Near-Infrared Genetically Encoded Positive Calcium Indicator Based on GAF-FP Bacterial Phytochrome

Int J Mol Sci. 2019 Jul 16;20(14):3488. doi: 10.3390/ijms20143488.


A variety of genetically encoded calcium indicators are currently available for visualization of calcium dynamics in cultured cells and in vivo. Only one of them, called NIR-GECO1, exhibits fluorescence in the near-infrared region of the spectrum. NIR-GECO1 is engineered based on the near-infrared fluorescent protein mIFP derived from bacterial phytochromes. However, NIR-GECO1 has an inverted response to calcium ions and its excitation spectrum is not optimal for the commonly used 640 nm lasers. Using small near-infrared bacterial phytochrome GAF-FP and calmodulin/M13-peptide pair, we developed a near-infrared calcium indicator called GAF-CaMP2. In vitro, GAF-CaMP2 showed a positive response of 78% and high affinity (Kd of 466 nM) to the calcium ions. It had excitation and emission maxima at 642 and 674 nm, respectively. GAF-CaMP2 had a 2.0-fold lower brightness, 5.5-fold faster maturation and lower pH stability compared to GAF-FP in vitro. GAF-CaMP2 showed 2.9-fold higher photostability than smURFP protein. The GAF-CaMP2 fusion with sfGFP demonstrated a ratiometric response with a dynamic range of 169% when expressed in the cytosol of mammalian cells in culture. Finally, we successfully applied the ratiometric version of GAF-CaMP2 for the simultaneous visualization of calcium transients in three organelles of mammalian cells using four-color fluorescence microscopy.

Keywords: calcium imaging; genetically encoded calcium indicator; near-infrared fluorescent bacterial phytochrome; protein engineering.

MeSH terms

  • Calcium / analysis
  • Calcium / metabolism*
  • Calcium Signaling
  • Calmodulin / genetics
  • Calmodulin / metabolism*
  • HeLa Cells
  • Humans
  • Infrared Rays
  • Optical Imaging / methods*
  • Phytochrome / genetics*
  • Phytochrome / metabolism
  • Protein Engineering / methods*
  • Recombinant Proteins / genetics
  • Recombinant Proteins / metabolism


  • Calmodulin
  • Recombinant Proteins
  • Phytochrome
  • Calcium