Synthesis and properties of Asante Calcium Red-A novel family of long excitation wavelength calcium indicators

Krzysztof L. Hyrc, Akwasi Minta, P. Rogelio Escamilla, Patrick P.L. Chan, Xenia A. Meshik, Mark P. Goldberg

Research output: Contribution to journalArticlepeer-review

12 Scopus citations


Although many synthetic calcium indicators are available, a search for compounds with improved characteristics continues. Here, we describe the synthesis and properties of Asante Calcium Red-1 (ACR-1) and its low affinity derivative (ACR-1-LA) created by linking BAPTA to seminaphthofluorescein. The indicators combine a visible light (450-540nm) excitation with deep-red fluorescence (640nm). Upon Ca2+ binding, the indicators raise their fluorescence with longer excitation wavelengths producing higher responses. Although the changes occur without any spectral shifts, it is possible to ratio Ca2+-dependent (640nm) and quasi-independent (530nm) emission when using visible (<490nm) or multiphoton (~780nm) excitation. Therefore, both probes can be used as single wavelength or, less dynamic, ratiometric indicators. Long indicator emission might allow easy [Ca2+]i measurement in GFP expressing cells. The indicators bind Ca2+ with either high (Kd=0.49±0.07μM; ACR-1) or low affinity (Kd=6.65±0.13μM; ACR-1-LA). Chelating Zn2+ (Kd=0.38±0.02nM) or Mg2+ (Kd~5mM) slightly raises and binding Co2+ quenches dye fluorescence. New indicators are somewhat pH-sensitive (pKa=6.31±0.07), but fairly resistant to bleaching. The probes are rather dim, which combined with low AM ester loading efficiency, might complicate in situ imaging. Despite potential drawbacks, ACR-1 and ACR-1-LA are promising new calcium indicators.

Original languageEnglish (US)
Pages (from-to)320-333
Number of pages14
JournalCell Calcium
Issue number4
StatePublished - Oct 2013
Externally publishedYes


  • Confocal microscopy
  • Dissociation constant
  • Emission spectra
  • Excitation spectra
  • Fluorescence
  • Intracellular free calcium concentration
  • Magnesium ions
  • Multiphoton microscopy
  • Neurons
  • Ratio imaging
  • Zinc ions

ASJC Scopus subject areas

  • Molecular Biology
  • Physiology
  • Cell Biology


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