PT  - JOURNAL ARTICLE
AU  - Nakatani, Yuya
AU  - Gaumer, Scott
AU  - Shechtman, Yoav
AU  - Gustavsson, Anna-Karin
TI  - Long axial-range double-helix point spread functions for 3D volumetric super-resolution imaging
AID  - 10.1101/2024.07.31.605907
DP  - 2024 Jan 01
TA  - bioRxiv
PG  - 2024.07.31.605907
4099  - http://biorxiv.org/content/early/2024/08/01/2024.07.31.605907.short
4100  - http://biorxiv.org/content/early/2024/08/01/2024.07.31.605907.full
AB  - Single-molecule localization microscopy (SMLM) is a powerful tool for observing structures beyond the diffraction limit of light. Combining SMLM with engineered point spread functions (PSFs) enables 3D imaging over an extended axial range, as has been demonstrated for super-resolution imaging of various cellular structures. However, super-resolving structures in 3D in thick samples, such as whole mammalian cells, remains challenging as it typically requires acquisition and post-processing stitching of multiple slices to cover the entire sample volume or more complex analysis of the data. Here, we demonstrate how the imaging and analysis workflows can be simplified by 3D single-molecule super-resolution imaging with long axial-range double-helix (DH)-PSFs. First, we experimentally benchmark the localization precisions of short- and long axial-range DH-PSFs at different signal-to-background ratios by imaging of fluorescent beads. The performance of the DH-PSFs in terms of achievable resolution and imaging speed was then quantified for 3D single-molecule super-resolution imaging of mammalian cells by DNA-PAINT imaging of the nuclear lamina protein lamin B1 in U-2 OS cells. Furthermore, we demonstrate how the use of a deep learning-based algorithm allows the localization of dense emitters, drastically improving the achievable imaging speed and resolution. Our data demonstrate that using long axial-range DH-PSFs offers stitching-free, 3D super-resolution imaging of whole mammalian cells, simplifying the experimental and analysis procedures for obtaining volumetric nanoscale structural information.Competing Interest StatementY.N., Y.S., and A.-K.G declare no competing financial interest. S.G. is an employee of Double Helix Optics Inc.