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    We developed a new pupil phase function for nanoscale 3D microscopy, significantly improving depth imaging. This innovation allows precise 3D localization over extended ranges, capturing cellular dynamics.

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    Area of Science:

    • Optics and Photonics
    • Biomedical Imaging
    • Nanotechnology

    Background:

    • Conventional 3D-localization microscopy faces limitations in axial imaging depth and precision.
    • Achieving high-resolution 3D imaging over extended axial ranges is crucial for studying complex biological systems.
    • Existing methods often struggle to maintain localization accuracy across large depth variations.

    Purpose of the Study:

    • To introduce an analytical pupil phase function for extended-depth nanoscale 3D-localization microscopy.
    • To enhance 3D localization precision and maintain it over significantly larger axial ranges.
    • To enable advanced imaging applications, such as capturing nanoscale dynamics within entire cells.

    Main Methods:

    • Development of a novel pupil phase function utilizing cropped secondary astigmatism.
    • Implementation and validation of the phase function in a 3D-localization microscopy setup.
    • Testing with real-time imaging of 3D fluid flow over a 40 µm depth range at NA 0.8.

    Main Results:

    • The proposed phase function achieves high localization precision in all three dimensions (X, Y, Z).
    • Localization precision is maintained over extended axial ranges, up to two orders of magnitude greater than conventional methods.
    • Successful demonstration of real-time 3D fluid flow imaging over a 40 µm depth.

    Conclusions:

    • The analytical pupil phase function offers a flexible and simple solution for extended-depth 3D-localization microscopy.
    • This method significantly improves axial imaging capabilities, making it attractive for various microscopy applications.
    • Enables new possibilities for observing nanoscale dynamics in biological samples across greater depths.