Multidimensional Super-resolution Imaging: Wasting Light to Learn New Things

Session

Session 4 - New Technologies: Recent advances from Acquisition to Analysis

Authors

Steven Lee (1)

Affiliations

1. Yusuf Hamied Department of Chemistry, University of Cambridge

Keywords

Abstract text

The talk will outline two single-molecule fluorescence approaches that can be used to determine orthogonal metrics about a single emitter.

The first half introduces "POLCAM," a simplified single-molecule orientation localization microscopy (SMOLM) method based on polarised detection using a polarisation camera. POLCAM's fast algorithm operates over 1000 times faster than the current state-of-the-art, allowing near-instant determination of molecular anisotropy. To aid adoption, open-source image analysis software and visualization tools were developed. POLCAM's potential was demonstrated in studying alpha-synuclein fibrils and the actin cytoskeleton of mammalian cells. (Nature Methods in press). 

The second approach focuses on "Single-Molecule Light Field Microscopy" (SMLFM), encoding 3D positions into 2D images for volumetric super-resolution microscopy. SMLFM shows an order-of-magnitude speed improvement over other 3D PSFs, resolving overlapping emitters through parallax. Experimental results reveal high accuracy and sensitivity in point detection, enabling whole-cell imaging of single membrane proteins in live primary B cells and high-density volumetric imaging in dense cytosolic tubulin datasets. (Nature Comms 2024)

References