Molecular relativity: tracking single molecule movement relative to cell structures

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.

In this project we aim to develop a method which can image both states of a photoswithable fluorophore simultaneously, giving data about the structure of the sample and the movement of individual molecules. We will then develop analysis techniques to track the movement of the structure and use it to correct the movement of individual molecules, so they are not influenced by sample motion or remodelling. This will allow us to accurately characterise the movement of molecules in motile cell structures. The performance of the software in correcting the positions of individual molecules will be characterised using simulations. Data will be collected using filopodia as a test system, and we will analyse the behaviour of the molecules to detect if there is any difference in the motion of molecules between fast moving and static filopodia, or between the tip and the main part of the filopodium.

There is information present in the time domain of the high density channel. We will use this in two ways, once the movement of the structure has been eliminated from the time series. Firstly, frame sequences can be analysed using high density super-resolution techniques, which allow enhanced resolution to be achieved by fitting the positions of fluorophores in the high density data sequence. Secondly, the average movement of molecules within the sample will be analysed using spatio temporal image correlation spectroscopy. This will allow us to quantify how material is being transported in the sample at a micron lengthscale. This information will be compared with the information on single molecule movement to examine the difference between the characteristics of bulk transport and individual molecule movement. We will create an integrated software package and experimental protocol which will allow other users to carry out this type of experiment.

The initial impact is primarily expected to be seen in an ability to perform single molecule tracking in systems which would previously have been considered too motile. There are a number of biological systems that could benefit from live cell localisation microscopy imaging, including but not limited to: fascin in filopodia (upregulated in all known human cancers, cannot be effectively imaged in fixed cells), the dynamics of proteins inside sarcomeres in muscle cells, and (for the two dimensional case) structures on the immune synapse in T cells and B cells. These are all systems of high biomedical importance and in the longer term the greatest impact of this project is likely to be enabling and accelerating new biomedical research. Professor Maddy Parsons (co-I) has strong links with pharmaceutical companies and would be well placed to help us take these results into an industrial context.

We have links to a number of microscopy companies, and on this project we have partnered with Leica. The preliminary results shown in the grant were obtained with a modified version of the Leica GSD system. Leica have expressed strong support for those developing open-source solutions for other researchers. They stand to benefit from our work because it would extend the experiments that could be carried out on their systems and give users greater confidence in their results. In turn, their users will benefit because we will be able to advise on changes to the hardware and software of the system which would optimise the experimental performance.

We also plan to clone the current microscope system into the Microscopy Innovation Centre at King's. The microscopes at the MIC will be supported by two full-time members of staff, and this will give us the capability to bring the method to users in a stable form without comporomising our research.

Dr Richard Marsh, the post-doctoral researcher on this project, has received training in both analysis of microscopy data and in carrying out experiments. Advanced microscopy is a rapidly growing area, with many jobs being created, and a shortage of people with in-depth training, and we anticipate that the training and experience he would acquire over the course of this project would be highly beneficial in enabling his future career choices.