Dissertation defense: Friday, May 30

May 16, 2025

Student Name: Sajjad Khan
Program: PhD NSME
Date: Friday, May 30, 2025
Time: 8:30 am
Place: PAIS 2540
Advisor: Dr. Keith Lidke
Title: "Point-Spread Function (PSF) Engineering and Adaptive Measurements for Single Molecule Super-Resolution Imaging"

Abstract
Super-resolution techniques developed over the past couple of decades enabled us to bypass the classical diffraction limit of light by exploiting the independent behavior of the sample tagged with fluorophores. The independence arises from fluorophores when they switch between the dark and fluorescent state. These fluorophores can then be localized using the emission pattern of the individual fluorophores with precision of localization much better than the diffraction limit of light. Finally, the resulting list of coordinates is used to generate the high-resolution images or extract the quantitative insights into the sub-cellular structures within the 10 to 200 nm range. At this scale, many interesting biological questions can be investigated by visualizing the protein-protein interactions or by performing the spatiotemporal analyses of the biological structure of interest. Collectively referred to as nanoscopy, these techniques play a crucial role in advancing our understanding of biological processes and solving complex biological questions.

Single-molecule localization microscopy (SMLM) enables the precise spatial localization of single molecules in cellular structures. A phenomenon called supercritical angle fluorescence (SAF) is utilized in supercritical angle localization microscopy (SALM) to estimate the axial positions of single fluorophores. It is based on the fact that the intensity of SAF is highly sensitive to the fluorophore-coverslip distance. Conventional SALM methods typically involve splitting the fluorescence emission into supercritical and undercritical components, which requires a complicated two-channel system and can lead to reduced light efficiency. In this work, we introduce a simplified approach to traditional SALM by directly detecting all fluorescence into a single channel. Through simulations, we found that by accurately modeling the point spread function (PSF) with SAF, a single-channel system achieves better localization precision than conventional two-channel based SALM systems. Furthermore, we developed a stage-tilt correction algorithm, incorporating stage tilt in the PSF model, to improve axial precision over the entire field of view. We applied our method experimentally by imaging F-actin filaments in HeLa cells. We demonstrate that our method efficiently exploits the information from SAF and achieves enhanced axial localization precision and accuracy compared to traditional SMLM localization methods for single-channel systems.