Super-resolution fluorescence microscopy (SuRFM)
From BioImagingUKWiki
Contents |
Introduction
Visualisation of sub-cellular structures and even single molecules is absolutely essential in biological and biomedical research for our understanding of complex biological morphologies and functions. Thus, light microscopy has developped into one of the most widely used imaging modality in the tool kit of bioscientists. Light microscopy is limited by the diffraction of light, defining a relatively low spatial resolution of 200 – 300 nm laterally and 500 – 700 nm in the axial direction. Although transmission electron microscopy (TEM) provides unsurpassed ultra resolution down to the nm range, it comes with a range of significant technical limitations in terms of molecular labelling, complex sample preparation and the lack of live cell imaging.
However, recent developments in light microscopy have successfully circumnavigated the diffraction-limited resolution barrier, closing the resolution gap between conventional fluorescence microscopy and EM by offering a whole range of novel approaches allowing super-resolution fluorescence microscopy (SuRFM) and improving the spatial resolution of up to a magnitude.
Topics
Tools for super-resolution fluorescence microscopy (SuRFM) and single molecule imaging
Members
Ann Wheeler, Gail McConnell, Dave Clarke, Ian Dobbie, Mark Neil, John Girkin, Andrew Carter, Rolly Wiegand, Martin Spitaler
SuRFM technologies
SuRFM includes a whole range of relatively novel emerging imaging techniques, which provide a huge potential to improve and revolutionise biological and biomedical research. The following list of techniques is in no way comprehensive, but only highlights the most important technologies that have been discussed at the last facility manager meeting (see link).
• Structured-illumination microscopy (SIM) • Stimulated depletion microscopy (STED) • Statistical localisation methods (PALM, STORM, FPALM, etc)
More detailed information can be found in the appropriate literature and a short summary can be found at this link (link). In general, these techniques require highly accurate control of the stage position and its environment. Thus, including ‘beacon’ molecules in the experiment that allow for correction of sample movement is recommended. Some of the data analysis algorithms also have a high computational demand for the data fitting and more freely available open-source analysis software has been published recently. With the right know-how, this in theory makes it possible to implement some of these techniques on a high-end widefield-TIRFM system with little or no additional add-ons, based on the use of freeware for data analysis.
How can BioImaging UK help to implement and foster SuRFM?
SuRFM represents a whole range of fast developing techniques that promise a huge improvement in subcellular and molecular biological imaging beyond the diffraction-limited resolution limit. To help assessing, implementing and foster these techniques in the UK, BioImaging UK have established the Super-Resolution Microscopy working group to define and facilitate this process. The following section discusses the aims laid out by this working group.
Assessment and implementation of SuRFM technology
The potential of the new super-resolution techniques seems enormous, but the process of providing easy-to-use systems to a wider microscopy community is in its infancy. As with other high-end technologies for biological research, implementation of novel techniques is time-consuming and, depending on the technique, expensive at varying degrees. To facilitate this process in particular for central imaging facilities, the Super-resolution Working Group has discussed the following options:
Discussion platform and forum – This will allow the exchange and dissemination of all vital information, the assessment of advantages and pitfalls of different techniques and the discussion of published data and novel developments.
SuRFM Workshop – This will be valuable for the direct comparison of all available systems. The manufacturers of turn-key SuRFM-system have in principle agreed to participate in this kind of event. The workshop could also be used to initially teach the basic use of the novel techniques and for presentation of published or ongoing work.
In addition, an annual SuRFM teaching course in the format of a Summer School or similar could help to foster the advanced use of SuRFM in the UK, and further educate technical staff of imaging facilities and members of the UK microscopy community.
Provision of high-end systems for SuRFM
Some of the above described technologies (e.g. statistical localisation methods, CW-laser based STED etc) seem affordable to be implemented in imaging facilities of all sizes (see above). However, specialist training of technical staff and more hands-on time for the more demanding assistance with SuRFM-techniques will be required.
Central UK SuRFM facility - The idea of large central SuRFM-research and development facilities in the UK would be desirable, but their funding would be critical and might prove difficult. Industrial partners could provide a solution and would in exchange for their funding and hardware donations profit from the research output of these facilities. In this environment, a cross-disciplinary direct collaboration of biologists, application specialists, physicists and IT experts at one site could significantly help to improve applications and developments and provide teaching about these novel technologies. The ability of central facilities to offer full service mode operation should be particularly useful to researchers who are new to SuRFM techniques, and would like to establish these technologies based on trial experiments carried out in central facilities. BioImaging UK should be a central player in the initiation and organisation of these facilities. It is proposed that the STFC Imaging Solutions Centre (ISC) on the Harwell Campus would offer a significant amount of SuRFM microscopy instrumentation. This would build on the already existing Lasers for Science Facility, which operates in the cross-disciplinary Research Complex at Harwell and offers access to single molecule TIRFM microscopes as part of its portfolio. This facility is centrally funded and offers free at the point of use access to the UK community through a peer-reviewed application procedure, and could be a model for a future SuRFM central facility if funding was made available. Furthermore, central facilities would be perfectly suited to hold the above mentioned workshops, discussion meetings and teaching events, which relate to SuRFM. They should be 'catalyser' units that quickly help expert staff, who are new to SuRFM, to learn these new technologies.
