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    Area of Science:

    • Optical microscopy
    • Super-resolution imaging
    • Nanotechnology

    Background:

    • Point spread function (PSF) engineering enhances single emitter localization in 3D.
    • Advanced PSF models are sensitive to optical aberrations, limiting precision.
    • Spatial light modulators (SLMs) offer potential for complex PSF generation but require precise calibration.

    Purpose of the Study:

    • To present a calibration and alignment protocol for fluorescence microscopes with SLMs.
    • To achieve wavefront error below the diffraction limit for optimal engineered PSF application.
    • To enable high-precision 3D single-emitter localization and parameter estimation.

    Main Methods:

    • Developed a calibration protocol for SLM-equipped fluorescence microscopes.
    • Utilized a separate light path for SLM pixel calibration and alignment.
    • Employed vectorial PSF model fitting to bead z-stacks for aberration retrieval and compensation.

    Main Results:

    • Achieved wavefront control below 20 mλ aberration over 30 minutes.
    • Demonstrated SLM alignment within 3 μm (x/y) and 100 μm (z).
    • Attained localization precisions below 10 nm (x, y, λ) and 20 nm (z) with engineered PSFs.

    Conclusions:

    • The presented protocol enables high-precision wavefront control in fluorescence microscopy.
    • Well-calibrated systems facilitate the creation of complex '3D+λ' PSFs.
    • This approach significantly improves single-emitter localization accuracy and parameter estimation.