pSMLM

pSMLM applies phasor-based algorithms to localize single molecules from engineered point spread functions (PSFs), providing sub-pixel three-dimensional localization for single-molecule localization microscopy.

Key Features:

• Engineered PSF Analysis: Handles engineered PSFs including double-helix, saddle-point, and tetra-pod that encode three-dimensional localization information in the Fourier plane and decodes that information.
• Phasor-Based Localization: Uses phasor-based methods to achieve high-precision localizations, identifying individual lobes and using their relative rotation for z-resolved localizations with double-helix PSFs and applying a phasor-based deconvolution method for saddle-point and tetra-pod PSFs.
• Performance Metrics: Delivers precision and recall rates comparable to SMAP at signal-to-noise ratios typical for organic fluorophores.
• Efficiency and Speed: Increases localization rates by a factor of 2 to 4 on standard CPUs and reports localization rates up to approximately 104 localizations per second for double-helix PSFs and 105 localizations per second for saddle-point or tetra-pod PSFs.

Scientific Applications:

• Structural Biology: Enables high-resolution molecular-scale mapping of macromolecular structures.
• Cell Biology: Provides three-dimensional single-molecule localization for imaging cellular components and processes.
• Nanotechnology: Supports visualization and analysis of nanoscale engineered structures using engineered PSFs.

Methodology:

Adapts phasor-based algorithms to engineered PSFs by analyzing spatial frequency components encoded in the PSFs, identifying individual lobes and their relative rotation for double-helix PSFs, and applying a phasor-based deconvolution method for saddle-point and tetra-pod PSFs.