Fluorescence lifetime imaging with distance and ranging using a miniaturised SPAD system.

In this work we demonstrate a miniaturised imaging system based around a time-gated SPAD array operating in a "chip-on-tip" manner. Two versions of the system are demonstrated, each measuring 23 mm × 23 mm × 28 mm with differing fields of view and working distances. Initial tests demonstrate contrast between materials in widefield fluorescence imaging (WFLIm) mode, with frame rates of > 2 Hz achievable. Following this, WFLIm images of autofluorescence in ovine lung tissue are obtained at frame rates of ~ 1 Hz. Finally, the ability of the second system to perform simultaneous WFLIm and time of flight (aka Flourescence Lifetime Imaging Distance and Ranging, FLImDAR) is also tested. This shows that the system is capable of 4 mm resolution of object separation when tested on 3D printed samples. It is further demonstrated as being able to perform scene reconstruction on autofluorescent lung tissue. This system is, to date, the smallest chip on tip WFLIm system published, and is the first demonstration of the FLImDAR technique in a compact, portable system.

Photon counting fibre optic distributed temperature sensing with a CMOS SPAD array.

Time-resolved fibre optic Raman distributed temperature sensing (DTS) measurements experience long measurement times due to a weak backscattered Raman signal inside optical fibres or limited detector count rates. Here, improvements to previous work based on individual detectors are demonstrated using a 512 pixel complementary-metal-oxide semiconductor (CMOS) single-photon avalanche diode (SPAD) line sensor array with integrated (on-chip) timing electronics. Multiplexed single photon counting increases count rate and decreases measurement time for practical applications. This allows temperature to be measured every 0.5 m with 0.7 °C accuracy and a 10 s measurement time using a 13.0 m optical fibre, performance over longer distance is also investigated.

Combined fluorescence lifetime and surface topographical imaging of biological tissue.

In this work a combined fluorescence lifetime and surface topographical imaging system is demonstrated. Based around a 126 × 192 time resolved single photon avalanche diode (SPAD) array operating in time correlated single-photon counting (TCSPC) mode, both the fluorescence lifetime and time of flight (ToF) can be calculated on a pixel by pixel basis. Initial tests on fluorescent samples show it is able to provide 4 mm resolution in distance and 0.4 ns resolution in lifetime. This combined modality has potential biomedical applications such as surgical guidance, endoscopy, and diagnostic imaging. The system is demonstrated on both ovine and human pulmonary tissue samples, where it offers excellent fluorescence lifetime contrast whilst also giving a measure of the distance to the sample surface.

Handheld wide-field fluorescence lifetime imaging system based on a distally mounted SPAD array.

In this work a handheld Fluorescent Lifetime IMaging (FLIM) system based on a distally mounted < 2 mm2 128 × 120 single photon avalanche diode (SPAD) array operating over a > 1 m long wired interface is demonstrated. The head of the system is ∼4.5 cm x 4.5 cm x 4.5 cm making it suitable for hand-held ex vivo applications. This is, to the best of the authors' knowledge, the first example of a SPAD array mounted on the distal end of a handheld FLIM system in this manner. All existing systems to date use a fibre to collect and relay fluorescent light to detectors at the proximal end of the system. This has clear potential biological and biomedical applications. To demonstrate this, the system is used to provide contrast between regions of differing tissue composition in ovine kidney samples, and between healthy and stressed or damaged plant leaves. Additionally, FLIM videos are provided showing that frame rates of > 1 Hz are achievable. It is thus an important step in realising an in vivo miniaturized chip-on-tip FLIM endoscopy system.

Ultrafast laser ablation of a multicore polymer optical fiber for multipoint light emission.

We demonstrate the use of ultrafast laser pulses to precisely ablate the side of polymer multicore optical fibres (MCF) in such a way that light is efficiently coupled out of a set of MCF cores to free space. By individually exciting sets of MCF cores, this flexible "micro-window" technology allows the controllable generation of light sources at multiple independently selectable locations along the MCF. We found that the maximum fraction of light that could be side coupled from the MCF varied between 55% and 73%.

