Development of optical projection tomography for mesoscopic 3-D biomedical imaging
File(s)
Author(s)
Chen, Lingling
Type
Thesis
Abstract
Optical projection tomography (OPT) is an exciting technique for imaging “mesoscopic” (1 – 10 mm) samples at a high resolution, providing three-dimensional (3-D) absorption and/or fluorescence distributions of chromophores in optically transparent specimens. This thesis describes the study, development and application of OPT technology for potential applications in biomedical research including developmental biology, tissue analysis (e.g. for histopathology), the study of disease mechanisms and therapies, dosimetry and drug discovery.
The thesis begins with an introduction to fluorescence, fluorescence lifetime imaging (FLIM) and OPT and then describes the experimental configurations and characterisation of in-house developed intensity-based OPT instruments. Using the measured characteristics of specific OPT set-ups, different tomographic reconstruction approaches for OPT are investigated through modelling and experiments and the achievable image quality and speed are compared to the performance obtained with the standard reconstruction algorithm.
The development of OPT using a novel angular multiplexing technique is then discussed. This employs multiple imaging systems in parallel that can acquire OPT data sets simultaneously. Their performances using different focusing arrangements to image either shifted focal planes or a common focal plane in the sample is investigated and shown to provide faster imaging with improved spatial resolution. The latter configuration is demonstrated to offer the capability to track feature motions such as cell trajectories with a time lapse resolution limited by the frame rate of the cameras and to provide 3-D feature tracking with significantly reduced light dose compared to standard OPT.
The application of OPT and its extension to incorporate wide-field time-gated FLIM, referred as FLIM-OPT, to a range of biological specimens is then discussed, including fixed and chemically cleared tissues and live animal models. These experiments demonstrate the efficacy of OPT as a 3-D imaging tool for biomedical research. The potential efficacy of FLIM-OPT to read out Förster resonance energy transfer (FRET) is then demonstrated, including in live zebrafish embryos. Thus OPT is shown to provide a 3-D tomographic imaging technique able to yield structural and functional information in intact organisms.
The thesis begins with an introduction to fluorescence, fluorescence lifetime imaging (FLIM) and OPT and then describes the experimental configurations and characterisation of in-house developed intensity-based OPT instruments. Using the measured characteristics of specific OPT set-ups, different tomographic reconstruction approaches for OPT are investigated through modelling and experiments and the achievable image quality and speed are compared to the performance obtained with the standard reconstruction algorithm.
The development of OPT using a novel angular multiplexing technique is then discussed. This employs multiple imaging systems in parallel that can acquire OPT data sets simultaneously. Their performances using different focusing arrangements to image either shifted focal planes or a common focal plane in the sample is investigated and shown to provide faster imaging with improved spatial resolution. The latter configuration is demonstrated to offer the capability to track feature motions such as cell trajectories with a time lapse resolution limited by the frame rate of the cameras and to provide 3-D feature tracking with significantly reduced light dose compared to standard OPT.
The application of OPT and its extension to incorporate wide-field time-gated FLIM, referred as FLIM-OPT, to a range of biological specimens is then discussed, including fixed and chemically cleared tissues and live animal models. These experiments demonstrate the efficacy of OPT as a 3-D imaging tool for biomedical research. The potential efficacy of FLIM-OPT to read out Förster resonance energy transfer (FRET) is then demonstrated, including in live zebrafish embryos. Thus OPT is shown to provide a 3-D tomographic imaging technique able to yield structural and functional information in intact organisms.
Version
Open Access
Date Issued
2013-10
Date Awarded
2014-02
Copyright Statement
Attribution NoDerivatives 4.0 International Licence (CC BY-ND)
Advisor
French, Paul
McGinty, James
Dunsby, Christopher
Sponsor
Lee Family Scholarship
Publisher Department
Physics
Publisher Institution
Imperial College London
Qualification Level
Doctoral
Qualification Name
Doctor of Philosophy (PhD)