Endoscopic FLIM for label-free tissue contrast

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This project aims to provide new label-free imaging tools for minimally invasive diagnosis of diseases including cancer. When irradiating tissue with light at an appropriate wavelength, many molecules absorb this excitation energy and emit new radiation called fluorescence . As these fluorescent molecules occur naturally in biological tissue, their emission is called autofluorescence. By analysing such autofluorescence signals, it is possible to detect the presence of particular kinds of molecule, e.g. providing information on the tissue structure, and also to learn about their local environment, e.g. whether they are bound to other molecules. Autofluorescence may therefore provide a means to detect the early onset of diseases that cause changes in the concentration, distribution and interaction of biological molecules. Because autofluorescence measurements do not require the addition of any chemicals, this approach is label-free and can be non-invasive, making it attractive for diagnostic applications. Biological tissue, however, often contains several kinds of fluorescent molecule in unknown quantities and strongly scatters optical radiation, making quantitative fluorescence measurements difficult. It is therefore desirable to analyse tissue autofluorescence in a way that avoids intensity artefacts and to acquire images so that variations in the autofluorescence signal can be correlated with the observed structures in the tissue. This is analogous to conventional histopathology, where diagnoses are made following biopsy using images of sections of biological tissue that have been stained with dyes to indicate the distributions of different types of molecule. In this proposed work, we will develop a novel endoscope to provide microscope-like images of biological tissue with the autofluorescence signal providing molecular contrast. To quantify the autofluorescence signal, we will exploit the fact that different molecular species radiate fluorescence at different rates and so it is possible to distinguish them by observing the fluorescence decay times (lifetimes) for each pixel in the field of view. By combining fluorescence lifetime imaging (FLIM) with a special endoscope that provides microscope images with depth resolution, we will be able to perform optical biopsy in situ, analysing optically sectioned images with fluorescence lifetime providing the molecular contrast.We have investigated FLIM of biological tissue since 1998, demonstrating some of the first label-free lifetime contrast of ex vivo disease in tissue (e.g. cancer, osteoarthritis and atherosclerosis) and have developed a range of sophisticated laboratory-based FLIM instrumentation including proof-of-principle FLIM endoscopy. It is now vital to progress to in vivo imaging using a clinically viable endoscope-based approach. The world-leading endoscopic confocal microscope developed by Mauna Kea Technologies (MKT) provides optically sectioned imaging with subcellular resolution and has been approved for clinical use in Europe and the USA. Currently it is limited to intensity imaging at a single excitation wavelength (488 nm). We aim to develop a FLIM version of this endoscope, initially using 488 nm excitation and then expanding to shorter wavelengths in order to excite more biological molecules. This instrument will be highly suitable for optical biopsy but the inherently limited field of view of confocal endoscopy will limit its application for screening of disease. We will therefore also develop a clinically viable wide-field FLIM endoscope to provide a larger viewing area, albeit without optical sectioning. This will permit a comparison of the performance of these two approaches to endoscopy. We will also correlate endoscopic FLIM with histopathology and with existing advanced FLIM instrumentation at Imperial, which will help elucidate the molecular origins of the autofluorescence contrast we observe between normal and diseased tissue.