Computational localization microscopy with extended axial range.

Area of Science:

• Microscopy and Imaging
• Biophysics
• Fluid Dynamics

Background:

• Accurate 3D particle localization and tracking are crucial for understanding dynamic biological processes.
• Existing methods often face limitations in depth range or resolution.
• A need exists for techniques that offer extended axial range without sacrificing optical performance.

Purpose of the Study:

• To introduce a new single-aperture 3D particle localization and tracking technique.
• To significantly enhance the depth range for 3D particle imaging.
• To validate the technique's performance in real-world applications.

Main Methods:

• Exploitation of an Airy beam's point spread function (PSF) for depth-dependent translation.
• Single-snapshot 3D localization over an extended volume.
• Application to bright-field and fluorescence microscopy modalities.
• Demonstration using real-time 3D velocity imaging of fluid flow in capillaries with fluorescent tracer beads.

Main Results:

• Achieved an axial localization precision of 50 nm.
• Covered an extended depth range of 120 μm.
• Demonstrated high axial range-to-precision ratio, exceeding previous reports.
• Validated applicability across various microscopy types and biological samples.

Conclusions:

• The developed Airy beam-based technique offers an order-of-magnitude increase in depth range for 3D particle localization and tracking.
• This method maintains high optical resolution and throughput.
• It presents a versatile tool for diverse applications, including real-time fluid dynamics imaging.