Bioimaging Seminar, 12th May 2010
Wednesday 12th May, 10:00 – 12:00
Warwick Mathematics Institute
Zeeman Building, Room B3.02
- Kai Wicker, King’s College London: "Resolution and Efficiency Enhancement for Scanning Fluorescence Microscopes Using Image Inversion Interferometers"
- Anne Straube, CMCB, Warwick: "Migrational behaviour of human epithelial cells – persistence, turns and collisions"
More information on the Bioimaging Seminar Series can be found here
Resolution and Efficiency Enhancement for Scanning Fluorescence Microscopes Using Image Inversion Interferometers
Kai Wicker1, Simon Sindbert1,2, Rainer Heintzmann1
1King’s College London, Randall Division of Cell and Molecular Biophysics, London, UK
2Faculty of Medicine Mannheim, University of Heidelberg, Mannheim, Germany
Fluorescence confocal microscopy has become an indispensable tool of modern biology, allowing the imaging of live fluorescent specimen with high lateral as well as axial resolution. Through the introduction of a sufficiently small confocal pinhole, the lateral resolution can be enhanced compared to that of a wide field microscope. However, this gain in resolution comes at the cost of a decrease in detection efficiency, as light blocked by the pinhole is lost.
Sandeau et al. proposed a way of improving the lateral resolution of a 4Pi microscope . We present a more general method for improving the lateral resolution and detection efficiency of scanning fluorescence microscopes by adding an interferometer with image inversion in one of its arms to the detection pathway . We show that the resulting detection-only transfer function, without a pinhole, is essentially the absolute square of the system’s amplitude transfer function, enlarged to twice its spatial frequency range. This way the limit for lateral resolution in a conventional confocal microscope (completely closed pinhole) can be surpassed while increasing the detection efficiency substantially.
We present first experimental results, recorded with a UZ-Interferometer (UZI) . The light in this setup follows three-dimensional U- and Z-shaped paths and relies on reflections off planar surfaces only in order to achieve image inversion. Point spread function measurement show excellent agreement with theory; first biological images exhibit a clear improvement in image quality and resolution.
 N. Sandeau and H. Giovannini, “Increasing the lateral resolution of 4Pi fluorescence microscopes”, J. Opt. Soc. Am. A 23, 1089-1095 (2006).
 K. Wicker and R. Heintzmann, “Interferometric resolution improvement for confocal microscopes”, Opt. Express 15, 12206-12216 (2007).
 K. Wicker, S. Sindbert, and R. Heintzmann, “Characterisation of a resolution enhancing image inversion interferometer”, Opt. Express 17, 15491-15501 (2009).
Anne Straube, CMCB, Warwick: Migrational behaviour of human epithelial cells – persistence, turns and collisions
My lab recently started to become interested in the cytoskeletal coordination of cell migration. We use a combination of RNAi and live cell imaging of human retinal pigment epithelial cells randomly migrating on a fibronectin-coated surface to understand how the cytoskeletal filament systems direct migrational behaviour. We find that interfering with the microtubule plus end complex or microtubule-based transport impairs the persistence of cell migration, but also affects the ability of cells to change the direction of cell movement in response to collisions with other cells, a phenomenon called contact inhibition. Our imaging data reveal complex cellular behaviours that are difficult to assess with standard analysis tools. I will discuss our needs for advanced imaging analysis algorithms paired with sophisticated data analysis to understand the observed changes in complex behaviour.