UK Research & Innovation Future Leader Fellowship awarded to develop a compact super-resolution microscopy technology

The principal investigator of the Nanoscale Microscopy Group, Dr Izzy Jayasinghe, has been awarded a UKRI Future Leader Fellowship to build a new portable imaging technology which will allow scientists, medical doctors, conservationists and industrial parties to visualise the smallest building blocks of any biological sample from any location.

The UK and other governments are currently making urgent investments into understanding the role of molecules and cells in some of the biggest challenges of today’s society which include the effects of climate change on food sources and lifestyle effects on major human diseases and ageing. Despite this urgency, we remain unable to visualise the relevant genes, proteins and cellular components in their natural environments or the geographical locations of the problem. Frustratingly, the technology for visualising such minute structures exists. It is called ‘super-resolution microscopy’ and we even hailed its invention with a Nobel Prize in Chemistry in 2014. However, it has remained beyond the reach of field scientists and clinicians because it has always relied upon specialist skill for its operations and expensive and bulky equipment for its implementation.

In this fellowship, we will use a radically new approach to make super-resolution microscopy portable, cheap and easy to use. We will harness a novel chemical reaction called ‘Expansion Microscopy’ which we have refined and mastered over the last three years (read more about our recent paper about it here). This method allows one to physically inflate a desired feature of a sample, for example a patient biopsy or a small organism, by over a 1000-fold in volume. We will build a set of chemical, biological and physical tools which allows this method to reveal minute cellular details in tissues or organism which were previously too small to be visualised with traditional, laboratory-based, optical microscopes. These developments will be carried out with a view to assemble a miniature super-resolution microscope that is both affordable and portable beyond the laser lab.

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A fluorescence micrograph of a sea-urchin embryo which will be the subject of a field-trial for the portable super-resolution microscopy technology, currently under development. Image credit: Dr Andrea Gaion

To refine and ensure that this device delivers this claimed imaging capability, we will carry out case studies in partnership with experts whose samples are collected outside of the academic laboratory (in Phase II). They include a field scientist who will use it to examine young sea urchins in the UK coast, doctors and sports scientists who will screen for the fine structure of needle biopsies taken in the clinic from human patients, and a member of the Worms in Space programme who will use it to remotely study the effect ‘zero gravity’ on the ageing of microscopic worms sent between earth and the international space station.

Our expectation is that by making super-resolution available beyond the laboratory, one unlocks the benefits of rapid visualisation of sub-cellular structures which underpin the life processes and pathology at a new spacial scale. For field scientists, it would accelerate research programmes; sample collection and high-end microscopic analyses would no longer be mutually exclusive processes. In the clinic, this could unlock faster decision-making.

The fellowship allows us to work more closely with two important industrial partners who have supported us over the last few years, Badrilla Ltd and Cairn Research.

New paper on “Enhanced Expansion Microscopy” and application on 3D and chemical in situ mapping of RyR

fig1.pngWe demonstrate the ability to exploit in-plane resolution of ~ 15 nm and axial resolution of ~ 35 nm by combining X10 Expansion Microscopy with Airyscan 3D imaging.

Expansion Microscopy is the newest of super-resolution imaging methods which allows finer details of samples to be visualised with relatively conventional fluorescence imaging techniques by physically expanding the sample. This is achieved by embedding the samples in an acrylamide hydrogel matrix, crosslinking the fluorescent probes, enzymatically clearing the sample and then osmotically swelling the hydrogel (this paper demonstrates ~ 1000-fold volume expansion). 

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Before and after EExM. Photo credit: Tom Sheard

The enhanced three-dimensional resolution achieved by combining this with Airyscan microscopy (hence, the name Enhanced Expansion Microscopy, or EExM) is better suited than conventional implementations of localisation microscopies, particularly for imaging cell interiors and ultrastructures in cell types with 3D complexity, more so than some of the more popular super-resolution techniques. We demonstrate this by imaging cytoskeletal alpha-actinin lattices and RyR nanodomains in ventricular cardiomyocytes. We go a step further to show how this single-channel resolution can reveal dispersed RyR array structures and the altered single-channel phosphorylation patterns which coincide with the fatal heart pathology – right ventricular failure. To better-understand the functional implications, we have teamed up with Dr Michael Colman (http://physicsoftheheart.com/) to simulate the local calcium signalling events based on the experimentally-mapped RyRs.

This is the first research paper for Tom Sheard, and marks the first home-publication for the Nanoscale Microscopy Group together with a multi-lateral collaboration. Well done, everyone! This work was funded by the MRC DiMeN and Wellcome Trust Seed Award.

Access the paper at https://pubs.acs.org/doi/10.1021/acsnano.8b08742

Tom Sheard reporting on doctoral research placement at the University of Auckland, New Zealand

Screen Shot 2018-12-07 at 22.33.56In October 2018 I flew across the world to the laboratory of Dr David Crossman at the University of Auckland in New Zealand. The goal: to study pathological remodelling in human heart biopsies from patients with idiopathic dilated cardiomyopathy (IDCM), using the super-resolution imaging technique expansion microscopy (ExM).

