A Super-resolution multiphoton and dynamic STORM imaging facility

Recent advances in fluorescence microscopy have led to significant improvements in our ability to locate and discriminate small objects. Super-resolution is the term used for technologies that can resolve objects that are smaller or closer together than was previously thought possible according to the laws of Physics. It is now possible to see objects with nanometre resolution and precision as well as image at depth in living tissue and examine rapid biological events at high speed. Achieving all of these improvements at the same time is the "holy grail" of optical microscopy but this has proved practically difficult. At the University of Leicester, we have developed a new hybrid technology called SuperRAMP that allows high speed, multicolour, multiphoton, super-resolution imaging deep in living tissues.

Super-resolution, Random Access MultiPhoton microscopy (SuperRAMP) is a unique technique that combines patterned illumination with mathematical methods to pinpoint discrete objects with nanometre precision at very high speeds. Devices that use sound waves passing across crystals are used to project patterns of infra-red light, which can pass through specimens better than visible wavelengths of light and produce better penetration into deep tissues. These patterns are processed mathematically to produce high-resolution images. SuperRAMP improves both the lateral (xy) and depth (z) resolution by 2-3-fold compared to standard microscopes to an absolute lateral resolution of 120 nm. It can collect two colour images at speeds of around 10 frames per second and from super-resolved points of interest at thousands of samples per second. No other microscope available today can operate at this resolution, at depth, and at this speed in living tissues. SuperRAMP will be combined with a second, complementary method called dSTORM to provide even higher resolution. dSTORM uses the same mathematical techniques used for SuperRAMP microscopy but without scanning. Although it takes longer to create an image, it has a higher resolution of 20 nm.

We will develop a super-resolution, multi-user facility that will make these exciting technologies available to researchers at Leicester and to the wider community. This will help us to drive academic excellence amongst existing BBSRC funded researchers in projects that range from studies of the synthesis of new proteins, the processes of cell division, mechanisms of cell-cell communication in the central nervous system, the neurological bases for development and behaviour - including circadian clocks that control daily rhythms - in model animal systems including locusts, zebra fish, fruit flies, rats and mice.

This facility will support research collaborations within the Midlands (Nottingham, Leicester and Warwick) and further afield (Cambridge) and allow us to strengthen our commercial impact by developing a show case facility for functional, super-resolution imaging. Having developed this revolutionary technology, we are uniquely placed to establish a world-leading centre of excellence for functional, super-resolution imaging.

Recent advances in microscopy have led to significant improvements in resolution. It is now possible to exceed the so-called diffraction barrier, image at depth in living tissue and examine dynamic events at high speed. Achieving all of these at the same time has, however, proved practically difficult. We have developed a new technology that allows high speed, multicolour, multiphoton super-resolution imaging deep in living systems.

Super-resolution, Random Access MultiPhoton microscopy (SuperRAMP) combines structured illumination with stochastic localisation methods to pinpoint objects with nanometre precision at high speed. Acousto-optic devices are used in conjunction with multiphoton lasers to produce structured illumination at depths up to ~500 micrometers, allowing functional images to be collected in living systems. SuperRAMP uniquely improves both the lateral (xy) and axial (z) resolution by between 2 and 3 fold to an absolute lateral resolution of 120 nm. It can collect two or four colour images at speeds of around 10 frames per second and from super-resolved points of interest at kHz speeds. dSTORM is an overlapping technology that can produce even higher resolution images, but at slower speeds.

We wish to develop a super-resolution, multi-user facility that will make these exciting technologies available to researchers at Leicester and to the wider community. This will help us to drive academic excellence amongst existing BBSRC funded researchers in a range of projects including single molecule studies of transcription, the structural and functional mechanisms of mitotic cell division, synaptic physiology, the neurobiological basis of development and behaviour in model animal systems and the molecular basis for circadian biology. This facility will also provide opportunities for collaborations outside the University and allow us to strengthen our commercial impact by developing a show case facility for functional, super-resolution imaging.

The beneficiaries of this research will extend well beyond the applicants. We will set up a multi-user facility for functional super-resolution imaging that will be accessible to anyone at the University of Leicester and within the Midlands as part of the M5 consortium. This consists of Leicester, Warwick, Birmingham, Leicester, Nottingham and Aston Universities. Within this group, equipment access will be provided on the same basis as that for internal users. Payment of VAT, for example, is not required. We have received expressions of interest from colleagues at Warwick, Nottingham and Cambridge Universities who would like to use this unique technology.

Our intention is to form an EU Bio-imaging hub for sharing distributed imaging infrastructure. By joining this network, we will allow external users from around the UK and Europe to access the facility. With this in mind, we wish to set up the facility as a "research hotel" that would not simply provide the necessary imaging facilities and expertise, but all the other necessary facilities for successful experiments including cell culture, animal holding facilities, insectaria, fish tanks etc. We will look for opportunities to become a hub once the microscopes have been commissioned.

We intend to pursue the commercial possibilities that this new technology brings. We have already secured follow-on-funding, the objective of which is to demonstrate the use of SuperRAMP technology in a range of experimental approaches from biology to engineering. The formation of this facility will directly benefit this aim by the fact that it will be used by wide range of scientists for a wide range of applications. We have already developed strong links with the Space Research Centre at Leicester who are interested in applying the sensor and image processing technologies developed for space research to biological and engineering applications. The College of Engineering has also developed a "life science interface" theme which is designed specifically to encourage collaboration between biologists, chemists, physicists and engineers and on of the Co-I's, Dr Andrew Hudson, plays a leading role in this theme through his collaborations with Eperon, Hartell, and Revyakin for example.

SuperRAMP technology is a combination of structured illumination and localisation based mathematical methods. Two patents have been filed which protect the two key processes, which are the illumination system and the processing of the data. The basic concepts can be applied to other imaging modalities. We are in the process of developing a system that uses visible laser light sources. It is also possible to use the mathematical algorithms on data from a Nipkov disk type microscope to convert it into a super-resolution microscope. We also intend to develop new detector technology, in collaboration with Leicester's Space Research Centre, to produce a "smart" sensor array that will apply the algorithms in real time to fluorescent data to transform a standard confocal microscope into a real time super-resolution microscope. Finding a simple way to convert existing technology into super-resolution technology is a very attractive prospect in terms of enhancing access to users and from a commercial point of view. We will aim to submit grant applications within 6 months of this award for work towards these aims.

The applicants who will use the facility and the support staff that will operate it will all benefit from additional training in the use of super-resolution technology. Some of the applicants and their research staff will benefit from learning how to construct microscopes.