Optimising structured illumination microscopy
File(s)
Author(s)
Liang, Meizhu
Type
Thesis
Abstract
Structured illumination microscopy (SIM) features high imaging speed, resolution beyond the diffraction limit and a large field of view, revolutionising conventional microscopy and is well-suited for long-term live-cell imaging and structural biology studies. The system presented in this thesis exploits hexagonal-patterned illumination using a spatial light modulator (SLM) to realise SIM.
The SIM imaging process is simulated and extended to cover various SIM geometries, where patterns of illumination are produced from polarised beams. All simulations are integrated as a plugin for the Napari platform and include sample movements such as defocusing, drift and Brownian motion.
In this SIM technique, the SLM provides the illumination beams by diffraction from displayed holograms. However, in practice, the diffracted orders were found to be extended due to the SLM not being perfectly flat. To measure and correct this aberration, a phase-recovery method is developed based on measurements of the diffracted PSF, which effectively corrects distorted diffracted orders to achieve diffraction-limited results. The resultant wavefront RMS 0.063 rad is smaller than the diffraction-limited value of 0.469 rad.
Three methods of hologram generation are compared. A fast method based on direct binarisation of beam interference (DBBI) is introduced. This method can generate holograms including phase corrections without compromising speed. Subtle features of the corresponding structured illumination can be further corrected by stepping an additional phase offset.
Our optical system is modified to allow remote and easy alignment. Phase recovery is performed in-situ to correct any changes in SLM flatness. The resultant PSF is diffraction-limited since the wavefront RMS is measured as 0.113 rad. Captured and reconstructed images with this correction show a complete field of view (FOV), while results without correction have a limited FOV. Additionally, phase stepping is applied to reduce patterned artefacts in the reconstructed images.
The SIM imaging process is simulated and extended to cover various SIM geometries, where patterns of illumination are produced from polarised beams. All simulations are integrated as a plugin for the Napari platform and include sample movements such as defocusing, drift and Brownian motion.
In this SIM technique, the SLM provides the illumination beams by diffraction from displayed holograms. However, in practice, the diffracted orders were found to be extended due to the SLM not being perfectly flat. To measure and correct this aberration, a phase-recovery method is developed based on measurements of the diffracted PSF, which effectively corrects distorted diffracted orders to achieve diffraction-limited results. The resultant wavefront RMS 0.063 rad is smaller than the diffraction-limited value of 0.469 rad.
Three methods of hologram generation are compared. A fast method based on direct binarisation of beam interference (DBBI) is introduced. This method can generate holograms including phase corrections without compromising speed. Subtle features of the corresponding structured illumination can be further corrected by stepping an additional phase offset.
Our optical system is modified to allow remote and easy alignment. Phase recovery is performed in-situ to correct any changes in SLM flatness. The resultant PSF is diffraction-limited since the wavefront RMS is measured as 0.113 rad. Captured and reconstructed images with this correction show a complete field of view (FOV), while results without correction have a limited FOV. Additionally, phase stepping is applied to reduce patterned artefacts in the reconstructed images.
Version
Open Access
Date Issued
2024-06
Date Awarded
2024-11
Copyright Statement
Creative Commons Attribution NonCommercial Licence
License URL
Advisor
Neil, Mark
Publisher Department
Physics
Publisher Institution
Imperial College London
Qualification Level
Doctoral
Qualification Name
Doctor of Philosophy (PhD)