors and from the spatio‐temporal compartmentalisation of the Ca2+ signal into subcellular nano‐ and microdomains. The measurement of such local and short‐lived Ca2+ signals presents a major experimental challenge. Ca2+ imaging with fluorescent indicators has initially involved synthetic small‐molecule chemical Ca2+ indicators. While a large choice exists among molecules with different hues and Ca2+ affinities and these molecules come in salt and ester forms for single‐cell or bulk loading, respectively, the subcellular targeting of Ca2+ indicators and their long‐term use in live animals have remained problematic. The advent of genetically encoded Ca2+ indicators that can be linked to different retention and targeting domains now permits the readout of local Ca2+ signals, the targeting to different subcellular regions or cell types in intact tissue, the measurement of organelle Ca2+ as well as long‐term recordings over months. For the detection of Ca2+ nanodomains, hybrid organic–inorganic Ca2+ nanobiosensors start to emerge as a promising alternative.

Key Concepts

  • Ca2+ signals are local, both in space and time, and their spread and kinetics are shaped by the presence of endogeneous Ca2+‐binding molecules (Ca2+ buffers).
  • Ca2+ indicators are fluorescent exogeneous Ca2+ buffers and as such distort the Ca2+ signal to be measured.
  • Ca2+‐related fluorescence does not equal Ca2+ concentration.
  • Genetically encoded Ca2+ indicators are a powerful alternative to chemical small‐molecule organic indicators.
  • Measured Ca2+ signals depend not only on the biological process studied and the properties of the fluorescent Ca2+ indicator but also on the microscope and image acquisition parameters.
  • The measurement of local (rather than temporally and spatially averaged) Ca2+ signals requires confinement of the dye volume, the fluorescence excitation volume, the fluorescence readout volume or a combination of these.

Keywords: calcium ions; small‐molecule chemical Ca2+ indicator; genetically encoded Ca2+ indicator; Ca2+ buffering; Ca2+ microdomain; Ca2+ nanobiosensor

Figure 1. Measuring the many [Ca2+]. (a) Schematic representation of a cell with the different organelles (M – mitochondrium, ER – endoplasmic reticulum, Nuc – nucleus, V – vesicle, LY – lysosome) all compartmentalising and shaping Ca2+ signal. (b) Zoom on a little stretch of the plasma and organelle membranes where ion channels, pumps and exchangers translocate Ca2+, leading to the formation of ephemerous Ca2+ microdomains. (c) Principle of particle‐based Ca2+ nanobiosensing. NC – photoluminescent nanocrystal (for ion‐channel localisation and as a FRET donor for local excitation), Ca2+‐indicator – spectrally matched small‐molecule Ca2+ probe, attached to the NC surface. Conveys sensing capability to the NC). The ensemble is functionalised so as to bind intracellular to a target site.


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Oheim, Martin(Aug 2015) Fluorescent Probes Used for Measuring Intracellular Calcium. In: eLS. John Wiley & Sons Ltd, Chichester. http://www.els.net [doi: 10.1002/9780470015902.a0002642.pub2]