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签成像工具。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体荧光。通过分析这种自身荧光信号，可以检测到特定种类分子的存在，例如提供有关组织结构的信息，也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引起生物分子浓度、分布和相互作用变化的疾病的早期发病。由于自体荧光测量不需要添加任何化学物质，这种方法是无标签的，可以是非侵入性的，使其对诊断应用具有吸引力。然而，生物组织中往往含有数种未知数量的荧光分子，并且对光辐射有强烈的散射，使得荧光定量测量变得困难。因此，需要以一种避免强度伪影的方式分析组织自身荧光，并获取图像，以便自荧光信号的变化可以与组织中观察到的结构相关联。这与传统的组织病理学类似，其中诊断是在活检后使用生物组织切片的图像，并用染料染色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内窥镜，通过提供分子对比的自身荧光信号来提供生物组织的显微镜样图像。为了量化自身荧光信号，我们将利用不同的分子种类以不同的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，分析具有荧光寿命的光学切片图像，提供分子对比度。自1998年以来，我们一直在研究生物组织的FLIM，展示了一些组织中体外疾病（如癌症、骨关节炎和动脉粥样硬化）的第一个无标签寿命对比，并开发了一系列复杂的实验室FLIM仪器，包括原理验证FLIM内窥镜。现在至关重要的是，使用临床可行的内窥镜为基础的方法进行体内成像。由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，并已被批准在欧洲和美国临床使用。目前仅局限于单一激发波长（488nm）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488nm激发，然后扩展到更短的波长，以激发更多的生物分子。该仪器将非常适合于光学活检，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，我们也将开发一种临床可行的宽视场FLIM内窥镜，提供更大的观察区域，尽管没有光学切片。这将允许对这两种内镜检查方法的性能进行比较。我们还将把内窥镜荧光显微镜与组织病理学和帝国理工学院现有的先进荧光显微镜仪器联系起来，这将有助于阐明我们在正常和病变组织之间观察到的自体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.