High-speed 3D structured illumination microscopy (SIM)
File(s)
Author(s)
Boualam, Abderrahim
Type
Thesis
Abstract
Super-resolution Structured Illumination Microscopy (SIM) is a microscopy technique which provides a two-fold enhancement in resolution over traditional microscopy, beyond the diffraction limit of light. In this thesis, I investigate the patiotemporal limits of SIM and explore new strategies to increase speed and throughput. I describe a new method, developed to assess the spatiotemporal resolution limits of SIM in silico. This method is based on tracking the magnitude of different spatial frequencies in the reconstructed SIM images while modulating the intensity of the illumination with different temporal frequencies. Notably, it was applied to determine the temporal resolution limits of a recent SIM reconstruction approach termed interleaving reconstruction. It was demonstrated that the latter does not provide the increase in temporal resolution that it claims over traditional SIM reconstruction approaches.
After identifying the factors limiting the performance of current SIM systems through a thorough study of the literature, a simple and robust optical design for SIM is proposed, which features a high power efficiency, imaging versatility and theoretically capable of live-cell imaging at speeds up to 800 two-dimensional SIM stacks/second and 740 three-dimensional SIM stacks/second. The potential enhancement in imaging speed provided by this new system relies on the use of a single moving part (galvo) to perform the rotations and phase shifts of the striped illumination with minimum switching delays, bringing the pattern switching speed to 2,600 frames/second. The first laboratory prototype was built successfully, and preliminary tests and characterisations were performed. Results show that the striped illumination pattern can be generated with high temporal stability. While the modulation contrast is yet not sufficient to obtain good-quality SIM images, strategies to reach the required contrast and improve the overall performance of the system in the future are proposed. Biological applications that will benefit from the microscope are explored.
After identifying the factors limiting the performance of current SIM systems through a thorough study of the literature, a simple and robust optical design for SIM is proposed, which features a high power efficiency, imaging versatility and theoretically capable of live-cell imaging at speeds up to 800 two-dimensional SIM stacks/second and 740 three-dimensional SIM stacks/second. The potential enhancement in imaging speed provided by this new system relies on the use of a single moving part (galvo) to perform the rotations and phase shifts of the striped illumination with minimum switching delays, bringing the pattern switching speed to 2,600 frames/second. The first laboratory prototype was built successfully, and preliminary tests and characterisations were performed. Results show that the striped illumination pattern can be generated with high temporal stability. While the modulation contrast is yet not sufficient to obtain good-quality SIM images, strategies to reach the required contrast and improve the overall performance of the system in the future are proposed. Biological applications that will benefit from the microscope are explored.
Version
Open Access
Date Issued
2022-09-03
Date Awarded
01/06/2023
License URL
Advisor
Rowlands, Christopher
Sponsor
Imperial College London
Engineering and Physical Sciences Research Council
Grant Number
EPSRC EP/S016538/1
Publisher Department
Bioengineering
Publisher Institution
Imperial College London
Qualification Level
Doctoral
Qualification Name
Doctor of Philosophy (PhD)