Regional SuRFM facilities - Another solution of centrally providing expensive SuRFM technology on a smaller scale and closer to the end user would be Regional Imaging Facilities. Rather than providing a large range of different technologies for large parts of the UK, these facilities would focus on some of the SuRFM systems and how to make their use available to scientists in the area. Costs for technical staff and maintenance can be met by charges for the usage of these facilities. This strategy could be implemented in existing imaging facilities and a very good example has been set by the purchase of an AP OMX microscope by the SULSA initiative in Scotland. This system is based in the College of Life Sciences at Dundee University, but is available to researchers from across Scotland. Regional facilities could be set up to complement rather than to compete with large central units.
Local facilities - Although centralised facilites could potentially provide the services as described above, it is important to implement super-resolution technologies at the local level to guarantee that scientists have easy access close to their biological research laboratories. Central facilities could provide and teach the know-how to facilitate and speed up the implementation of SuRFM techniques.
The existing inventory of UK Light Microscopy Facilities (link) will help scientists to find equipment they do not have access to, but that is being made available by other service units. This inventory should also include expertise provided by members of the mentioned facilities, in particular if this relates to SuRFM.
Central and regional facilities should be planned and organised so that they complement local imaging facilities and BioImaging UK should be a key player in co-ordinating this development.
The sustainability of the implementation and maintenance of these novel technologies at all three above mentioned levels is crucial and essential for keeping the UK imaging community internationally at the cutting edge. You will find more information about this topic on this website on the Sustainability [1] working group page.
Future requirements for SuRFM
Turn-key SuRFM systems - Most central imaging facilities rely on commercial turn-key microscope systems for the services they offer. This guarantees the smooth delivery of imaging services with little down-times, highly efficient equipment and a predictable cost structure. It is expected that this trend will continue whilst SuRFM is being introduced commercially. First systems have been released (Zeiss Elyra, AP OMX, Leica STED) and in the interest of the whole imaging community, it is hoped that more systems will be entering the market and that patenting of novel technical developments will not have a limiting effect. In addition, quick solutions for the outstanding lack of a high axial resolution for most of the so far released technologies would be required for true 3D imaging at super-resolution.
Computational capacity for data storage and data processing - Since some of the mentioned SuRFM techniques require an increasing amount of data handling and processing, the rising demand in data computing, storage and archiving should be taken into account. Although data analysis for SuRFM can be carried out on affordable high-end work stations, the use of grid computing and decentralised SANs might be recommendable and speed up data processing significantly. Some Universities already provide grid computing facilities internally and for external users. Imaging facilities that start using SuRFM should make sure they provide enough data storage capacity, locally or centrally. Furthermore, it has been suggested that BioImaging UK could have a central, UK-wide agreement with the Rutherford Appleton Lab with regard to large scale data storage, if local solutions cannot be provided sufficiently.
Fluorophore development for SuRFM - Some of the SuRFM techniques are based on special fluorophores (STED, localisation methods) and further fluorophore development is crucial for the improvement of the technology. BioImaging UK should play a supporting role in inter-disciplinary discussions about the development of novel fluorophores between molecular biologists, chemists and application specialists.
Multi-disciplinary collaboration – Developing and supplying complex microscopes for SuRFM involves the integration of several very different disciplines, such as biology, physics, informatics and manufacturing. It is in the interest of all experts and end-users involved to generate an optimal, mutual exchange of information to improve future developments of SuRFM technologies. BioImaging UK is the ideal body to facilitate this exchange and already, the participation of many representatives from microscope manufacturing at the annual facility manager meetings provide a versatile discussion platform. This exchange could be extended to the above mentioned forum and workshops.
BioImaging UK Website for SuRFM
As an extension of the above mentioned singular events for discussion and teaching of novel SuRFM technologies, a new website, stand-alone or as part of the existing BioImaging UK website, should be established and serve as a comprehensive resource for microscopists with an interest in SuRFM. This could be integrated with the SuRFM discussion forum.
Future goals in brief
Implementation of SuRFM through
• Discussion platform and forum
• Workshops
• Summer schools
Provision of high-end systems for SuRFM by
• Central UK facilities
• Regional SuRFM facilities
• Local imaging facilities
Future requirements for SuRFM
• Turn-key SuRFM systems
• Computational capacity for data storage and data processing
• Fluorophore development for SuRFM
• Multi-disciplinary collaboration
Dissemination of SuRFM information
• BioImaging UK website
