Multidisciplinary Super Resolution Microscopy Facility at Nottingham University

Lead Research Organisation: University of Nottingham
Department Name: Sch of Molecular Medical Sciences

Abstract

For many years there has been a gap in the resolution of Confocal Laser Scanning Microsopy and even with perfect lenses, optimal alignment, and large numerical apertures the optical resolution of this form of light microscopy has been limited to approximately half of the wavelength of the light used meaning that only cellular structures and objects at least 200 to 350 nm apart could be resolved as distinct separate structures. However, much of the fundamental biology of the cell, occurs at the level of micro-molecular complexes in the size range of tens to few hundred nm, i.e., beyond the reach of conventional light microscopy. Super resolution microscopes (SRM) have been developed to break or bypass the classical diffraction limits and shift the optical resolution down to macro-molecular or even molecular levels. It can locate single molecules down to 20nm precision. SRM can also provide particle tracking to follow single molecules, e.g. proteins, DNA, drugs, in a sample without compromising resolution.
Although the University of Nottingham (UoN) houses a range of microscopy facilities there is a clear gap in resolution that requires the cutting edge technology SRM provides to take the research into areas that are not currently achievable in Nottingham Life Sciences at present. Some of these areas include: (i) Microbiology: to understand better how bacterial cells interact with each other and how they can transfer molecules and signals within and between cells in bacterial communities known as biofilms; (ii) Bioenergy: to understand how some bacterial cellular energy reservoirs are transformed into biofuels and hence be able to optimize of these processes; (iii) Synthetic Biology: to understand how artificial cells and specialized DNA structures are developed as this can have a significant impact within different industrial contexts; (iv) Stem cell biology and tissue engineering: the SRM will have a significant impact on understanding the development of specialized cells from stem cells and the development of tissue scaffolds which will be paramount in regenerative medicine required for the repair of different damaged human tissues; (v) Plant sciences: to understand some of the signaling processes in root development and the mechanisms of pollen development which impact on crop production and sustainable agriculture; (vi) Food security: to unravel essential mechanisms of interaction between pathogens and host cells of farm animals which are responsible for significant loses in the farm industry with the ultimate aim of targeting some of these to reduce the negative economic impact they have and (vii) Molecular pharmacology: to facilitate the understanding of drug-target interactions enabling their optimization with the consequent improvements to health.
Hence the acquisition of a SRM facility will enable UoN to address key biological questions in the above areas which will have a significant impact on science, the economy and society.

Technical Summary

The objective of this grant application is to provide the University of Nottingham with a Super Resolution Microscopy Unit in a Class 2 containment facility which can be used for both Class 1 and Class 2 experiments. The SR microscope will combine super resolution structured illumination microscopy (SR-SIM) and single molecule localisation techniques (PALM & dSTORM) to enable the localization of single molecules with precision down to 20 nm. Using this technology, an optical grid leading to a large number of images (e.g. 10,000) with very low levels of blinking fluorescence can be collected from single molecules by localising the dots in each of the 10,000 images. Because of the large number of acquired images required for the localisation data, samples in general need to be fixed unless lower localization precision is required. It will also provide particle tracking to follow single molecules in a sample without compromising resolution. Amongst the applications will be the ability to track single molecules (proteins, DNA, drugs etc). This microscope will be combined with a sophisticated confocal microscope with a 32-channel GaAsP detector for better quantum efficiency to improve the detection of weak signals and signal-to-noise ratio and which also enables the observation of molecules at high resolution in real time and the potential to image the same cell with SRM after fixation. This combination is essential for the acquisition of the molecular details of e.g. protein transport mechanisms, bacterial and viral proteins interacting with eukaryotic molecules, localization of small molecules in plant cells, biofilm architecture, tracking molecules in stem cell biology, DNA oregami structures, bacterial communication within biofilms, the activation of multiple nanosensors in specific areas of a bacterial biofilm and in tissue scaffolds etc.

