Primed Conversion Oblique Plane Microscopy

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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.