Endoscopic FLIM for label-free tissue contrast

该项目旨在为包括癌症在内的疾病的微创诊断提供新的无标记成像工具。当用适当波长的光照射组织时，许多分子吸收这种激发能量并发出称为荧光的新辐射。由于这些荧光分子自然存在于生物组织中，它们的发射被称为自发荧光。通过分析这种自发荧光信号，可以检测特定种类分子的存在，例如提供关于组织结构的信息，并且还可以了解它们的局部环境，例如它们是否与其他分子结合。因此，自体荧光可以提供一种手段来检测疾病的早期发作，导致生物分子的浓度，分布和相互作用的变化。由于自发荧光测量不需要添加任何化学物质，因此这种方法是无标记的，并且可以是非侵入性的，因此对于诊断应用具有吸引力。然而，生物组织通常含有未知数量的几种荧光分子，并且强烈散射光辐射，使得定量荧光测量困难。因此，期望以避免强度伪影的方式分析组织自发荧光，并且获取图像，使得自发荧光信号的变化可以与组织中观察到的结构相关联。这类似于传统的组织病理学，其中诊断是在活检后使用已经用染料染色的生物组织切片的图像进行的，以指示不同类型分子的分布。在这项拟议的工作中，我们将开发一种新型的内窥镜，以提供生物组织的显微镜般的图像与自体荧光信号提供分子对比度。为了量化自发荧光信号，我们将利用不同分子种类以不同速率辐射荧光的事实，因此可以通过观察视场中每个像素的荧光衰减时间（寿命）来区分它们。通过将荧光寿命成像（FLIM）与提供具有深度分辨率的显微镜图像的特殊内窥镜相结合，我们将能够进行原位光学活检，分析具有荧光寿命提供分子对比度的光学切片图像。我们自1998年以来一直在研究生物组织的FLIM，展示了组织中离体疾病的一些第一个无标记寿命对比度（例如癌症、骨关节炎和动脉粥样硬化），并开发了一系列复杂的基于实验室的FLIM仪器，包括原理验证FLIM内窥镜。现在至关重要的是，使用临床上可行的基于内窥镜的方法进行体内成像。Mauna Kea Technologies（MKT）开发的世界领先的内窥镜共聚焦显微镜提供亚细胞分辨率的光学切片成像，并已在欧洲和美国被批准用于临床。目前，它仅限于在单一激发波长（488 nm）的强度成像。我们的目标是开发这种内窥镜的FLIM版本，最初使用488 nm激发，然后扩展到更短的波长，以激发更多的生物分子。该仪器将非常适合光学活检，但共聚焦内窥镜固有的有限视野将限制其在疾病筛查中的应用。因此，我们还将开发一种临床上可行的宽视场FLIM内窥镜，以提供更大的观察区域，尽管没有光学切片。这将允许比较这两种内窥镜检查方法的性能。我们还将内镜FLIM与组织病理学和现有的先进FLIM仪器在帝国，这将有助于阐明我们观察到的正常和病变组织之间的自发荧光对比的分子起源。

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.