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Having collected all of this data it was important to decide on the best way to build up the super-resolution image map of structures we had imaged. The image processing for PALM/STORM is a new and evolving field; powerful statistical analysis of the PALM/ STORM dye blinks is needed for generation of a robust super-resolution image. Several of these alogrithms were compared to find out which was the most suitable for the spinning disk super-resolution microscopy technique. Since spinning disk microscopy acquires images slightly differently from other PALM/STORM methods and images parts of the cell no other microscopes can reach we expected slightly different results from those others had seem. Happily we were able to achieve an image resolution with our current setup of 80nm. This is around threefold improvement on standard confocal microscopy. It allowed us to see interactions between chromatin and the nuclear membrane and small changes in the arrangement of the cytoskeleton which occur when the cells physical environment is altered.
One of the major advances of the technique is, following the precision engineering the spinning disk system is very straight-forwards for cell biologists to use. Once all the important components are attached the only really important thing is to ensure the system is stabilised at a standard temperature. This means that the technique can be used by anyone with a bit of microscopy experience. “Creating an easy to use super-resolution microscopy technique with broad application was one of the major aims of this project,” said Dr Ann Wheeler. “As head of the light microscopy facility I see all sorts of different interesting Biomedical research every week. Although stem cell research is vitally important it’s great to have generated a technique which can be used in applications ranging from the organisation of the bacterial membrane, to HIV viral restriction in infection and cancer cell invasion. Using spinning disk microscope we have truly created a technology that opens a window on structure and organisation in the centre of the cell.
Nuclear Membrane and Chromatin - Here we show the membrane around the nucleus in Red and organisation of Chromatin, which packages up DNA in green in the nucleus at super- resolution. This image shows that there are regions of Chromatin which specifically interact with the nuclear membrane and the very fine structure of the chromatin in the cell.
Spinning disk super-resolution relies on a set or technologies called Photoactivated Localization Microscopy (PALM) / Stochastic Optical Reconstruction Microscopy (STORM). Both of these technologies make use of fluorescent probes which can be switched on and off using light or chemicals in the sample environment. The PALM/STORM probes each individually blink on and off randomly. Localisation of each random blink allows a very detailed map of a structure which has been labelled by the probes to be built up. The more images of random blinks that can be acquired the better the resolution of the output image. This is where the spinning disk comes into its own. It is able to collect many focussed images of PALM/STORM dye blinks from anywhere in the cell including the nucleus. More excitingly we found that it was possible to use more than one PALM/STORM dye at a time on the spinning disk. This meant that we were able to find out how two separate components in the nucleus were interacting with one another, which is a useful tool in investigations of stem cell fate specification.
The group picture l-r is Dr Ann Wheeler, Dr Neveen Hosny and Professor Martin Knight
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Focal Adhesions: This image shows the interaction between actin (green), which gives the cell structure and paxillin which is a protein involved in cell adhesion (red). The super-resolution image gives unprecedented detail of structure of the interactions between these two proteins.
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