Molecular detection of Gram-positive bacteria in the human lung through an optical fiber-based endoscope.

PURPOSE: The relentless rise in antimicrobial resistance is a major societal challenge and requires, as part of its solution, a better understanding of bacterial colonization and infection. To facilitate this, we developed a highly efficient no-wash red optical molecular imaging agent that enables the rapid, selective, and specific visualization of Gram-positive bacteria through a bespoke optical fiber-based delivery/imaging endoscopic device. METHODS: We rationally designed a no-wash, red, Gram-positive-specific molecular imaging agent (Merocy-Van) based on vancomycin and an environmental merocyanine dye. We demonstrated the specificity and utility of the imaging agent in escalating in vitro and ex vivo whole human lung models (n = 3), utilizing a bespoke fiber-based delivery and imaging device, coupled to a wide-field, two-color endomicroscopy system. RESULTS: The imaging agent (Merocy-Van) was specific to Gram-positive bacteria and enabled no-wash imaging of S. aureus within the alveolar space of whole ex vivo human lungs within 60 s of delivery into the field-of-view, using the novel imaging/delivery endomicroscopy device. CONCLUSION: This platform enables the rapid and specific detection of Gram-positive bacteria in the human lung.

Core crosstalk in ordered imaging fiber bundles.

Coherent fiber bundles are used widely for imaging. Commonly, disordered arrays of randomly sized fiber cores avoid proximity between like-cores, which would otherwise result in increased core crosstalk and a negative impact on imaging. Recently, stack-and-draw fiber manufacture techniques have been used to produce fibers with a controlled core layout to minimize core crosstalk. However, one must take manufacturing considerations into account during stack-and-draw fiber design in order to avoid impractical or unachievable fabrication. This comes with a set of practical compromises, such as using only a small number of different core sizes. Through characterization of core crosstalk patterns, this Letter aims to aid the understanding of crosstalk limitations imposed by such compromises in the core layout made for ease of fabrication.

Time-Resolved Spectroscopy of Fluorescence Quenching in Optical Fibre-Based pH Sensors.

Numerous optodes, with fluorophores as the chemical sensing element and optical fibres for light delivery and collection, have been fabricated for minimally invasive endoscopic measurements of key physiological parameters such as pH. These flexible miniaturised optodes have typically attempted to maximize signal-to-noise through the application of high concentrations of fluorophores. We show that high-density attachment of carboxyfluorescein onto silica microspheres, the sensing elements, results in fluorescence energy transfer, manifesting as reduced fluorescence intensity and lifetime in addition to spectral changes. We demonstrate that the change in fluorescence intensity of carboxyfluorescein with pH in this "high-density" regime is opposite to that normally observed, with complex variations in fluorescent lifetime across the emission spectra of coupled fluorophores. Improved understanding of such highly loaded sensor beads is important because it leads to large increases in photostability and will aid the development of compact fibre probes, suitable for clinical applications. The time-resolved spectral measurement techniques presented here can be further applied to similar studies of other optodes.

A hydrogel-based optical fibre fluorescent pH sensor for observing lung tumor tissue acidity.

Technologies for measuring physiological parameters in vivo offer the possibility of the detection of disease and its progression due to the resulting changes in tissue pH, or temperature, etc.. Here, a compact hydrogel-based optical fibre pH sensor was fabricated, in which polymer microarrays were utilized for the high-throughput discovery of an optimal matrix for pH indicator immobilization. The fabricated hydrogel-based probe responded rapidly to pH changes and demonstrated a good linear correlation within the physiological pH range (from 5.5 to 8.0) with a precision of 0.10 pH units. This miniature probe was validated by measuring pH across a whole ovine lung and allowed discrimination of tumorous and normal tissue, thus offering the potential for the rapid and accurate observation of tissue pH changes.

