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    Area of Science:

    • Biochemistry
    • Molecular Biology
    • Cell Biology

    Background:

    • Green fluorescent protein (GFP)-based Ca(2+) indicators are valuable tools for monitoring calcium dynamics in diverse biological contexts.
    • Genetically encoded probes offer advantages like cofactor independence, intracellular targeting, and suitability for single-cell imaging.
    • Fluorescence resonance energy transfer (FRET) probes, such as Yellow Cameleons (YCs), utilize CFP and YFP variants for quantitative calcium sensing.

    Purpose of the Study:

    • To describe the application of an enhanced Yellow Cameleon (YC3.60) for imaging rapid intracellular free Ca(2+) concentration ([Ca(2+)]i) changes.
    • To demonstrate the utility of YC3.60 in HeLa cells using advanced microscopy techniques.

    Main Methods:

    • Utilized Yellow Cameleons (YCs), a FRET-based biosensor composed of CFP, calmodulin, M13 peptide, and YFP.
    • Engineered YC3.60 for improved performance as a Ca(2+) indicator.
    • Employed laser-scanning confocal microscopy for FRET imaging of [Ca(2+)]i in HeLa cells.

    Main Results:

    • YC3.60 effectively imaged rapid changes in intracellular free Ca(2+) concentration ([Ca(2+)]i).
    • The FRET-based emission ratioing technique provided quantitative measurements of Ca(2+) dynamics.
    • Demonstrated successful application in HeLa cells, highlighting the probe's efficacy.

    Conclusions:

    • YC3.60 is a powerful tool for real-time imaging of intracellular calcium dynamics.
    • FRET-based indicators offer quantitative and sensitive measurements of Ca(2+) signaling.
    • This technology advances the study of calcium signaling in cellular environments.