Long-Axial-Range Double-Helix Point Spread Functions for 3D Volumetric Super-Resolution Imaging

Area of Science:

• Biophysics
• Microscopy
• Cell Biology

Background:

• Single-molecule localization microscopy (SMLM) offers super-resolution imaging beyond light diffraction limits.
• Engineered point spread functions (PSFs) extend SMLM to 3D imaging, but challenges remain for thick samples like mammalian cells.
• Current 3D SMLM for thick samples often requires complex multi-slice acquisition and post-processing.

Purpose of the Study:

• To simplify 3D super-resolution imaging workflows for thick biological samples.
• To demonstrate the utility of long-axial-range double-helix (DH)-PSFs for stitching-free 3D SMLM.
• To improve imaging speed and resolution in 3D SMLM of cellular structures.

Main Methods:

• Experimental benchmarking of DH-PSF localization precision using fluorescent beads.
• Quantification of DH-PSF performance in 3D SMLM of mammalian cells (U-2 OS) using DNA-PAINT.
• Application of a deep-learning algorithm for localizing dense emitters.

Main Results:

• Long-axial-range DH-PSFs were experimentally validated for 3D SMLM.
• Performance was quantified for imaging nuclear lamina protein lamin B1 in mammalian cells.
• Deep learning significantly enhanced imaging speed and resolution by localizing dense emitters.

Conclusions:

• Long-axial-range DH-PSFs enable stitching-free 3D super-resolution imaging of entire mammalian cells.
• The developed method simplifies experimental and analysis procedures for volumetric nanoscale imaging.
• This approach facilitates obtaining nanoscale structural information in 3D within thick biological samples.