Ultrafast-laser-ablation-assisted spatially selective attachment of fluorescent sensors onto optical fibers.

A robust method to selectively attach specific fluorophores onto the individual cores of a multicore fiber is reported in this Letter. The method is based on the use of ultrafast laser pulses to nanostructure the facet of the fiber core, followed by amine functionalization and sensor conjugation. This surface-machining protocol not only enables precise spatial selectivity, but it also facilitates high deposition densities of the sensor moieties. As a proof of concept, the successful deposition of three different fluorophores onto selected cores of a multicore fiber is demonstrated. The protocol was developed to include attachment of a fluorescence-based pH sensor using the ratiometric carboxynapthofluorescein.

Frugal filtering optical lenses for point-of-care diagnostics.

Infectious diseases are the leading cause of morbidity and mortality in low and middle income countries (LMICs). Rapid diagnosis of infections in LMICs presents many challenges, especially in rural areas where access to health care, including diagnostics, is poor. Microscopy is one of the most commonly used platforms to diagnose bacterial infections on clinical samples. Fluorescence microscopy has high sensitivity and specificity but to date is mostly performed within a laboratory setting due to the high-cost, low portability and highly specialist nature of equipment. Point-of-care diagnostics could offer a solution to the challenge of infection diagnosis in LMICs. In this paper we present frugal, easy to manufacture, doped polydimethylsiloxane filtering optical lenses that can be integrated into smartphone microscopes for immediate detection of fluorescently labelled bacteria. This provides a breakthrough technology platform for point-of-care diagnostics.

High fidelity fibre-based physiological sensing deep in tissue.

Physiological sensing deep in tissue remains a clinical challenge. Here a flexible miniaturised sensing optrode providing a platform to perform minimally invasive in vivo in situ measurements is reported. Silica microspheres covalently coupled with a high density of ratiometrically configured fluorophores were deposited into etched pits on the distal end of a 150 µm diameter multicore optical fibre. With this platform, photonic measurements of pH and oxygen concentration with high precision in the distal alveolar space of the lung are reported. We demonstrated the phenomenon that high-density deposition of carboxyfluorescein covalently coupled to silica microspheres shows an inverse shift in fluorescence in response to varying pH. This platform delivered fast and accurate measurements (±0.02 pH units and ±0.6 mg/L of oxygen), near instantaneous response time and a flexible architecture for addition of multiple sensors.

Fibre-based spectral ratio endomicroscopy for contrast enhancement of bacterial imaging and pulmonary autofluorescence.

Fibre-based optical endomicroscopy (OEM) permits high resolution fluorescence microscopy in endoscopically accessible tissues. Fibred OEM has the potential to visualise pathologies targeted with fluorescent imaging probes and provide an in vivo in situ molecular pathology platform to augment disease understanding, diagnosis and stratification. Here we present an inexpensive widefield ratiometric fibred OEM system capable of enhancing the contrast between similar spectra of pathologically relevant fluorescent signals without the burden of complex spectral unmixing. As an exemplar, we demonstrate the potential of the platform to detect fluorescently labelled Gram-negative bacteria in the challenging environment of highly autofluorescent lung tissue in whole ex vivo human lungs.

Polymer Microarrays for the Discovery and Optimization of Robust Optical-Fiber-Based pH Sensors.

Polymer microarrays were utilized for the high-throughput screening and discovery of optimal polymeric substrates capable of trapping functional ratiometric fluorescence-based pH sensors. This led to the identification of poly(methyl methacrylate- co-2-(dimethylamino) ethyl acrylate) (PA101), which allowed, via dip coating, the attachment of fluorescent pH sensors onto the tips of optical fibers, resulting in robust, rapid, and reproducible sensing of physiological pHs.

High-speed dual color fluorescence lifetime endomicroscopy for highly-multiplexed pulmonary diagnostic applications and detection of labeled bacteria.

