This talk will describe ratiometric imaging that can enhance visualisation of imaging probes against tissue autofluorescence. The Proteus project (www.proteus.ac.uk) aims to improve the detection and diagnosis of pulmonary infection and inflammation by employing targeted fluorescent molecules (Smartprobes) for labelling specific pathologies in tissue. However, imaging Smartprobes using a widefield fibred imaging system within the human lung can be challenging, in part, because both lung tissue and imaging probes have broad and overlapping emission spectra.. Weak signals from pathogens labelled with probes are easily missed due to the strong autofluorescent signatures of elastin and collagen that are abundantly present in the human lung. In addition to resolving probes from intrinsic fluorescence, multiple probes may have overlapping emission spectra themselves. This is particularly true for many well-established fluorophores that reside in the green region of the spectrum. If imaging with fluorophores that have distinct emission spectra is not possible or desirable, then spectral sorting of the signals can be carried out.
To successfully resolve probes from healthy tissue or to resolve similar probes from each other, acquiring full spectral information is not necessarily a requirement. We describe a simple widefield fibred imaging system consisting of a single colour LED illumination source (480nm) that enables ratiometric methods to enhance contrast between different fluorescent sources. Fluorescence from 480nm excitation of tissue as well as Smartprobes present on the tissue is split into two optical paths, above and below a cut-off wavelength, by a dichroic mirror. A triggered system of a monochrome CMOS camera and optical chopper allows collection of dual images of the same field of view from different parts of the spectrum. Contrast enhancement is carried out by post processing of the images, enabling us to interpret better the images produced both in autofluorescence and molecular imaging contexts.
Our widefield fibred imaging system is enabled by a novel optical fibre bundle developed by the University of Bath. The imaging fibres consist of 8100 cores with a 450µm corner to corner field of view and allows for multiplexed visualisation of pathologies within the lung. Biological targets, such as bacteria, that are of interest to clinicians, occupy one or two cores within the imaging fibre. We use 6µm Inspeck microspheres to demonstrate that the technique is shown to be able to distinguish targets analogous to bacteria. Also presented and demonstrated, is imaging and enhanced contrast of a biological model of labelled cells.
We present a multifunctional endoscope capable of imaging, fluid delivery and fluid sampling in the alveolar space. The endoscope consists of an imaging fibre bundle fabricated from cost effective OM1 PCVD graded index preforms made for the telecommunications market. These low-cost fibres could potentially make our endoscope disposable after a single use. The performance of our low-cost imaging fibre bundle is shown to be comparable to the current commercial state-of-the-art. The imaging fibre bundle is packaged alongside two channels for the delivery and extraction of fluids. The fluid delivery channels can be used to deliver fluorescent smart probes for the detection of pathogens and to perform a targeted alveolar lavage without the removal of the imaging fibre as is currently standard procedure. Our endoscope is fully biocompatible and with an overall outer diameter of 1.4 mm allowing it to fit into the standard working channel of a bronchoscope. We demonstrate the use of our endoscope in ex-vivo human lungs. We show alveolar tissue and bacterial imaging over two wavelength bands 520 nm – 600 nm and 650 nm – 750 nm both commonly used for bacterial smart probe detection.
In healthy humans, the physiological state in the distal lung alveolar acinar units is tightly regulated by normal homeostatic mechanisms. Pulmonary abnormalities such as chronic obstructive pulmonary disease, that are characterized by recurrent cycles of inflammation and infection involving dense infiltration by myeloid derived peripheral blood cells, may result in significant perturbation of the homeostatic baselines of physiology in addition to host tissue damage. Therefore, the ability to quantify and monitor physiology (e.g. pH, glucose level, oxygen tension) within the alveolar acinar units would provide a key biomarker of distal lung innate defence. Although in vitro modeling of fundamental biological processes show remarkable sensitivity to physiological aberrations, little is known about the physiological state of the distal lung due to the inability to concurrently access the alveolar sacs and perform real-time sensing. Here we report on previously unobtainable measurements of alveolar pH using a fiber-optic optrode and surface enhanced Raman spectroscopy (SERS) and show that alveolar pH changes in response to ventilation. The endoscope-deployable optrode consisted of para-mercaptobenzoic acid functionalized 150 nm gold nanoshells located at the distal end, and an asymmetric dual-core optical fiber designed for spatially separated optical pump delivery and SERS signal collection in order to circumvent the unwanted Raman signal originating from the fiber itself. We demonstrate a ~ 100-fold increase in SERS signal-to-fiber background ratio and pH sensing at multiple sites in the respiratory acinar units of a whole ex vivo ovine lung model with a measurement accuracy of ± 0.07 pH units.