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Primed Conversion Oblique Plane Microscopy

启动转换斜平面显微镜

基本信息

批准号：
BB/T011947/1
负责人：
Christopher Rowlands
金额：
$ 19.25万
依托单位：
Imperial College London
依托单位国家：
英国
项目类别：
Research Grant
财政年份：
2020
资助国家：
英国
起止时间：
2020 至无数据
项目状态：
已结题

来源：
https://gtr.ukri.org/projects?ref=BB%2FT011947%2F1
关键词：
Primed Conversion Oblique Plane Microscopy

项目摘要

One of the most fundamental questions asked by even small children is 'where do I come from?' One interpretation of this question might be to explain how a single fertilized egg can grow into a fully functioning person. The intricate dance of chemical signals and cell motions that governs this process is complex, difficult to understand and, perhaps most surprisingly of all, reliable: for approximately 350,000 generations of humans in the 7 million years or so since humans evolved, this process has proceeded more or less successfully. Clearly, it is worthwhile understanding this process, not only from a medical perspective (in which we seek to treat diseases that occur during the growth of a baby) but also out of simple curiosity; the need to understand how the processes which formed us work.One crucial aspect of this question is how a ball of cells knows which cells should form which parts of the body; why we don't normally end up with two heads, for example. One way to tackle this question is to label the cells when there are only a few of them, and then watch as this ball of cells develops into an embryo. If the label persists as the cell splits into more and more cells, we can follow the generations back to the original cell by looking for those which have this label. Currently there are a few ways to label a cell in this manner, but one of the most common is to add fluorescent molecules (i.e. molecules which glow when you shine light on them) to the cell; when it splits, these molecules end up in the two 'daughter' cells, and the process repeats. Dr Pantazis has pioneered a way to label individual cells in just this way by a technique called Primed Conversion. In Primed Conversion, the cell produces fluorescent proteins, but these proteins can be switched from green to red by shining two different coloured lights on them at the same time. Only regions where these two colours overlap undergo labelling.Despite the power of Primed Conversion to label cells, its use has been limited to date, not because the technique is hard to use, but because to work most effectively a new type of microscope needs to be developed. This is where the Rowlands lab can help; this lab specializes in creating new types of microscopes and other optical systems. Dr Rowlands has designed a system that not only can make sure the two coloured lights overlap in exactly the right point in space, but can also image the cells as they split. A particularly powerful advantage of doing both processes on the same microscope is that, ordinarily, the red proteins get diluted when the cell splits. Using the new microscope, the signal can be 'topped up' every generation, so the cells can be traced over much longer periods of time. In addition, because the method for imaging the cells in this microscope (known as light-sheet fluorescence microscopy) is particularly kind to cells (it uses very low light levels so that the cells do not get exposed to too much light) it is very suitable for studying embryos which are very sensitive to light and other perturbations.Ultimately this microscope will be used for other applications outside of embryology as well. For example, the same system can be used to perform super-resolution imaging, allowing it to see beyond the so-called 'diffraction limit' which prevents microscopes from seeing very small things like viruses. It can be used to track immune cells as they fight off an infection, to quantify blood flow, and investigate how cancer invades the body. The whole system was designed to work as an add-on to a normal microscope, letting scientists work with the kinds of tools they are familiar with, and probably already have in their labs. Finally, because we are strong believers in open access to science, all the plans, software and data will be released for anyone to use.

即使是小孩子也会问的最基本的问题之一是“我从哪里来？”对这个问题的一种解释可能是解释一个受精卵如何成长为一个功能齐全的人。控制这一过程的化学信号和细胞运动的错综复杂的舞蹈是复杂的，难以理解的，也许最令人惊讶的是，可靠的：自从人类进化以来的大约700万年里，大约35万代人，这个过程或多或少成功地进行了。显然，不仅从医学角度，（我们试图治疗婴儿成长过程中发生的疾病），但也出于简单的好奇心;需要了解形成我们的过程是如何工作的。这个问题的一个关键方面是细胞球如何知道哪些细胞应该形成身体的哪些部分;为什么我们通常不会有两个头解决这个问题的一种方法是在只有几个细胞时标记它们，然后观察这个细胞球发育成胚胎。如果这个标签随着细胞分裂成越来越多的细胞而持续存在，我们可以通过寻找具有这个标签的细胞来追踪这些代回到原始细胞。目前有几种方法可以用这种方式标记细胞，但最常见的方法之一是向细胞中添加荧光分子（即当你照射它们时会发光的分子）;当它分裂时，这些分子最终会进入两个“子”细胞，并且这个过程会重复。Pantazis博士已经开创了一种方法，通过一种称为“引发转换”的技术，以这种方式标记单个细胞。在引发转换中，细胞产生荧光蛋白，但这些蛋白质可以通过同时照射两种不同颜色的光而从绿色转换为红色。只有这两种颜色重叠的区域才会被标记。尽管Primed Conversion具有标记细胞的能力，但迄今为止它的使用受到限制，这并不是因为该技术难以使用，而是因为需要开发一种新型的显微镜才能最有效地工作。这就是罗兰兹实验室可以提供帮助的地方;该实验室专门研究新型显微镜和其他光学系统。罗兰兹博士设计了一个系统，不仅可以确保两种颜色的光在空间的正确点重叠，而且还可以在细胞分裂时成像。在同一显微镜下进行这两个过程的一个特别强大的优势是，通常，当细胞分裂时，红色蛋白质会被稀释。使用新的显微镜，信号可以在每一代中“加满”，因此可以在更长的时间内跟踪细胞。此外，由于这种显微镜中的细胞成像方法（称为光片荧光显微镜）对细胞特别友好（它使用非常低的光水平，因此细胞不会暴露在太多的光下），因此非常适合研究对光和其他干扰非常敏感的胚胎。最终，这种显微镜也将用于胚胎学以外的其他应用。例如，相同的系统可用于执行超分辨率成像，使其能够超越所谓的“衍射极限”，这使得显微镜无法看到病毒等非常小的东西。它可以用来跟踪免疫细胞，因为它们对抗感染，量化血流，并研究癌症如何侵入身体。整个系统被设计为作为普通显微镜的附加组件，让科学家使用他们熟悉的工具，并且可能已经在他们的实验室中使用。最后，因为我们是开放获取科学的坚定信徒，所有的计划，软件和数据将被发布给任何人使用。