The focus of our research is a super-resolution fluorescence microscopy technique called localisation microscopy (also known as photoactivatable localisation microscopy (PALM) or stochastic optical reconstruction microscopy (STORM)). Localisation microscopy is a method in which many thousands of images are taken. In each image only a few fluorophores are emitting light. The position of each fluorophore is fitted in each frame, building up an image of the sample. Our focus is on decreasing the number of frames required, increasing the speed and making the technique applicable to live cell imaging. This requires improved analysis algorithms which allow the number of frames required to be decreased to a few hundred.
We have investigated a number of cell structures using super-resolution techniques, including podosomes (in collaboration with the group of Gareth Jones). Podosomes are cytoskeletal structures associated with cell adhesion, migration and degradation of the extracellular matrix They consist of an actin core surrounded by a ring of integrin-associated proteins such as talin and vinculin. This ring was thought to be roughly round, but our super-resolution images revealed them to have a polygonal shape. Other structures of interest include reticular adherens junctions between endothelial cells (in collaboration with Anne Ridley, Randall Division and Jaime Millán Martinez, Centro de Biología Molecular Severo Ochoa, CSIC-UAM, Madrid), and polarity factors in yeast (in collaboration with Rafael Carajo-Salas, University of Cambridge).
Livecell widefield (left) and Bayesian analysis of blinkng and bleaching (right) images of a truncated talin construct in podosomes. This image is generated from 200 frames (4 seconds) of raw data. The widefield image is created by averaging 200 frames. Scalebar is 2 Μμ.