We present a dual-color laser scanning endomicroscope capable of fluorescence lifetime endomicroscopy at one frame per second (FPS). The scanning system uses a coherent imaging fiber with 30,000 cores. High-speed lifetime imaging is achieved by distributing the signal over an array of 1024 parallel single-photon avalanche diode detectors (SPADs), minimizing detection dead-time maximizing the number of photons detected per excitation pulse without photon pile-up to achieve the high frame rate. This also enables dual color fluorescence imaging by temporally shifting the dual excitation lasers, with respect to each other, to separate the two spectrally distinct fluorescent decays in time. Combining the temporal encoding, to provide spectral separation, with lifetime measurements we show a one FPS, multi-channel endomicroscopy platform for clinical applications and diagnosis. We demonstrate the potential of the system by imaging SmartProbe labeled bacteria in ex vivo samples of human lung using lifetime to differentiate bacterial fluorescence from the strong background lung autofluorescence which was used to provide structural information.

Ultra-low background Raman sensing using a negative-curvature fibre and no distal optics.

Measuring Raman spectra through an optical fibre is usually complicated by the high intrinsic Raman scatter of the fibre material. Common solutions such as the use of multiple fibres and distal optics are complex and bulky. We demonstrate the use of single novel hollow-core negative-curvature fibres (NCFs) for Raman and surface-enhanced Raman spectroscopy (SERS) sensing using no distal optics. The background Raman emission from the silica in the NCF was at least 1000× smaller than in a conventional solid fibre, while maintaining the same collection efficiency. We transmitted pump light from a 785-nm laser through the NCF, and we collected back the weak Raman spectra of different distal samples, demonstrating the fibre probe can be used for measurements of weak Raman and SERS signals that would otherwise overlap spectrally with the silica background. The lack of distal optics and consequent small probe diameter (<0.25 mm) enable applications that were not previously possible.

Dual purpose fibre - SERS pH sensing and bacterial analysis.

The exploitation of fibre based Raman probes has been challenged by often complicated fabrication procedures and difficulties in reproducibility. Here, we have demonstrated a simple and cost-effective approach for sensing pH through an optical fibre, by employing a wax patterned filter paper-based substrate for surface enhanced Raman spectroscopy (SERS). Through this method, high reproducibility between fibres was achieved. In addition to sensing pH, it was possible to extract fluid samples containing P. aeruginosa for further analysis. This dual purpose fibre is bronchoscope deployable, and is able to gather information about both the host and pathogen, which may lead to an improved treatment plan in future in vivo applications.

Controlled core-to-core photo-polymerisation - fabrication of an optical fibre-based pH sensor.

The fabrication of fluorescence-based pH sensors, embedded into etched pits of an optical fibre via highly controllable and spatially selective photo-polymerisation is described and the sensors validated.

Characterization and modelling of inter-core coupling in coherent fiber bundles.

Recent developments in optical endomicroscopy (OEM) and associated fluorescent SmartProbes present a need for sensitive imaging with high detection performance. Inter-core coupling within coherent fiber bundles is a well recognized limitation, affecting the technology's imaging capabilities. Fiber cross coupling has been studied both experimentally and within a theoretical framework (coupled mode theory), providing (i) insights on the factors affecting cross talk, and (ii) recommendations for optimal fiber bundle design. However, due to physical limitations, such as the tradeoff between cross coupling and core density, cross coupling can be suppressed yet not eliminated through optimal fiber design. This study introduces a novel approach for measuring, analyzing and quantifying cross coupling within coherent fiber bundles, in a format that can be integrated into a linear model, which in turn can enable computational compensation of the associated blurring introduced to OEM images.

High-extinction ratio integrated photonic filters for silicon quantum photonics.

We present the generation of quantum-correlated photon pairs and subsequent pump rejection across two silicon-on-insulator photonic integrated circuits. Incoherently cascaded lattice filters are used to provide over 100 dB pass-band to stop-band contrast with no additional external filtering. Photon pairs generated in a microring resonator are successfully separated from the input pump, confirmed by temporal correlations measurements.