Department of Biological and Medical Sciences

Methods and techniques

  • Optical trapping

    Optical trappingOptical trapping is a technique in which an intensely focused laser beam is used to physically move micron-sized dielectric particles, which might be molecules, organelles or even whole cells. These particles are attracted to the narrowest part of the laser beam which contains a very strong light gradient.

    Once particles are trapped in this electric gradient, the optical trap (also known as laser tweezers) is controlled via computer software and the particle movement can be observed simultaneously with a confocal laser scanning microscope. In plants optical tweezers have for example been used to micromanipulate the cytoplasm or the nucleus (see Hawes et al. 2010).

    We perform our experiments with the commercially available “ mmi CellManipulator” in collaboration with Tijs Ketelaar and Norbert de Ruijter at Wageningen University, The Netherlands.

    Using this system we have trapped Golgi bodies in arabidopsis and tobacco leaf epidermal cells in wild type and mutant lines. When Golgi bodies are manipulated with the optical trap, the endoplasmic reticulum (ER) remodels quickly and a tubular extension forms, which is following Golgi movement. This shows that in leaves Golgi bodies are firmly tethered to the ER network (Sparkes et al. 2009).

    On rare occasions we observed Golgi bodies “breaking free”, but they easily reconnected with the ER. We are currently studying the effects of mutations in putative Golgi tethering factors (= golgins) on the connection between Golgi bodies and the ER.

    High pressure freezing

    High pressure freezing apparatus

    Dr Ulla Neumann and Barry Martin with the BAL-TEC HPM010 high pressure freezing apparatus

    Now that biology has truly entered the post genomics era, there is an ever-increasing demand for both structural and spatial information on the myriad of proteins encoded by the genomes being sequenced.

    Technological developments in light microscopy such as confocal and two photon microscopy, combined with developments in immunolabelling and fluorescent protein technology, have revolutionised the study of cell structure. However, all these techniques are limited in resolution and for fine detail at the sub-cellular level, electron microscopy (EM) is still essential.

    Faithful preservation of cell structure is imperative if electron microscope images are to be correctly interpreted, and the use of ultra-rapid freezing techniques prior to any chemical fixation is the ideal procedure for preparing tissue for electron microscopy.

    A recent BBSRC grant awarded to Chris Hawes in the School of Life Sciences at Oxford Brookes University has funded the purchase of a BAL-TEC HPM 010 High Pressure Freezer, one of only four such machines in the UK.

    Plant golgi stacks

    Plant Golgi stacks in a tobacco root cap cell that was high pressure frozen

    This machine permits the cryo-preservation of relatively large samples of biological material by subjecting specimens to 2100 bar during the freezing procedure, thus preventing the nucleation of ice crystals and specimen damage.

    A postdoctoral research assistant Dr Eric Hummell, an expert in plant electron microscopy, has been employed to help run the facility alongside Barry Martin, the Cell Biology Laboratory Manager at Oxford Brookes University who has many years of experience in cryo-microscopy techniques.

    The High Pressure Freezing Facility at Oxford Brookes University, combined with freeze-substitution processing is available to BBSRC grantholders wishing to prepare specimens for structural and immunocytochemical studies.