Evaluation of a cheap ultrasonic stage for light source coherence function measurement, optical coherence tomography and dynamic focusing

We evaluate the performance of a cheap ultrasonic stage in setups related to optical coherence tomography. The stage was used in several configurations: (1) optical delay line in an optical coherence tomography (OCT) setup; (2) as a delay line measuring coherence function of a low coherence source (e. g. superluminescent diode) and (3) in a dynamic focusing arrangement. The results are as follows: the stage is suitable for coherence function measurement (coherence length up to 70 mu m) of the light source and dynamic focusing. We found it unsuitable for OCT due to an unstable velocity profile. Despite this, the velocity profile has a repeatable shape (4% over 1000 A-scans) and slight modifications to the stage promise wider applications

Deconvolution and Restoration of Optical Endomicroscopy Images

Optical endomicroscopy (OEM) is an emerging technology platform with preclinical and clinical imaging applications. Pulmonary OEM via fibre bundles has the potential to provide in vivo, in situ molecular signatures of disease such as infection and inflammation. However, enhancing the quality of data acquired by this technique for better visualization and subsequent analysis remains a challenging problem. Cross coupling between fiber cores and sparse sampling by imaging fiber bundles are the main reasons for image degradation, and poor detection performance (i.e., inflammation, bacteria, etc.). In this work, we address the problem of deconvolution and restoration of OEM data. We propose a hierarchical Bayesian model to solve this problem and compare three estimation algorithms to exploit the resulting joint posterior distribution. The first method is based on Markov chain Monte Carlo (MCMC) methods, however, it exhibits a relatively long computational time. The second and third algorithms deal with this issue and are based on a variational Bayes (VB) approach and an alternating direction method of multipliers (ADMM) algorithm respectively. Results on both synthetic and real datasets illustrate the effectiveness of the proposed methods for restoration of OEM images

A time-resolved multifocal multiphoton microscope for high speed FRET imaging in vivo

Imaging the spatio-temporal interaction of proteins in vivo is essential to understanding the complexities of biological systems. The highest accuracy monitoring of protein-protein interactions is achieved using FRET measured by fluorescence lifetime imaging with measurements taking minutes to acquire a single frame, limiting their use in dynamic live cell systems. We present a diffraction limited, massively parallel, time-resolved multifocal multiphoton microscope capable of producing fluorescence lifetime images with 55 ps time-resolution giving improvements in acquisition speed of a factor of 64. We present demonstrations with FRET imaging in a model cell system and demonstrate in vivo FLIM using a GTPase biosensor in the zebrafish embryo

New high-speed centre of mass method incorporating background subtraction for accurate determination of fluorescence lifetime

We demonstrate an implementation of a centre-of-mass method (CMM) incorporating background subtraction for use in multifocal fluorescence lifetime imaging microscopy to accurately determine fluorescence lifetime in live cell imaging using the Megaframe camera. The inclusion of background subtraction solves one of the major issues associated with centre-of-mass approaches, namely the sensitivity of the algorithm to background signal. The algorithm, which is predominantly implemented in hardware, provides real-time lifetime output and allows the user to effectively condense large amounts of photon data. Instead of requiring the transfer of thousands of photon arrival times, the lifetime is simply represented by one value which allows the system to collect data up to limit of pulse pile-up without any limitations on data transfer rates. In order to evaluate the performance of this new CMM algorithm with existing techniques (i.e. Rapid lifetime determination and Levenburg-Marquardt), we imaged live MCF-7 human breast carcinoma cells transiently transfected with FRET standards. We show that, it offers significant advantages in terms of lifetime accuracy and insensitivity to variability in dark count rate (DCR) between Megaframe camera pixels. Unlike other algorithms no prior knowledge of the expected lifetime is required to perform lifetime determination. The ability of this technique to provide real-time lifetime readout makes it extremely useful for a number of applications

Optical Computed Tomography Instrumentation For Read-Out of 3-D Dosimeters.

The main topic of this thesis is 3-D measurement of absorbed dose in radiotherapy using advanced optical imaging methods. The main ideas behind the implementation presented in this thesis come from X ray computed tomography, 3-D microscopy and schlieren techniques. Every tumour has different shape and radiotherapy aims to conform the absorbed dose to tumour shape thus sparing healthy tissue as much as possible. If a complex treatment plan is tested on tissue-equivalent 3-D dosimeter prior to treating a patient then the quality of treatment can be improved. 3-D dosimeters (often called gel dosimeters) have been developed extensively over the last 20 years. They consist of radiosensistive chemicals evenly distributed across the volume of the supporting matrix (e. g. gelatin). They are designed with the readout technique in mind, which can be Magnetic Resonance Imaging, X ray computed tomography or optical computed tomography (optical-CT). This PhD project focuses on optical-CT readout. Optical-CT can be performed with either a laser light source coupled to a photodiode or broadbeam light source coupled to camera - most often a charged coupled detector (CCD). Both options have been either improved or designed from scratch. The main results are as follows: 1) detailed analysis of improved focusing optics of CCD based optical-CT shows telecentric focusing enables low noise measurements with a simple design; 2) characterization of CCD based optical tomography instrument (optical-CT) shows signal-to-noise ratio to be greater than 80:1 for 1mm voxel; 3) a fast and novel laser scanning architecture is demonstrated and characterized. Both of the instruments developed are a significant improvement to the field of optical-CT in 3-D dosimetry paving the way, hopefully, for a clinical application