Planned Impact

The RCUK have identified an urgent need to invest in imaging technology through the RCUK Strategic Framework for Capital Investment. The establishment of next generation microscopy platforms are key to answering questions involving the multi-disciplinary research that underpins biology that utilises cutting-edge light microscopy and dynamic cell imaging. In this context the Super Resolution Microscope (SRM) facility will provide a unique dimension to the microscopy facilities at the University of Nottingham (UoN). Due to the multidisciplinarity of the research areas for which the SRM unit will be used, the main objective for this facility will be to impact on research, researchers and industry from the following disciplines:
-Microbiology: the research proposed will have a significant impact in a wide range of industrial sectors where biofilms have either positive or detrimental effects.
-Bioenergy Industry: it will enable a better understanding of how cellulosomes are transformed into biofuels resulting in the optimization of these processes and how to reduce product toxicity.
-Synthetic Biology: the applications proposed will have an impact on the development of artificial cells and DNA nanostructures relevant to different industrial contexts.
-Stem cell biology and tissue engineering: the SRM will have a significant impact on understanding the development of specialized cells from stem cells and the development of tissue scaffolds which will be paramount in regenerative medicine
-Plant sciences: the proposed research will be important to understand signaling processes in root development and the mechanisms of pollen development which impact on crop production and sustainable agriculture.
-Food security: the SRM will be used to unravel essential mechanisms of interaction between pathogens and host cells from farm animals which are responsible for significant losses in the farm industry with the ultimate aim of targeting these and reduce their negative economic impact.
-Molecular pharmacology: the use of SRM will facilitate the understanding of drug-target interactions enabling their optimization with the subsequent impact on patients suffering so contributing to health.
The SRM facility will also have an impact on:
a. Researchers: Through the acquisition of formalized training on the use of the SRM facility. These will include not only researchers at UoN but also visitors from Nottingham collaborators needing to use SRM.
b. The University of Nottingham: It will support the advancement of current research areas and the development of new research possibilities. Having a SRM will attract new collaborations to Nottingham, especially in areas where the current microscopy facilities do not provide the appropriate level of resolution
c. The Midlands: as it will enable researchers from this region, especially those collaborating with the UoN to have access to a local SRM facility and associated expertise.
d. The international relationships between UoN and researchers outside the UK through collaborations between Nottingham researchers and the international research community and industrial companies.
The wider public will also benefit in the longer term from the research conducted at the SRM facility through the increased ability of different industrial sectors to respond to their customer needs from the environment, to agriculture and health.
The research achievements from the SRM facility will be communicated to a range of audiences via presentations through to discussions and workshops with industry contacts, publications in journals targeting a wide range of audiences and conferences and a dedicated website. The research from the SRM facility and its potential will also be communicated to the general public through the yearly University of Nottingham May Fest and through the 'Nottingham Potential' outreach activities.

Publications

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Ramsawhook AH (2017) Immunostaining for DNA Modifications: Computational Analysis of Confocal Images. in Journal of visualized experiments : JoVE

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Wojciechowska M (2018) Quantitative Methods to Monitor RNA Biomarkers in Myotonic Dystrophy. in Scientific reports

 
Title Competition 
Description We have launched an image competition where users of the microscopes submit their best work to the imaging team. Selected images are printed on mugs and prints and displayed on the walls too. 
Type Of Art Image 
Year Produced 2018 
Impact Images attract new users and advertise microscopy as a tool for research. 
URL https://www.nottingham.ac.uk/life-sciences/facilities/slim/slim-image-gallery.aspx
 
Title High resolution prints of super resolution microscopy images 
Description A selection of super resolution images have been printed and mounted on the corridors of the University. The images were captured using the instrument (Elyra PS.1 super resolution microscope) acquired using the founds of this grant. 
Type Of Art Image 
Year Produced 2015 
Impact Each image has a short description, explaining the biology and the super resolution technique applied. 
 
Title Super resolution images printed and displayed 
Description The winning entry of by Robert Markus was printed and displayed permanently in the building hosting the microscope. 
Type Of Art Image 
Year Produced 2018 
Impact The image has an excellent visual impact, therefore fellow researchers ask about it, how it was achieved and where, thus we have another chance to advertise our super resolution microscope and propose them to use and do research with this high end instrument. 
 
