Developing and validating a new tool for simultaneous multi-channel wide-field imaging

对生物系统进行成像可以直接了解它们是如何工作的。随着越来越多的系统被研究，越来越有必要标记和可视化更多的组件。然而，尽管现在有各种各样的颜色来标记蛋白质，但我们同时看到它们的能力是有限的。有两种主要的多色成像方法，宽视场和共聚焦。前一种方法包括一次拍摄整个样本的照片，但每张照片仅限于四种颜色。后一种方法包括在一个地方拍照，然后移动到另一个位置拍摄第二张照片，以此类推，直到整个样本被编目。显然，这种共聚焦方法意味着整个样品不能同时成像，但它确实允许许多颜色被成像。在这个提议中，我们正在开发一种在宽视场中同时成像六种颜色的新方法，希望这可以在未来进一步扩展到更多的颜色。为了实现这一目标，我们将建立一个简单的设备，连接到一个标准的显微镜上，这将允许连接两个市售的三色分光镜和两个相机。这些相机需要相互通信，这样它们才能同时拍照，我们将使用软件或硬件构建这个接口。然后，这里开发的六色成像仪将使用生物系统进行测试。在我们的实验室中，我们使用三色成像设备研究细菌核苷酸切除DNA修复（NER）过程的摘录。六色成像仪将使我们能够同时研究所有共同修复DNA损伤的NER蛋白。我们的方法是使用DNA绳索，它是悬浮在显微镜盖盖上的微观平台之间的DNA纳米线。我们将加入所有与NER相关的蛋白质，但每种类型都将被标记为不同颜色的标签。然后我们将研究DNA是如何一次修复一个分子复合体的。作为对这里开发的新技术的测试，这个模型系统是理想的，因为它需要多色成像和最高水平的检测灵敏度。我们预计这项工作将为研究具有多组分的许多不同生物系统铺平道路，不仅在单分子水平上，而且在细胞水平上，由于宽视场成像的结果。这是一个非常重要的工具，需要开发和测试。

Imaging biological systems provides a direct insight into how they work. As more systems are being studied there is an increasing necessity to label and visualise more components. However, despite there now being a large variety of colours with which to label proteins our ability to simultaneously visualise them is limited. There are two major methods to image in multiple colours, wide-field and confocal. The former approach involves taking a picture of the whole sample at once, but is limited to only four colours per image. The latter approach involves taking pictures in one location before moving to another position to take a second image and so on until the whole sample is catalogued. Clearly this confocal approach means that the whole sample cannot be imaged simultaneously, however it does permit many colours to be imaged. In this proposal we are developing a new method to image six-colours simultaneously in wide-field, with the hope that this could be further extended in the future to many more colours. To achieve this, we will build a simple device that attaches onto a standard microscope, this will allow two commercially available 3-colour splitters to be attached and then two cameras. These cameras need to talk to each other so that they take images at the same time, we will build this interface using software or hardware. The six-colour imager developed here will then be tested using a biological system. In our lab we study excerpts from the bacterial nucleotide excision DNA repair (NER) process using a three colour imaging device. A six-colour imager will enable us to study simultaneously all of the NER proteins that act together to repair DNA damage. Our approach is to use DNA tightropes, which are nanowires of DNA suspended between microscopic platforms on a microscope coverslip. To these tightropes we will add all of the proteins involved in NER but each type will be labelled with a different coloured tag. We will then study how DNA is repaired one molecular complex at a time. As a test for the new technology developed here this model system is ideal because it requires multiple colour imaging and the highest level of detection sensitivity. We anticipate that this work will pave the way for the study of many different biological systems with multiple components, not only at the single molecule level but also at the cellular level as a consequence of wide-field imaging. This is a hugely important tool that needs to be developed and tested.