Polarisation-based approaches to quantitative phase and hyperspectral imaging
File(s)
Author(s)
Liu, Huihui
Type
Thesis
Abstract
This thesis presents the development and application of polarisation-based hyperspectral and quantitative phase imaging (QPI) techniques featuring fast acquisition and overall simple and cost-effective implementation. These techniques, either already open-source or in preparation for public sharing, are suitable for both manual microscopy, including in lower resourced settings, and integration in high-content analysis (HCA) platforms.
PolSpec, a polarisation-based spectral imaging technique, was developed to provide fast, versatile, cost-effective and data-efficient hyperspectral imaging with improved signal levels compared to conventional “push-broom” approaches. PolSpec employs Lyot filter-based set-ups to directly acquire data for calculating spectral modulation vectors (SMVs), which provide an efficient graphical representation of spectral signatures to facilitate spectral classification and linear unmixing. PolSpec data acquisition requires only several images using normal cameras, or a single shot using a polarisation-resolving (Polarsens™) camera. Multiple potential PolSpec configurations are proposed, of which two have been implemented, with their measured instrument response functions demonstrating consistency with the theoretical curve shapes. Spectral classification and linear unmixing were performed using PolSpec data acquired from test objects and biological samples.
Open-source software and hardware tools were also developed for polarisation-resolved differential phase contrast (pDPC) microscopy, a single-shot, wavelength-agnostic (semi)quantitative phase imaging technique using the Polarsens™ camera. Utilising these tools, condenser-based pDPC was implemented on a customized openFrame microscope. The phase reconstruction accuracy and spatial resolution were benchmarked using a quantitative phase target, and its applications to diverse biological samples were demonstrated. pDPC was also integrated into an existing HCA platform configured on a commercial microscope frame to provide single-cell tracking during long time-lapse multiwell plate fluorescence assays probing cancer cell response to drug treatment and, combined with machine learning, to realise label-free identification of cell cycle stages. Noting the spatial resolution limits of condenser-based pDPC, low-cost condenser-less pDPC and Polarsens™-based phase-stepping holography were explored for high-NA single-shot QPI.
PolSpec, a polarisation-based spectral imaging technique, was developed to provide fast, versatile, cost-effective and data-efficient hyperspectral imaging with improved signal levels compared to conventional “push-broom” approaches. PolSpec employs Lyot filter-based set-ups to directly acquire data for calculating spectral modulation vectors (SMVs), which provide an efficient graphical representation of spectral signatures to facilitate spectral classification and linear unmixing. PolSpec data acquisition requires only several images using normal cameras, or a single shot using a polarisation-resolving (Polarsens™) camera. Multiple potential PolSpec configurations are proposed, of which two have been implemented, with their measured instrument response functions demonstrating consistency with the theoretical curve shapes. Spectral classification and linear unmixing were performed using PolSpec data acquired from test objects and biological samples.
Open-source software and hardware tools were also developed for polarisation-resolved differential phase contrast (pDPC) microscopy, a single-shot, wavelength-agnostic (semi)quantitative phase imaging technique using the Polarsens™ camera. Utilising these tools, condenser-based pDPC was implemented on a customized openFrame microscope. The phase reconstruction accuracy and spatial resolution were benchmarked using a quantitative phase target, and its applications to diverse biological samples were demonstrated. pDPC was also integrated into an existing HCA platform configured on a commercial microscope frame to provide single-cell tracking during long time-lapse multiwell plate fluorescence assays probing cancer cell response to drug treatment and, combined with machine learning, to realise label-free identification of cell cycle stages. Noting the spatial resolution limits of condenser-based pDPC, low-cost condenser-less pDPC and Polarsens™-based phase-stepping holography were explored for high-NA single-shot QPI.
Version
Open Access
Date Issued
2025-08-15
Date Awarded
01/01/2026
License URL
Advisor
French, Paul
Sponsor
Imperial College London
Publisher Department
Department of Physics
Publisher Institution
Imperial College London
Qualification Level
Doctoral
Qualification Name
Doctor of Philosophy (PhD)