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

开发并验证同步多通道宽视场成像的新工具

基本信息

批准号：
BB/M019144/1
负责人：
Neil Kad
金额：
$ 19.01万
依托单位：
University of Kent
依托单位国家：
英国
项目类别：
Research Grant
财政年份：
2015
资助国家：
英国
起止时间：
2015 至无数据
项目状态：
已结题

来源：
https://gtr.ukri.org/projects?ref=BB%2FM019144%2F1
关键词：
Developing validating new tool simultaneous

项目摘要

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.

成像生物系统可以直接了解它们的工作方式。随着越来越多的系统的研究，标记和可视化更多组件的必要性越来越多。但是，尽管现在有多种颜色可以标记蛋白质同时可视化它们的能力是有限的。有两种主要的方法可以以多种颜色，宽场和共焦点形象形象。前者的方法涉及一次拍摄整个样品的照片，但每图像仅限于四种颜色。后一种方法涉及在一个位置拍照，然后再移到另一个位置拍摄第二张图像，依此类推，直到整个样本被分类为止。显然，这种共聚焦方法意味着整个样本不能同时成像，但是它确实可以成像许多颜色。在此提案中，我们正在开发一种新方法，以宽场同时对六色进行图像，希望将来可以将其进一步扩展到更多颜色。为了实现这一目标，我们将构建一个简单的设备，该设备连接到标准显微镜上，这将允许将两个商业可用的3色拆分器连接，然后允许两个摄像机。这些相机需要相互交谈，以便它们同时拍摄图像，我们将使用软件或硬件构建此接口。然后，此处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，我们使用三种彩色成像装置研究了细菌核苷酸切除DNA修复（NER）工艺的摘录。六色成像仪将使我们能够同时研究所有起作用以修复DNA损伤的NER蛋白。我们的方法是使用DNA Wightropes，它们是显微镜盖玻片上显微镜平台之间悬浮的DNA的纳米线。在这些绳索上，我们将添加NER中涉及的所有蛋白质，但每种类型都会用不同的彩色标签标记。然后，我们将研究一次DNA如何修复一个分子复合物。作为对这里开发的新技术的测试，该模型系统是理想的选择，因为它需要多种颜色成像和最高水平的检测灵敏度。我们预计，这项工作将为研究许多不同组分的生物系统的研究铺平道路，不仅在单分子水平上，而且由于宽场成像的结果，在细胞水平上也是如此。这是一个非常重要的工具，需要开发和测试。