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Updated: Mar 12, 2026

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Time-Resolved Genetically Encoded Indicators toward Quantitative Imaging of Calcium Dynamics in Living Cells.

Zizhu Tan1,2, Yadan Hou1, Yanan Huang1

  • 1Department of Chemistry, Westlake University, Hangzhou 310030, P. R. China.

ACS Sensors
|March 11, 2026
PubMed
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New genetically encoded calcium indicators (tr-GECIs) use fluorescence lifetime to accurately measure cellular calcium levels, overcoming limitations of traditional biosensors for precise signaling network analysis.

Area of Science:

  • Biophysics
  • Cellular Biology
  • Molecular Imaging

Background:

  • Quantitative imaging of cellular signaling relies on biosensors, but intensity-based methods face challenges with probe concentration and excitation intensity variations.
  • Accurate measurement of dynamic ion and metabolite concentrations is crucial for understanding cellular signaling networks.

Purpose of the Study:

  • To develop a novel class of genetically encoded calcium indicators (tr-GECIs) that measure calcium levels via fluorescence lifetime, making them insensitive to probe concentration and excitation intensity.
  • To engineer and characterize tr-GECIs with broad dynamic ranges and complementary affinities for precise calcium concentration determination.

Main Methods:

  • Development of time-resolved genetically encoded calcium indicators (tr-GECIs) by converting calcium levels into fluorescence lifetime changes.
Keywords:
calcium exchangeconcentration quantificationfluorescence lifetime imaging microscopy (FLIM)fluorescent proteingenetically encoded calcium indicators (GECIs)

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  • Engineering of key residues and linker optimization to achieve broad lifetime dynamic ranges (2.14 ns for tr-CCaMP, 1.32 ns for tr-GCaMP, 1.57 ns for tr-RCaMP).
  • Characterization of sensor affinities (Kd from 23.8 nM to 416 nM) and application in fluorescence lifetime imaging microscopy (FLIM).

    • Main Results:

    • Demonstrated that tr-GECIs are intrinsically insensitive to probe concentration and excitation intensity variations.
    • Achieved broad lifetime dynamic ranges and complementary Ca2+ affinity ranges, enabling concentration determination from tens of nanomolar to micromolar.
    • Mapped resting Ca2+ near organelles, revealing localized microdomains, and demonstrated simplified multiplexed imaging using a single optical channel.

    Conclusions:

    • tr-GECIs offer a robust platform for quantitative analysis of cellular signaling events with high spatial precision.
    • This technology overcomes limitations of intensity-based biosensors, facilitating more accurate cellular calcium imaging.
    • The developed sensors enable precise mapping of intracellular calcium dynamics and facilitate multiplexed imaging applications.