Dr. Steven F. Lee is a Royal Society University Research Fellow & a Fellow of Sidney Sussex College, Cambridge.
NEWS: PhD Studentships available in the area of Multi-Dimentional Super-Resolution microscopy - Starting October 2017 - Any interested students are encouraged to contact SFL directly by email.
Background.There is a fundamental limit to how small we can see objects, caused by the diffraction of light itself, this is called Abbe's diffraction limit. This innate 'blurriness' means that optical microscopy cannot resolve anything smaller than ~250 nanometres. Unfortunately the spatial scale that many biological processes occur on is smaller than this limit, therefore it has been difficult to directly probe these events.
Super-resolution microscopy is an advanced, interdisciplinary optical imaging technique currently attracting immense interest as a new and exciting way to break this theoretical diffraction limit of visible light. Using single molecule control, super-resolution microscopy retains the non-invasive advantages of fluorescence imaging but has the ability to resolve biological structures more compatible with the spatial scale that these events actually take place on, typically attaining resolutions of ~15nm or better.
We use novel approaches of super-resolution microscopy in both 2D and 3D, to directly visualise biological processes going on inside living cells at two physical areas: in the eukaryotic plasma membrane and in the nucleus, highlighted by two key areas:
1) The molecular origins of human immunity by studying the spatial distribution and interaction of individual membrane protein complexes on the plasma membrane of live T-cells during triggering.
2) How the molecular composition, stoichiometry and spatial arrangement of histones assemble during DNA replication and repair, all inside the nucleus of fission yeast in three dimensions.
Figure 1. (Upper) The key idea of super-resolution imaging of a structure. (a) It is not possible to resolve the underlying structure in a conventional widefield fluorescence image because the fluorescent labels are in high concentration and the individual emitters overlap. (b) Using controllable fluorophores, it is possible to ‘turn on’ and image a sparse subset of molecules which can then be localised with nanometre precision (grey line is the underlying structure being sampled). Once the first subset of molecules photobleaches, another subset is turned on and localised. This process is repeated and the resulting localisations are summed to give a super-resolution image of the underlying structure. (Lower) Example of 3D two colour SR imaging of the helical cytoskeletal protein crescentin in the live bacterium caulobacter crescentus (shown in orange/red) in relative context to the cellular membrane (grey) attaining lateral and axial precisions of 15 and 20nm respectively. Conventional diffraction limited imaging is shown (far left) as a reference, compared with the exploded view of the two component channels (middle images) and then combined (far right). A white light image (inset) and a reference grid (green) made up of 1µm squares is shown for scale.
These imaging strategies can be applied to a host of other problems where there is need for watching key biological process unfold at high spatial precision at both the membrane and the nucleus. This can only be achieved by attaining a fundamental understanding of the physical and chemical processes behind many aspects of the technology, including: kinetic understanding of fluorophores, theoretical fitting of impulse response functions and detailed knowledge of image reconstruction algorithms .
For an up-to-date publications list, please see our google scholar page
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