Description We further improved the super resolution imaging platform by screening and selecting new chemicals for sample preparation (ms. in prep), implemented new data analysis techniques, trained more scientists to use the instrument independently, started new imaging projects and initiated new collaborations, and at outreach events engaged with the public. Overall last year (2018) we run 48 different imaging projects. Key projects and findings: 1. Discovered new molecules involved in the life cycle (division) of the malaria parasite in a research which aims to identify molecular targets combat infections (Rita Tewari lab) 2. Developed a method to visualise and analyse very small (nanometre sized) vesicles, which are shed from cells. These contain molecular messages governing neuronal development (Virginie Sottile lab). 3. Several projects working in molecular pharmacology identified and visualised nano-scale structures used in drug delivery systems, these have been characterised with super resolution microscopy too (Maria Marlow lab, David Amabilino lab, Mischa Zelzer lab). 4. Continued working on RNA biology in relation to genetically inherited diseases (David Brook lab). 5. Continued the work on biofilm models including human tissue (Miguel Camara, Ahmad Elsahn, Manuel Romero). 6. Screened for chemicals to be used in sample preparation in order to achieve the best possible resolution results are implemented in the projects, a manuscript is in preparation on this (Robert Markus & Tim Self). 7. Continued the work on localisation of nano-particles in cells and investigated drug delivery system in vitro. (Virginie Sottile, Cameron Alexander) 8. Developed a new nano-calibration test slide sample for super resolution microscopy. The prototypes are ready and we are working on IP protection and accelerate commercialisation. This sample is aimed to serve as a standard to compare various super resolution microscopy systems. (Robert Markus, Veeren Chauhan, Amjad Abouselo). 9. We got new molecular imaging insight (single molecule imaging) into murine spinal cord and brain; the research aims to understand pain at the highest resolution possible using our multi tool microscope and different imaging modalities. Results are used to attract further funding. We helped companies (commercial work) to investigate, visualise, and demonstrate how their product works. 10. We designed experiments, investigated and recorded (high res movies) how gloves impregnated with a smart (light activated) antimicrobial agent works. The experiment was performed on stage as well in London, at a launch event of the product.


We have developed a number of techniques which are enabling us to visualise biological process which could not be resolved in such a high definition.
Using the Super Resolution Microscopy Facility researchers at Nottingham we have:
1. Design tracer molecules which bind to the surface of the human pathogen Staphylococcus aureus. The density and the surface localisation of the tracer molecules have been validated using super resolution microscopy. This pathogen relies upon surface expressed proteins to help initiate infection. Super resolution microscopy will help to investigate these proteins at 10 times more resolution than before enabling us to gain a better understanding of the infections caused by this bacterium (Phill Hill).

2.Localised proteins in E. coli involved in disease. We have constructed a three dimensional super resolution model which has enabled us to measure the mobility these proteins inside the bacterial cell using super resolution microscopy alongside in situ ?uorescent labelling of individual molecules. We have been able to visualize and precisely localise structures within the bacterial cell with minimal or no disturbance of those structures (Kim Hardie).

3. Demonstrated the binding of peptides to bacterial DNA using multicolour single molecule imaging and super resolution microscopy. We have found that this interaction results in changes in the virulence of the bacterial pathogens. (Jafar Mahdavi)

4.Generated high resolution three dimensional models to study the distribution of lipids in the microalgae (C. reinhardtii) used as functional food and feed. The super resolution methodology applied is suitable to assess lipid production in algae and to assess the efficiency of the extraction of these lipids (David Gray).

