Spectrally and temporally resolved single photon counting in advanced biophotonics applications

Biomedicine requires highly sensitive and efficient light sensors to analyse light-tissue or light-sample interactions. Single-photon avalanche diode (SPAD) sensors implemented with complementary metal-oxide-semiconductor (CMOS) technology have a growing range of applications in this field. Single-photon detection coupled with integrated timing circuits enables us to timestamp each detected photon with high temporal resolution (down to picoseconds). Arrays of SPAD based pixels and CMOS technology offer massively parallel time-resolved single-photon counting for spectrally and temporally resolved analysis of various light phenomena.This thesis examines how time-resolved CMOS SPAD based line sensors with per pixel timing circuits can be utilized to advance biophotonic applications. The study focuses on improving the existing techniques of fluorescence and Raman spectroscopy, and demonstrates for the first time CMOS SPAD based detection in optical coherence tomography (OCT). A novel detection scheme is proposed combining low-coherence interferometry and time-resolved photon counting. In this approach the interferometric information is revealed from spectral intensity measurements, which is supplemented by time-stamping of the photons building up the spectra.Two CMOS SPAD line sensors (Ra-I and its improved version, Ra-II) were characterized and the effect of their parameters on the selected techniques was analysed. The thesis demonstrates the deployment of the Ra-I line sensor in time-resolved fluorescence spectroscopy with indications of the applicability in time-resolved Raman spectroscopy. The work includes integration of the sensor with surrounding electrical and optical systems, and the implementation of firmware and software for controlling the optical setup. As a result, a versatile platform is demonstrated capable of micro- and millisecond sampling of spectral fluorescence lifetime changes in a single transient of fast chemical reactions.OCT operating in the spectral domain traditionally uses CMOS photodiode and charge-coupled device (CCD) based detectors. The applicability of CMOS SPAD sensors is investigated for the first time with focus on the main limitations and related challenges. Finally, a new detection method is proposed relying on both the wave and particle nature of light, recording time-resolved interferometric spectra from a Michelson interferometer. This method offers an alternative approach to analyse luminous effects and improves techniques based on the light’s time of flight. As an example, a proof of concept study is presented for the removal of unwanted reflections from along the sample and the optical path in an OCT setup

Sub millimetre flexible fibre probe for background and fluorescence free Raman spectroscopy

Using the shifted-excitation Raman difference spectroscopy technique and an optical fibre featuring a negative curvature excitation core and a coaxial ring of high numerical aperture collection cores, we have developed a portable, background and fluorescence free, endoscopic Raman probe. The probe consists of a single fibre with a diameter of less than 0.25 mm packaged in a sub-millimetre tubing, making it compatible with standard bronchoscopes. The Raman excitation light in the fibre is guided in air and therefore interacts little with silica, enabling an almost background free transmission of the excitation light. In addition, we used the shifted-excitation Raman difference spectroscopy technique and a tunable 785 nm laser to separate the fluorescence and the Raman spectrum from highly fluorescent samples, demonstrating the suitability of the probe for biomedical applications. Using this probe we also acquired fluorescence free human lung tissue data

Demonstrating the Use of Optical Fibres in Biomedical Sensing:A Collaborative Approach for Engagement and Education

This paper demonstrates how research at the intersection of physics, engineering, biology and medicine can be presented in an interactive and educational way to a non-scientific audience. Interdisciplinary research with a focus on prevalent diseases provides a relatable context that can be used to engage with the public. Respiratory diseases are significant contributors to avoidable morbidity and mortality and have a growing social and economic impact. With the aim of improving lung disease understanding, new techniques in fibre-based optical endomicroscopy have been recently developed. Here, we present a novel engagement activity that resembles a bench-to-bedside pathway. The activity comprises an inexpensive educational tool ($70) adapted from a clinical optical endomicroscopy system and tutorials that cover state-of-the-art research. The activity was co-created by high school science teachers and researchers in a collaborative way that can be implemented into any engagement development process

Dataset for "Tri-mode optical biopsy probe with fluorescence endomicroscopy, Raman spectroscopy, and time resolved fluorescence spectroscopy"

Data supporting "Tri-mode optical biopsy probe with fluorescence endomicroscopy, Raman spectroscopy, and time resolved fluorescence spectroscopy", including figures from the article, supplementary figures and diagrams of the experimental setups used, bend loss data from the fibre used for Time Resolved Fluorescence Spectroscopy (TRFS), and the raw Optical EndoMicroscopy (OEM) video files used to produce figure 5. This body of work characterises the performance of an optical fibre probe that was designed and built to provide three complimentary optical methods of endoscopically interrogating human tissue: Optical EndoMicroscopy (OEM), Time Resolved Fluorescence Spectroscopy (TRFS), and Raman spectroscopy. Its ability to characterise standard targets was evaluated, both when subjected to a sequence of bends, and then within a model of a human lung. Ex-vivo human lung tissue was also used as a target to explore its tissue characterisation capabilities. These data sets form part of the pre-clinical testing and validation required for progression to its intended application in pulmonology.Full details of the methodology can be found in the associated paper

Fibre-optic based exploration of lung cancer autofluorescence using spectral fluorescence lifetime

Fibre-optic based time-resolved fluorescence spectroscopy (TRFS) is an advanced spectroscopy technique that generates sample-specific spectral-temporal signature, characterising variations in fluorescence in real-time. As such, it can be used to interrogate tissue autofluorescence. Recent advancements in TRFS technology, including the development of devices that simultaneously measure high-resolution spectral and temporal fluorescence, paired with novel analysis methods extracting information from these multidimensional measurements effectively, provide additional insight into the underlying autofluorescence features of a sample. This study demonstrates, using both simulated data and endogenous fluorophores measured bench-side, that the shape of the spectral fluorescence lifetime, or fluorescence lifetimes estimated over high-resolution spectral channels across a broad range, is influenced by the relative abundance of underlying fluorophores in mixed systems and their respective environment. This study, furthermore, explores the properties of the spectral fluorescence lifetime in paired lung tissue deemed either abnormal or normal by pathologists. We observe that, on average, the shape of the spectral fluorescence lifetime at multiple locations sampled on 14 abnormal lung tissue, compared to multiple locations sampled on the respective paired normal lung tissue, shows more variability; and, while not statistically significant, the average spectral fluorescence lifetime in abnormal tissue is consistently lower over every wavelength than the normal tissue.</p

Dataset for "Sub millimetre flexible fibre probe for background and fluorescence free Raman spectroscopy"

This dataset contains data supporting the results presented in the paper "Sub millimetre flexible fibre probe for background and fluorescence free Raman spectroscopy ". It includes the data used to plot each figure (in .xlsx format). Using the shifted-excitation Raman difference spectroscopy technique and an optical fibre featuring a negative curvature excitation core and a coaxial ring of high numerical aperture collection cores, we have developed a portable, background and fluorescence free, endoscopic Raman probe. The probe consists of a single fibre with a diameter of less than 0.25 mm packaged in a sub-millimetre tubing, making it compatible with standard bronchoscopes. The Raman excitation light in the fibre is guided in air and therefore interacts little with silica, enabling an almost background free transmission of the excitation light. In addition, we used the shifted-excitation Raman difference spectroscopy technique and a tunable 785 nm laser to separate the fluorescence and the Raman spectrum from highly fluorescent samples, demonstrating the suitability of the probe for biomedical applications. Using this probe we also acquired fluorescence free human lung tissue data.The different data collection methods used for the collection of the data are described in the paper Sub millimetre flexible fibre probe for background and fluorescence free Raman spectroscopy