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    |September 9, 2020
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    We reveal how structured illumination microscopy can image fluorescent molecule rotation. The number of measurable rotational components depends on diffusion, decay, and acquisition times.

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    Area of Science:

    • Optics and Photonics
    • Biophysics
    • Microscopy

    Background:

    • Fluorescent molecule rotation is crucial for understanding biological processes.
    • Imaging these rotational dynamics with high resolution presents significant challenges.
    • Conventional microscopy limits the detection of angular information.

    Purpose of the Study:

    • To investigate the rotational diffusion of fluorescent molecules within angular potential wells.
    • To analyze the excitation and emission of these diffusing molecules.
    • To model the imaging of these emissions using high numerical aperture (NA) aplanatic optical microscopes.

    Main Methods:

    • Utilizing angular structured illumination to enhance the imaging of molecular emissions.
    • Analyzing the transmission of angular components through the optical system.
    • Developing a model that considers rotational diffusion time, fluorescence decay time, and acquisition time.

    Main Results:

    • Demonstrated that angular structured illumination can effectively alias higher-frequency angular components into the imaging system's passband.
    • Identified that the number of measurable angular components is fundamentally limited by the interplay of three key time scales.
    • Successfully simulated the model using a numerical phantom under various conditions (fast/slow diffusion, weak potentials).

    Conclusions:

    • The study provides a theoretical framework for imaging molecular rotational diffusion.
    • Angular structured illumination is a viable technique for overcoming limitations in detecting angular components.
    • The findings offer insights into optimizing imaging parameters for studying molecular dynamics.