5. Localised nanoparticles after treatment. These experiments show the efficiency of various drug delivery strategies. With super resolution microscopy were able to visualise and measure fluorescent nanoparticles inside different cell lineages (Virginie Sottile, Phil Williams)

6. Developed a nano calibration sample for super resolution microscopy. This sample is aimed to serve as a standard to compare various super resolution microscopy systems. The prototype samples will be sent out to other UK based imaging facilities (Robert Markus and Leo Marques)
Exploitation Route Our findings will be applicable to researchers working in protein localisation, antimicrobials, drug delivery, lipid localisation and more general in super resolution microscopy.
Sectors Agriculture, Food and Drink,Chemicals,Environment,Healthcare,Manufacturing, including Industrial Biotechology,Pharmaceuticals and Medical Biotechnology

 
Description Development of Nano calibration sample for super-resolution microscopy (UNICAS programme)
Amount £5,000 (GBP)
Organisation University of Nottingham 
Sector Academic/University
Country United Kingdom
Start 04/2015 
End 09/2015
 
Description National Biofilms Innovation Centre
Amount £12,801,513 (GBP)
Funding ID BB/R012415/1 
Organisation Biotechnology and Biological Sciences Research Council (BBSRC) 
Sector Public
Country United Kingdom
Start 12/2017 
End 11/2022
 
Title Nanoscale calibration samples for super-resolution microscopy & Nanoscale photoactivatable particles for localisation and tracking of molecules of interest 
Description A new type of nanoscale calibration sample for super-resolution microscopy has been developed, that enables photo-activation and photo-switching via defined lsers. Detection of single molecule signals enable localisation and image reconstruction of nanoscale fluorescent objects of 100 nm. The nanoscale features, nanobeads, are modified with photoswitchable dyes, like spiropyran. They can be resolved via photo-activated localization microscopy (PALM) and stochastic optical reconstruction microscopy (STORM) techniques in super-resolution microscopy due to their optical properties. The device can be easily manufactured at low cost and re-used over time for more than one experiment. The entire worldwide microscopy community using super-resolution microscopy could use such a sample in their laboratory to improve the calibration of their microscopes. The prototype will be finailsed in 2017. An Intellectual Property Review Form has been filed with the University of Nottingham. 
IP Reference  
Protection Protection not required
Year Protection Granted 2016
Licensed No
Impact At first glance the companies that could be interested in such technology would be companies that develop and work on the generation of new super resolution microscopes like Zeiss, Leica, Olympus or Nikon. Authors already have a contact in Zeiss that would be interested in testing the new samples. Beside, every laboratory in academia having a super resolution microscope could be interested in testing \ having such a sample.Authors have contacts over the country, research officers and academics, who would be happy to test the sample.
 
Title Implementation of image data base and sharing platform 
Description We have implemented an image storage and sharing interface, where microscope users store their imaging data. The interface is using the Open microscopy environment (OMERO). Researchers use their university credentials to access, analyze, download or share their data. A backup system is also available, with a copy of all data. Access to this computer is restricted, permission can be obtained via the imaging team of School of Life Sciences, University of Nottingham. 
Type Of Technology Software 
Year Produced 2015 
Impact Initially this image server has been implemented for the super resolution microscope, but in long term it will serve as a cross-disciplinary imaging platform. 
URL https://omero.nottingham.ac.uk/
 
Description 1st Elyra Super Resolution Microscopy Workshop London 
Form Of Engagement Activity A formal working group, expert panel or dialogue
Part Of Official Scheme? No
Geographic Reach National
Primary Audience Professional Practitioners
Results and Impact Since we have the Elyra super resolution microscope we have initiated a meeting between managers of similar microscope and the experts from the manufacturer (Zeiss). Experts of Super resolution microscopy from UK and Germany (Zeiss), met for the first Elyra Super Resolution meeting. The purpose of the meeting was to share our experience and difficulties and technical know-how we accumulated on the Super Resolution Microscope, which was acquired within this award. The attendees all work with similar or older instrument. Crucial technical details were shared, ideas exchange was very useful, additionally the representative of the manufacturer answered the questions of the audience. Robert Markus, gave a talk on his latest developments on refractive index of the mounting media of samples.
Year(s) Of Engagement Activity 2017
 
