A Super-resolution multiphoton and dynamic STORM imaging facility

Lead Research Organisation: University of Leicester
Department Name: Cell Physiology and Pharmacology

Abstract

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.

Technical Summary

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.

Planned Impact

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.

Publications

10 25 50
 
Description We have used this funding to develop a research facility for use by researchers at the University of Leicester and the Midlands. We are now in the process of working with users to ensure that this technology is made available as widely as possible. The developments associated with this facility and the grants that preceded the award have led to further developments in other related technologies that we are currently exploiting.
First Year Of Impact 2016
Sector Education,Pharmaceuticals and Medical Biotechnology,Other
Impact Types Economic

 
Description ALERT 14
Amount £638,019 (GBP)
Funding ID BB/M012034/1 
Organisation Biotechnology and Biological Sciences Research Council (BBSRC) 
Sector Public
Country United Kingdom
Start 11/2014 
End 11/2015
 
Title SuperRAMP 
Description We have developed a technique called SuperRAMP or SuperRAM which involves a new method of scanning light over samples, and subsequent image processing, to produce superresolution images. The method can be used with visible or infra-red light and is applicable to live samples. 
Type Of Material Improvements to research infrastructure 
Year Produced 2015 
Provided To Others? Yes  
Impact We were able to obtain funding to develop a superresolution facility to offer this methodology to the University and wider academic sector. The hardware and software necessary to implement these techniques have been protected by fling patents in Europe, Japan and the USA. Preliminary publications describing the software methods have been published and we are shortly due to publish the work that will describe the characteristics and utility of the method in live cells. 
 
Title Improved Optical Arrangement 
Description This patent describes the use of a digital micromirror device and arrangement of mirrors that enable the system to act as a digital confocal microcsope. 
IP Reference US9383562 
Protection Patent granted
Year Protection Granted 2016
Licensed No
Impact The device is currently in use within my lab and we are currently in discussions regarding licensing the technology
 
Title Improvements in or relating to super-resolution microscopy 
Description This invention relates to a method of processing images captured following structured illumination of a sample through which significant improvements in lateral and axial resolution can be achieved. 
IP Reference EP3060953 
Protection Patent application published
Year Protection Granted 2016
Licensed No
Impact This method forms the basis for the development of a new form of microscopy called SuperRAMP which is a super-resolution form of multiphoton microscopy. It is currently in use at Leicester University as part of a Super-resolution facility
 
Title Improvements in or relating to super-resoluton microcopy 
Description This patent describes a new form of super-resolution microscopy that uses multiphoton microscopy in combination with acousto-optic devices. The method produces a 2 fold improvement in imaging compared to standard mutliphoton images at high speed and at depth in living tissues 
IP Reference US9946060 
Protection Patent granted
Year Protection Granted 2017
Licensed No
Impact This development builds on work from previous grants. As a result of this invention, we have been able to apply succesful for funding from the BBSRC to set up a facility for the use of this new technology within and beyond the University. This facility is now functional and we are building a group of collaborators who are now making use of this and related technolgoy for their research projects.
 
Title Super-resolution microscopy 
Description The present invention relates to a method of processing images captured following structured illumination of a sample, the method comprising the steps of: identifying emission spots within each captured image; verifying the emission spots; and reconstructing an enhanced image of the sample from the emission spots. The method may comprise identifying only in focus emission spots. By identifying and processing only in focus spots, whether or not they are centered on expected illumination positions, improvements in resolution can be achieved compared to known SIM methods. In particular, by suitable selection of in focus spots, significant improvements in lateral and axial resolution can be achieved 
IP Reference US9946060 
Protection Patent granted
Year Protection Granted 2016
Licensed No
Impact Enabled funding to build and produce a facility utilising this technology
 
Title NiMan 
Description A suite of software that allows time resolved analysis of large stacks of images but also including a novel method for creating and analysing super-resolution images from structured illumination microscopy 
Type Of Technology Software 
Year Produced 2015 
Impact The software has been developed over many years and is used by members of my laboratory and collaborators to analyse data collected from the various research projects we have undertaken over the last 10 years 
 
Title NiMaq 
Description A suite of software that allows the control of imaging equipment including cameras, light sources, digital RF synthesisers, microscopes and scanning devices 
Type Of Technology Software 
Year Produced 2016 
Impact This software has been developed over many years and is the basis for most of the technical developments my lab has made over many years. We hope that it will either be released as an open source tool or form part of a spin out product that we can licence or sell 
 
Title RF Synthesiser External Operation (XOP) for Igor Pro 
Description A driver that allows control of a radiofrequency digital synthesiser for control of acousto-optic devices 
Type Of Technology Software 
Year Produced 2016 
Impact This is central to the development of our Super-resolution microscopes that use acousto-optics and forms the basis behind any future commercial products based upon this technolgy. It is central to the operation of our super-resolution facility that uses technology invented through BBSRC funding and which is currently used by members of our university 
 
Title SuperResolution Imaging Faciltiy 
Description A suite of microsccope that allow super-resolution imaging using tools and technologies developed in my laboratory with BBSRC support 
Type Of Technology Systems, Materials & Instrumental Engineering 
Year Produced 2016 
Impact We have developed a facility for members of our University, It is the first opportunity for scientists to have access to super-resolution microscopy. The technology was initially developed with BBSRC support leading to ALERT 14 funding to equip and setup the faclity. It is now starting to be used by members of the College 
URL http://www2.le.ac.uk/colleges/medbiopsych/facilities-and-services/cbs/AIF/Equipment/super-resolution...
 
Title Visible wavelength acousto-optical based super-resolution microscope 
Description A novel super-resolution microscope that produced improvements in lateral and axial resolution through a combination of patterned illumination and mathematical processing of images. 
Type Of Technology New/Improved Technique/Technology 
Year Produced 2018 
Impact Ongoing