Molecular relativity: tracking single molecule movement relative to cell structures

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Fluorescence microscopy is a crucial tool for cell biologists because it allows them to label different proteins with fluorescent molecules (fluorophores) and observe them in live cells. This yields information which can help us to understand diseases, and find new drugs to treat them. To understand how molecules in a cell behave, individual molecules labelled with fluorophores can be imaged and their speed measured. However, currently this can only be done on the membrane of cells, or on parts of the cell that stay very still. If the part of the cell which the molecule is attached to moves, it isn't possible to work out what is due to the movement of the molecule and what is due to the movement of the structure the molecule is attached to.We propose to solve this problem, so that tracking studies can be carried out in moving structures. To do this we will use the properties of photoswitchable fluorophores. These are fluorophores which can be switched between emitting light in two different colours. Normally only one colour is recorded, in which only a few fluorophores emit light. This means that individual molecules can be tracked. We want to develop a system to simultaneously image both of the emitting states. This will let us image the structure of the sample in one channel, and individual molecules in another. This can be done by illuminating the sample with two lasers simultaneously and splitting the emitted light across two sections of a camera chip, with the split being based on colour.To get useful information from this system, we will need to develop a new data analysis method. Both the positions of the single molecules and the structure of the sample will need to be fitted and tracked. The movement of the structure of the sample can then be used to correct the tracks of individual molecules. We will try two different methods to model the structure, first a simple method where it is approximated as a series of straight lines, and then a more accurate model where it is fitted as a curve.The images of the structure of the sample contain additional information which we will also try to extract. This is because the molecules in these images, while densely packed, are moving over time and switching between states in which they emit or don't emit light. This information can be used in two ways. First, to get enhanced resolution of the structure of the sample by analysing a short sequence of images and using information from fluorophore fluctuations to improve knowledge of the fluorophore positions. Second, to get information about how the molecules are moving around the cell structure on average. This is done by comparing the brightness of image patches between different frames. These two methods will allow us to visualise the cell structure more accurately, and to compare how individual molecules move to how the molecules in the structure are moving on average.This method would allow cell biologists to track single molecules in rapidly moving structures. Single molecule tracking has produced important results for those structures with which it can be used, and our work would make this technique applicable to a much wider range of cell biology problems.