Primed Conversion Oblique Plane Microscopy

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.

We propose development of a unique light-sheet microscope tailored specifically for the needs of Primed Conversion, which is a technique for changing a green photoconvertible protein into a red one with high spatial selectivity and low phototoxicity. While this ability to label an individual cell in a cluster has great promise in cell lineage tracing for embryology (along with many other fields), its adoption has been hampered by a lack of microscopes that can selectively label and image the developing embryo. This proposed system will function as an add-on to a conventional microscope, allowing researchers to maintain their existing sample mounting and culturing protocols, while integrating a highly optimized light-sheet fluorescence illumination modality and the ability to perform Primed Conversion with high three-dimensional resolution.

The project will focus on the construction and characterization of the instrument, as well as its use in embryology. All of the mechanical and optical design work has already been performed, and both the theoretical imaging and Primed Conversion resolutions are below one micron. After the instrument is constructed, it will be characterized using fluorescent nanospheres, as well as HeLa expressing H2B-pr-mEosFP (a photoconvertible protein). A major capability of the instrument will then be developed, that of 'dilution-unlimited' labelling. Unlike in normal labelling for lineage tracing, in which the label gets diluted as the cells divide (consequently reducing the signal to the point where it is lost), this system can photoconvert new proteins in every generation, thus boosting the signal and ensuring that it is not lost. This allows the lineage of a single cell to be traced through arbitrarily-many generations. Once all characterizations are complete and the instrument is ready for use, it will be tested on mouse embryos ordinarily discarded as a part of existing research activities.

Aside from the academic beneficiaries of this research, this research will also provide tangible benefits to industry and society. From an industrial perspective, one of the major difficulties in high-tech manufacturing is hiring well-trained workers who are experienced in precision work (such as the alignment of an optical system). This project directly provides this kind of training, as project students in the Department of Bioengineering will take small aspects of its construction under the supervision of the Postdoctoral Research Associate. It also supports the development of programmers in the same way; particularly programmers who are experienced in programming high-performance hardware, which is a niche skill and subject to constraints which are not encountered by the vast majority of software engineers. Training is also supported by the Rowlands lab policy of supporting short visits by external researchers who wish to learn how to build the microscope, some of the intricacies of using it, talk to the people who developed it and so on. Further industrial benefit comes from the availability of the system to other users; interested external users can apply to access the microscope to image their samples, as well as consulting with the researchers who built it. This is particularly suitable for small--to-medium enterprises who may not be able to afford a microscope with these capabilities; measurements taken using primed OPM (or even just normal OPM imaging) help support these companies to the benefit of the whole UK economy.

From the perspective of society, the research opens up interesting questions about how a single unthinking cell can turn into a person. Importantly, it does so in a very visual manner, which helps drive societal engagement. The images and videos produced during this project will be used both for artistic purposes, and to produce media intended for public consumption, with the goal of sharing our discoveries and excitement with a wider community. In this way artists, presenters, government officials and the whole general public can benefit from the resources produced. Finally, because the work studies the development process, it indirectly supports studies into reproductive disorders and developmental abnormalities. In the long term, we anticipate studies supported by this and similar instruments to be incredibly beneficial for couples struggling to have children, people with developmental disorders, and the general population as well.