Description 2nd Elyra Super Resolution Microscopy Workshop Norwich 
Form Of Engagement Activity A formal working group, expert panel or dialogue
Part Of Official Scheme? No
Geographic Reach National
Primary Audience Professional Practitioners
Results and Impact Imaging facility managers having similar microscope we have in Nottingham, met and discussed microscopy related topics. Representative of the manufacturer Zeiss also attended, and answered our questions related to the system. Novel super resolution microscopy technology was presented and debated.
Year(s) Of Engagement Activity 2019
 
Description Creating the image - filtering STORM data. Talk given by Robert Markus, on the implemenation of single molecule imaging (STORM). A case study and the software and analysis approaches were presented. 
Form Of Engagement Activity Participation in an activity, workshop or similar
Part Of Official Scheme? No
Geographic Reach Regional
Primary Audience Professional Practitioners
Results and Impact Around 70 people attended, the talk. We had a froutful discussion on the development of the super resolution techniques, uset in our multidisciplinary imaging facilities. I ve got valuable input in further developint the projects, namely in the recorting frequency of the single molecules. Additionally we have discussed the technical details of our imaging system, I've engaged new collaborations on image analysis, and we will implemet the clustering algorithm in our projects. The audiance apprciated the techniques ive used to detirmine the negative control, and the techniques to eliminate the false positive hits, on our datatsets.
Year(s) Of Engagement Activity 2017
URL https://sheffieldstorm.wordpress.com/
 
Description Frontiers if BioImaging 
Form Of Engagement Activity Participation in an activity, workshop or similar
Part Of Official Scheme? No
Geographic Reach International
Primary Audience Professional Practitioners
Results and Impact We attended the Frontiers if BioImaging meeting in Glasgow, where we met the experts in the filed of Super resolution microscopy and image analysis. Talks and one to one discussions expanded our knowledge, and we implemented the know-how on our return to Nottingham.
Year(s) Of Engagement Activity 2018
URL https://www.rms.org.uk/discover-engage/event-calendar/frontiers-in-bioimaging-2018.html
 
Description Frontiers of BioImaging 
Form Of Engagement Activity Participation in an activity, workshop or similar
Part Of Official Scheme? No
Geographic Reach International
Primary Audience Professional Practitioners
Results and Impact "Frontiers in BioImaging 2016 focused on the latest developments in applications of optical microscopy and image analysis across a range of biomedical fields. Sessions covered both technical developments and applications of these microscopy-based approaches to key cell and molecular biology questions. The meeting covered the key challenges in microscopy today: super-resolution imaging, phototoxicity and light sheet based methods, detection of in situ protein interactions and new tools for fluorescence visualisation and analysis." We met the leading experts of the field. Robert Markus learned about new computational methods for super resolution microscopy, the method was implemented in the facility. As well as interpenetration of super resolution results were discussed, and used in our publications regarding single molecule imaging.
Year(s) Of Engagement Activity 2016
URL https://www.rms.org.uk/discover-engage/event-calendar/frontiers-in-bioimaging.html
 
Description INTERNATIONAL SIMPOSIUM ON " GENE REGULATION IN BACTERIA WITH MEDICAL AND INDUSTRIAL INTEREST' Invited international speaker 
Form Of Engagement Activity Participation in an activity, workshop or similar
Part Of Official Scheme? No
Geographic Reach International
Primary Audience Postgraduate students
Results and Impact The title of the invited talk was: 'Exploiting the therapeutic potential of targeting quorum sensing in Pseudomonas aeruginosa'. The symposium was organised by the Universidad Nacional Autonoma de Mexico in Mexico City. It was attended by master students, PhD students and research group leaders.
Year(s) Of Engagement Activity 2018
 
Description International Seminar at the Universidad National Autonoma Mexico 
Form Of Engagement Activity Participation in an activity, workshop or similar
Part Of Official Scheme? No
Geographic Reach International
Primary Audience Postgraduate students
Results and Impact This was a seminar to promote the results from the SENBIOTAR programme and identify new collaborations from the results of these programme as well as promote NBIC.
The title of the seminar was: Versatility and targeting of quorum sensing signalling mechanisms
There was a mixed audience of scientists
Year(s) Of Engagement Activity 2018
 
