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    Area of Science:

    • Biophysics
    • Spectroscopy
    • Microscopy

    Background:

    • Dual focus fluorescence correlation spectroscopy (2fFCS) enables absolute diffusion coefficient measurements.
    • Existing methods require complex setups for fast-moving molecules at nanomolar concentrations.

    Purpose of the Study:

    • Introduce two novel, simplified experimental realizations of 2fFCS.
    • Enhance the accessibility and applicability of 2fFCS for studying molecular diffusion.

    Main Methods:

    • Developed a polarization rotation approach using pulsed lasers to achieve independent focus readouts.
    • Implemented a polarization modulation approach with continuous wave (cw) lasers and FLCS-like analysis for clean correlation curves.

    Main Results:

    • Both methods were tested with various lasers and samples, demonstrating their efficacy.
    • Identified the necessity for intensity cross-talk corrections and discussed artifacts like polarization misalignment and detector afterpulsing.

    Conclusions:

    • The polarization rotation method requires only one pulsed laser per wavelength.
    • The polarization modulation method utilizes standard cw lasers and allows flexible DIC prism placement.
    • These advancements facilitate easier integration of 2fFCS into commercial laser scanning microscopes.