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Far-red fluorescent genetically encoded calcium ion indicators.

Rochelin Dalangin1,2, Bill Z Jia3,4, Yitong Qi3

  • 1Department of Chemistry, University of Alberta, Edmonton, AB, Canada.

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|April 8, 2025
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Researchers developed new far-red genetically encoded calcium ion (Ca2+) indicators (GECIs) for advanced neuroscience research. These novel GECIs enable sensitive detection of neuronal activity and cellular processes in vivo.

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Area of Science:

  • Neuroscience
  • Molecular Biology
  • Biotechnology

Background:

  • Genetically encoded calcium ion indicators (GECIs) are essential for observing calcium dynamics and neuronal activity at the single-cell level.
  • Existing GECIs often have limitations in spectral range and sensitivity, necessitating the development of new tools.

Purpose of the Study:

  • To design and develop novel far-red fluorescent GECIs for enhanced functional imaging.
  • To characterize the performance of these new GECIs in terms of brightness, Ca2+ affinity, and response kinetics.

Main Methods:

  • Development of two far-red fluorescent GECIs, FR-GECO1a and FR-GECO1c, utilizing monomeric far-red fluorescent proteins mKelly1 and mKelly2.
  • In vitro characterization of Ca2+ responses, including dynamic range (ΔF/F0) and apparent dissociation constants (Kd).
  • Evaluation of GECI performance under one-photon and two-photon illumination, and assessment of their ability to detect single action potentials in neurons.

Main Results:

  • FR-GECO1a and FR-GECO1c exhibit excitation/emission maxima at ~596 nm and ~644 nm, respectively.
  • Demonstrated significant Ca2+ responses in vitro (ΔF/F0 = 6 for FR-GECO1a, 18 for FR-GECO1c) with high affinities (Kd = 29 nM for FR-GECO1a, 83 nM for FR-GECO1c).
  • FR-GECOs provide bright signals under both one-photon and two-photon microscopy and enable sensitive, fast detection of neuronal action potentials.

Conclusions:

  • FR-GECO1a and FR-GECO1c represent advanced far-red GECIs with improved sensitivity and brightness.
  • These tools facilitate in vivo imaging of neuronal activity and cellular processes.
  • The developed GECIs are suitable for all-optical manipulation and measurement of cellular activities when combined with optogenetic actuators.