Supervisors: Prof Jason Swedlow and Dr David McGloin
My participation in the PHOQUS: PHOtonic tools for Quantitative imaging in tissUeS project as an Early Stage Researcher started on 11th August 2014. My project is titled “Super-Resolution Analysis of the Human Mitotic Spindle” and its main goal is to successfully design, build and implement the state of the art 3D Stimulated Emission Depletion (STED) super-resolution confocal fluorescence microscope. There is still a lot to discover and unravel in biological structures, but those structures have not been fully explored due to the resolution barrier of the diffraction-limited microscopy. The last 20 years however showed a rapid development in super-resolution techniques that surpass the diffraction limit and enable to image structures in nanoscale. In year 2014 the Nobel Prize for Chemistry was awarded to three of the founders of super-resolution: Stefan Hell, Eric Betzig and William E. Moener.
The STED microscope is a fluorescent confocal microscope with an additional depletion laser that “switches off” some of the fluorophores. This laser beam has a “donut” shape with zero intensity in the centre, and therefore the fluorophores located in the centre are kept lit, while the fluorophores located in the area where depletion intensity is nonzero are turned off. Thanks to this trick we can effectively decrease the fluorophore excitation area which results in the increase in the resolution .
Since the start of my project I have been working on the design of a STED microscope that will be able to sequential optical sections to generate a 3D stack. This task is not trivial, as the STED depletion laser uses very high power that quickly photobleaches the sample. However, I am to use ultra fast scanning and elongated depletion beam pulses to extend the fluorophore lifetime.
During this first year I also spent 5 months at the Centre for Neural Circuits and Behaviour (CNCB) of the University of Oxford and worked closely with Prof. Martin’s Booth group, which specialises in adaptive optics in microscopy. I was working there with the state-of-the-art homebuilt STED microscope and learnt a lot about building such microscope, how to operate it and what to be cautious with. My time at CNCB gave me a lot of unique and valuable experience which will help me with building the STED microscope.
The next steps that will be taken are:
- Set up a new microscopy lab within the new Physics and Life Sciences Laboratory (PaLS Lab) of the School of Life Sciences of the University of Dundee
- Build the ultra fast scanning STED microscope with the ability to do imaging of 3D samples
- Use the STED microscope to do the 3D super-resolution imaging of structural deformations of the human kinetochore.
The human kinetochore is a critical machine, as it is mediates the generation of force that is used to segregate chromosomes at mitosis. The molecular components and interactions of proteins that build the kinetochore are largely established, but how this structure changes during the application of force in the mitotic spindle is not yet known. I aim to directly visualise these deformations in the 3D STED system I build in my PHOQUS project, to reveal the structural dynamics of this important biomolecular machine.
-  http://www.nobelprize.org/nobel_prizes/chemistry/laureates/2014/hell-lecture.html
-  Bianchini, P., Peres, C., Oneto, M., Galiani, S., Vicidomini, G., & Diaspro, A. (2015). STED nanoscopy: a glimpse into the future. Cell and Tissue Research,360(1), 143–150.
Journal and Conference Papers and Posters
[October 2017] An adaptive optics 3D STED microscope for uncovering mitotic spindle structure. Poster presented at the EMBO/EMBL Symposia Seeing is Believing – Imaging the Processes of Life, EMBL Heidelberg, 4th-7th October 2017.
[August 2016] Challenges in building a fast scanning Super-Resolution STED microscope. Oral presentation at the Biophotonic approaches: From molecules to living systems conference, Dundee, Scotland, 23rd August 2016.
[July 2015] Diamond as a multi-role fluorescent marker for bioimaging by Brian Patton, Piotr Zdankowski, Mirella Koleva and Martin Booth. Poster presented at The 66th Diamond Conference, Warwick University, UK.