Methods development for the measurement of single-cell molecular state

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Tissue systems are made up of a complex mixture of cells each with unique behaviours and regulatory mechanisms controlling them. Interestingly, even when purified groups of these cells with theoretically the same roles have been examined closely, they too have been shown to have a mix of individual behaviours/regulation. Such complexity is likely important in maintaining proper balance in the tissue system and for adaptation to changing conditions. Despite this, most common techniques used to measure how cells function are only able to measure the population average. While these bulk techniques can give extremely useful benchmarks of the general characteristics of a group of cells, they necessarily ignore much of the regulatory complexity which control the system behaviour. Techniques exist to look at some of the control mechanisms of single cells, however, they tend to be restricted to one type of control where cells use multiple mechanisms to ensure proper regulation. These techniques also tend to be both difficult to perform and expensive. We will develop new ways to measure multiple mechanisms of cell regulation at the same time in single cells to give a more complete picture of their decision-making process. To facilitate the use of these techniques we will also develop automated ways of performing these protocols while decreasing costs by reducing the scale of reactions down to sub-microliter volumes. Finally, we will apply nano-scale electronic detectors to create a new method for measuring proteins in single cells. Overall, the proposed program will provide valuable fundamental tools which will be applicable to a wide range of basic cell biological research.