Understanding the Fluorescence Change in Red Genetically Encoded Calcium Ion Indicators

Area of Science:

• Biophysics
• Molecular Biology
• Cell Biology

Background:

• Genetically encoded calcium indicators (GECIs) are vital tools for studying dynamic calcium signaling in living systems.
• Red fluorescent GECIs offer advantages for deep-tissue imaging due to reduced light scattering at longer wavelengths.

Purpose of the Study:

• To systematically investigate the photophysical properties of various red GECIs.
• To identify the key factors contributing to fluorescence changes in response to calcium binding.
• To classify red GECIs based on their underlying photophysical mechanisms.

Main Methods:

• Analysis of photophysical properties of nine red GECIs (jRGECO1a, K-GECO1, jRCaMP1a, R-GECO1, R-GECO1.2, CAR-GECO1, O-GECO1, REX-GECO1, jREX-GECO1).
• Evaluation of factors influencing fluorescence change: protonation equilibrium, quantum yield, extinction coefficient, and two-photon cross-section.
• Comparison of one-photon and two-photon excitation modes.

Main Results:

• Red GECIs were classified into three distinct groups based on the dominant factors affecting their fluorescence.
• The magnitude of fluorescence change varied significantly between indicators and excitation modes.
• Two-photon excitation sometimes yielded up to a twofold larger fluorescence change compared to one-photon excitation, attributed to altered two-photon cross-sections.

Conclusions:

• Understanding the photophysical basis of red GECI fluorescence is crucial for optimizing their application in biological imaging.
• Indicator classification provides a framework for selecting appropriate red GECIs based on experimental needs and excitation strategies.
• The observed differences in performance between one- and two-photon excitation underscore the importance of considering excitation modality when choosing a GECI.