Primed Conversion Oblique Plane Microscopy

即使是小孩子都会问的最基本的问题之一就是“我从哪里来？”对这个问题的一种解释可能是解释单个受精卵如何长成一个功能齐全的人。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，它是可靠的：在人类进化以来的700万年左右的时间里，大约35万代的人类，这个过程进行得或多或少是成功的。显然，理解这个过程是值得的，不仅从医学角度（我们寻求治疗婴儿成长过程中发生的疾病），而且出于简单的好奇心；需要了解形成我们的过程是如何运作的。这个问题的一个关键方面是，一团细胞如何知道哪些细胞应该形成身体的哪些部位；例如，为什么我们通常不会得到两个正面。解决这个问题的一种方法是在细胞数量不多的时候给它们贴上标签，然后观察这些细胞球发育成胚胎的过程。如果当细胞分裂成越来越多的细胞时，这个标签仍然存在，我们就可以通过寻找那些有这个标签的细胞来跟踪这几代细胞回到原来的细胞。目前，有几种方法可以用这种方式标记细胞，但最常见的一种是在细胞中添加荧光分子（即当光线照射时发光的分子）；当它分裂时，这些分子最终进入两个“子”细胞，并重复这个过程。Pantazis博士开创了一种用这种方法标记单个细胞的技术，这种技术被称为“引物转换”。在引物转换中，细胞产生荧光蛋白，但通过同时照射两种不同颜色的光，这些蛋白可以从绿色变为红色。只有这两种颜色重叠的区域才会被标记。尽管Primed Conversion有能力标记细胞，但迄今为止，它的使用一直受到限制，不是因为这项技术很难使用，而是因为为了更有效地工作，需要开发一种新型显微镜。这就是罗兰兹实验室可以提供帮助的地方；这个实验室专门制造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位置重叠，还可以拍摄细胞分裂时的图像。在同一台显微镜上进行这两种过程的一个特别强大的优势是，通常情况下，当细胞分裂时，红色蛋白质会被稀释。使用这种新型显微镜，每一代的信号都可以被“加满”，这样就可以在更长的时间内追踪细胞。此外，由于这种显微镜对细胞成像的方法（称为光片荧光显微镜）对细胞特别友好（它使用非常低的光照水平，这样细胞就不会暴露在太多的光线下），因此非常适合研究对光和其他扰动非常敏感的胚胎。最终，这种显微镜将用于胚胎学以外的其他应用。例如，同样的系统可以用于进行超分辨率成像，使其能够看到所谓的“衍射极限”之外的东西，这使得显微镜无法看到像病毒这样的非常小的东西。它可以用来追踪免疫细胞抵抗感染的过程，量化血流量，并研究癌症是如何侵入人体的。整个系统被设计为普通显微镜的附加组件，让科学家们可以使用他们熟悉的工具，可能已经在他们的实验室里了。最后，因为我们是开放获取科学的坚定信徒，所有的计划、软件和数据都将被发布给任何人使用。

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.