A comparison of fluorescent Ca²⁺ indicators for imaging local Ca²⁺ signals in cultured cells

Cell Calcium. 2015 Dec;58(6):638-48. doi: 10.1016/j.ceca.2015.10.003. Epub 2015 Oct 29.


Localized subcellular changes in Ca(2+) serve as important cellular signaling elements, regulating processes as diverse as neuronal excitability and gene expression. Studies of cellular Ca(2+) signaling have been greatly facilitated by the availability of fluorescent Ca(2+) indicators. The respective merits of different indicators to monitor bulk changes in cellular Ca(2+) levels have been widely evaluated, but a comprehensive comparison for their use in detecting and analyzing local, subcellular Ca(2+) signals is lacking. Here, we evaluated several fluorescent Ca(2+) indicators in the context of local Ca(2+) signals (puffs) evoked by inositol 1,4,5-trisphosphate (IP3) in cultured human neuroblastoma SH-SY5Y cells, using high-speed video-microscopy. Altogether, nine synthetic Ca(2+) dyes (Fluo-4, Fluo-8, Fluo-8 high affinity, Fluo-8 low affinity, Oregon Green BAPTA-1, Cal-520, Rhod-4, Asante Calcium Red, and X-Rhod-1) and three genetically-encoded Ca(2+)-indicators (GCaMP6-slow, -medium and -fast variants) were tested; criteria include the magnitude, kinetics, signal-to-noise ratio and detection efficiency of local Ca(2+) puffs. Among these, we conclude that Cal-520 is the optimal indicator for detecting and faithfully tracking local events; that Rhod-4 is the red-emitting indicator of choice; and that none of the GCaMP6 variants are well suited for imaging subcellular Ca(2+) signals.

Keywords: Calcium; Fluorescent indicators; GCaMP6; Genetically encoded calcium indicators; Local calcium signals.

Publication types

  • Comparative Study
  • Research Support, N.I.H., Extramural

MeSH terms

  • Calcium / metabolism*
  • Calcium Signaling / physiology*
  • Cells, Cultured
  • Fluorescent Dyes / analysis*
  • Humans
  • Inositol 1,4,5-Trisphosphate / metabolism*
  • Inositol 1,4,5-Trisphosphate Receptors / metabolism*
  • Kinetics


  • Fluorescent Dyes
  • Inositol 1,4,5-Trisphosphate Receptors
  • Inositol 1,4,5-Trisphosphate
  • Calcium