focus on Microscopy & Microtechniques


Spinning Disk Super-Resolution Microscopy – Bringing Super-Resolution in Focus for the Cell Biologist.


Ann P. Wheeler, Blizard Institute, Barts and the Royal London School of Medicine and Dentistry, Queen Mary University London, UK: Neveen A. Hosny and Martin M. Knight, Institute of Bioengineering, School of Engineering and Materials Science, Queen Mary University London, UK.


Email: a.p.wheeler@qmul.ac.uk Website: www.icms.qmul.ac.uk/imaging/index.html


A team from QMUL, headed by Dr Ann Wheeler and Professor Martin Knight , has recently taken on this challenge successfully by generating a new microscopy technology, Spinning disk super-resolution imaging. This work was published in October of 2013 in Plos ONE: www.plosone.org/article/info%3Adoi%2F10.1371%2Fjournal.pone.0074604


The nucleus is the control centre of the cell and is well known for being the organelle which determines the fate of cells and their behaviour. However how the components in the nucleus interact together to determine fate is really diffi cult to see. This is because structures in the nucleus can be very small, often less than 100 nm in size. Unfortunately even the best light microscope can only image structures clearly larger than 200 nm. To make matters worse the nucleus is very dense and scatters light making it diffi cult to obtain a focussed image. Similarly the primary cilium is a narrow hair-like cellular projection, only 200nm in diameter. This makes it very diffi cult to visualise the spatial and temporal protein dynamics within the cilium and which are believed to control many aspects of cell function.


A team from the Advanced light microscopy facility, at the Blizard Institute in QMUL (BALM), headed by Dr Ann Wheeler, has recently taken on these challenges by successfully generating a new microscopy technology, Spinning disk super-resolution imaging. “We were motivated to do this because Professor Martin Knight and Dr John Connelly both work on different aspects of how the cells’ environment regulate cell behaviour through changes in the structure and proteome of cellular organelles. Dr John Connelly, from the Blizard Institute QMUL, has recently shown that a cells’ fate can be determined by controlling how a cell spreads out as it attaches to a surface. Dr Martin Knight, from the School of Engineering and Materials Science at QMUL, looks at how the primary cilium senses changes in the environment and causes changes in cell behaviour. Both of these projects needed a technique which would be able to visualise cellular structures nanometres in size,” said Dr Ann Wheeler.


Microtubules - This image shows the organisation of DNA and Microtubules in a cell which is about to divide. Microtubules are thin fi laments which generate the force needed to separate the Chromosomes during cell division.


Recent advances in optical physics have led to the development of a new set of light microscopy methodologies which allow the resolution limit of light microscopy to be broken. These microscopy methodologies are called super-resolution imaging and allow visualisation of structures as small as 20 nm. Although, there are limitations to these methodologies; some require complex microscopy setups which are very costly, others can obtain amazing improvements in resolution for structures which are no more than 200 nm away from the coverslip. However, neither of these solutions were appropriate for the Knight or Connelly lab research because the parts of the cell they needed to image were more than 200 nm away from the coverslip, in the middle of the cell. As the UK was in a recession at the beginning of the project, causing a squeeze on research budgets, a new way forwards was needed.


2 ColEosActin Super-Resolution - This shows the organisation of Actin, which is a protein needed to give the cell structure, at the edge of the cell. The cell edge is highly dynamic and so actin can be enriched in specifi c domains here and SDSI maps at high resolution the structure of these actin domains.


To solve these problems a new type of super-resolution microscopy was developed by Dr Ann Wheeler and Dr Neveen Hosny a postdoctoral research associate. With support from the EPSRC QMUL Discipline bridging fund, together they developed a low cost, easy to use, super-resolution microscope which was capable of imaging the nucleus and other organelles in the centre of the cell. The technology relies on the use of a spinning disk confocal microscope. Spinning disk microscopy technology, coincidentally, was originally brought to the west by another QMUL microscopist, Professor Alan Boyde.


LAB ASIA - MARCH/APRIL 2014


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