A new method for single molecule tracking of fluorescent molecules promises a more accurate and efficient approach

Single-molecule spectroscopy can provide new insights into the dynamics of biological and material science, but analysing the data from this technique is no simple task. To this end, scientists in Germany have developed a new method for tracking single fluorescent molecules, using linear programming solvers.

Whereas previous methods have involved tracking by hand or with semi-manual systems, this new algorithm means standard computers can be used instead, freeing up the researchers’ time and reducing the risk of mistakes. This new technique is essential for the on-going miniaturization of devices to the nanoscale.

Single-molecule spectroscopy can be used to observe the translational and rotational motion of single fluorescent colloids, particles, viruses or antibodies. However, following the motion of single fluorescent molecules is more challenging. They generally have a low signal-to-noise ratio, the signal intensity can vary dramatically (so-called 'blinking') and photo bleaching from the analysis conditions can occur. ‘You could make the analogy that our task is comparable to that of a big shopping mall owner,’ says Dominik Wöll, who co-lead the team from the University of Konstanz. ‘He would like to know the shopping behaviour of different people and also distinguish different groups. The best way would be if he could track each single person and see how they move in the mall, how fast they go through different areas, where they stop and so on.’ 

The key to Wöll’s algorithm is performing a global optimization of the connections between the positions of the molecules, which are based on a set of parameters and can be adapted for different scenarios. The main parameter is the maximum distance between these positions, which must maintain a balance between false negative and positive connections and limit the number of possibilities. The result of this process is a fast and feasible way of tracking single fluorescent molecules using a standard computer.

Daniela T?uber, an expert in single molecule detection at Chemnitz University of Technology, Germany, told Chemistry World ‘The evaluation of positions is very convincing and a considerable improvement to the tracking programs which I personally know and use.’

The authors are hoping to develop their method further to allow the study of heterogeneous diffusion.