Description Poster at 12th International Conference on Renewable Resources and Biorefineries, taking place in Ghent, Belgium on May 30-31 & June 1, 2016. 
Form Of Engagement Activity Participation in an activity, workshop or similar
Part Of Official Scheme? No
Geographic Reach International
Primary Audience Professional Practitioners
Results and Impact A Poster presentation has been given with the following title:

Evaluation of Microalgae (Chlamydomas reinhardtii) for Human Consumption
Patchaniya Eakpetch, Darwish Randa, Esra Kaya, Robert Markus, David Gray

Images cerated using the supre resolution microscope had been presented.
Year(s) Of Engagement Activity 2016
URL http://www.rrbconference.com
 
Description Poster at Neuroscience @ Nottingham, 2016 
Form Of Engagement Activity Participation in an activity, workshop or similar
Part Of Official Scheme? No
Geographic Reach Local
Primary Audience Postgraduate students
Results and Impact A poster had been presenetd with the following title:

""Immunofluorescent Imaging of RNP Markers in differentiated Neuronal Cell Lines using Super Resolution Microscopy"
Braulio Martinez, Chris Gell, Robert Markus, Ian Macdonald, and Helen Miranda Knight

Images created at the super resolution microscope were included.
Year(s) Of Engagement Activity 2016
URL http://www.nottingham.ac.uk/neuroscience/index.aspx
 
Description Poster at Sheffield STORM Symposium 
Form Of Engagement Activity Participation in an activity, workshop or similar
Part Of Official Scheme? No
Geographic Reach Regional
Primary Audience Professional Practitioners
Results and Impact A poster competition has been taking place at the symposium. We have presented our multi colour STORM data.
"Localization of Proteins of interest in E coli bacteria using
two colour STORM" Robert Markus, Tim Self & Jafar Mahdavi
Year(s) Of Engagement Activity 2017
URL https://sheffieldstorm.wordpress.com/
 
Description Poster at Young Life Scientists' Symposium 2016 - Advanced Light Microscopy 
Form Of Engagement Activity Participation in an activity, workshop or similar
Part Of Official Scheme? No
Geographic Reach National
Primary Audience Postgraduate students
Results and Impact A poster with the following titles had been presented

"Immunofluorescent Imaging of differentiated Neuronal Cell Lines using Wide-Field, Confocal, and Super Resolution Microscopy"
Braulio Martinez, Chris Gell, Robert Markus, and Helen Miranda Knight
Year(s) Of Engagement Activity 2016
URL https://www.bps.ac.uk/news-events/future-scientific-meetings/2016/young-life-scientists-symposium-20...
 
Description Refractive index of the sample - get the best out of your microscpe 
Form Of Engagement Activity A talk or presentation
Part Of Official Scheme? No
Geographic Reach National
Primary Audience Professional Practitioners
Results and Impact Robert Markus, gave a talk on his latest developments on refractive index of the mounting media of samples. This is crucial to obtain the highest error free super resolution microscopy images and accurate data. Audience had useful comments and they had professional and very useful comments. Overall the ideas and the data met positive response therefore more work is invested to write up a manuscript with the aim to publish the technical results which were obtained at our super resolution microscope.
Year(s) Of Engagement Activity 2017
 
Description Talk preseneted at The Microbiology Society Annual Conference 2016, 21-24 March at the Arena and Convention Centre (ACC), Liverpool, UK. 
Form Of Engagement Activity A talk or presentation
Part Of Official Scheme? No
Geographic Reach International
Primary Audience Professional Practitioners
Results and Impact Result obtained using the super resolution microcope were presented in the Prokaryotic Cell Biology Forum on the afternoon of March 23rd in Liverpool at the Microbiology Society annual conference.

"Localizing Autotransporter EspC during secretion to identify potential targets for novel antimicrobials"
Mahmoud Ashawesh, Robert Markus, Christopher Penfold, C. and Kim R. Hardie.
Year(s) Of Engagement Activity 2016
URL http://www.microbiologysociety.org/events/annual-conferences/index.cfm/annual-conference-2016