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.