Our interest lies in seeing whether nanodomain remodelling observed in a rat model of heart failure in Leeds, including reorganisation of the internal calcium compartments and functional modification to calcium-handling proteins, is also present in end-stage human heart failure. Understanding the mechanisms of remodelling is one of the first steps towards investigating whether they can be targeted for preventative therapies.

ExM is novel imaging technique, enabling super-resolution imaging by spatially separating fluorophores within a swellable hydrogel. The compatibility of ExM gels with standard microscopes enables greater imaging depth and improved axial resolution over competing super-resolution techniques. ExM therefore provides a practical tool to observe remodelling within dyadic calcium release clusters. I was responsible for starting ExM experiments from scratch in a new lab across the world, requiring efficient independent work to obtain meaningful data in the space of just 4 weeks.

It was fantastic to take this journey and work in a laboratory that is home to a strong consortium of leading cardiovascular researchers. In my final week I gave a 30-minute seminar, in which I presented work to the physiology department and the wider bio-imaging facility. This allowed me to reach an international audience and receive valuable feedback on the progression of my research.

Many thanks to the MRC and DiMeN flexible fund grant which made this trip possible, and special thanks to David Crossman for welcoming me into his lab.

Follow Tom on Twitter: @TMDSheard.

New review out now: Shining New Light on the Structural Determinants of Cardiac Couplon Function

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Our new review article to mark 10 years since the first super-resolution imaging experiments on cardiac muscle: 
Abstract: Remodelling of the membranes and protein clustering patterns during the pathogenesis of cardiomyopathies has renewed the interest in spatial visualisation of these structures in cardiomyocytes. Coincidental emergence of single molecule (super-resolution) imaging and tomographic electron microscopy tools in the last decade have led to a number of new observations on the structural features of the couplons, the primary sites of excitation-contraction coupling in the heart. In particular, super-resolution and tomographic electron micrographs have revised and refined the classical views of the nanoscale geometries of couplons, t-tubules and the organisation of the principal calcium handling proteins in both healthy and failing hearts. These methods have also allowed the visualisation of some features which were too small to be detected with conventional microscopy tools. With new analytical capabilities such as single-protein mapping, in situ protein quantification, correlative and live cell imaging we are now observing an unprecedented interest in adapting these research tools across the cardiac biophysical research discipline. In this article, we review the depth of the new insights that have been enabled by these tools toward understanding the structure and function of the cardiac couplon. We outline the major challenges that remain in these experiments and emerging avenues of research which will be enabled by these technologies.
Read the full text using this link

New review chapter: “Advances in the Visualization of Molecular Assemblies Within Cellular Signaling Nanodomains: Insights From a Decade of Mapping of Ryanodine Receptor Clusters”

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Nanodomains are naturally assembled signaling stations, which facilitate fast and highly regulated signaling within and between cells. Calcium (Ca2+) nanodomains known as junctional membrane complexes (JMCs) transduce fast and highly synchronized intracellular signals, which are required by a variety of cell types. Common to most such nanodomains are clustered assemblies of the principal intracellular Ca2+ release channels, ryanodine eceptors (RyRs). JMCs found in cardiac muscle cells have been studied extensively as self-assembled clusters of RyR. While known to form crystalline arrays in vitro, the organization of RyRs in situ within the JMCs has been less clear. The development of single-molecule localization microscopy (SMLM or super-resolution) optical methods have transformed our ability to visualize and accurately quantify the spatial geometries and sizes of RyR clusters. The recent application of the novel DNA-PAINT super-resolution technology has exploited an unprecedented optical resolution of 10–15 nm to visualize the natural arrays of RyRs within JMCs. In this chapter, we review the key insights into the in situ RyR assembly within cardiac nanodomains that have been gained over the last decade with the utility of super-resolution microscopy and the major considerations in interpreting and validating such image data.

To request a preprint version of the chapter, please contact the authors via Researchgate.

Full text of the chapter can be accessed via this direct link.

 

Showcasing the recent and ongoing DNA-PAINT experiments at the Edinburgh Super Resolution Imaging Consortium Symposium

Isuru attended the second annual Edinburgh Super Resolution Imaging Consortium (ESRIC) symposium, held this year at the Institute of Genetics and Molecular Medicine (IGMM) of the University of Edinburgh (UoE). His talk on the Molecular-scale imaging of ryanodine receptors at both the cell surfaces and interiors with the adaptation of DNA-PAINT was well-received by a range of researchers based in Edinburgh and regionally in Europe.

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Highlights from this meeting included a number of world class investigations led by research fellows and academics in UoE and Heriot Watt University. Of note, were Dr Colin Rickman’s talk on using naturally occurring enzymes as super-resolution imaging probes, Dr Lynn Paterson’s adaptation of optofluidic devices and optical tweezers for developing novel optical tools for cell biology. The plenary speaker was Prof Christophe Zimmer (from Institut Pasteur) who spoke about the adaptation of artificial neuronal networks (a tool called ANNA-PALM) to speed up super-resolution microscopy and demonstrate high throughput imaging of structures such as microtubules, nuclear pore complexes and mitochondria. We now eagerly anticipate his paper on ANNA-PALM out in press very soon.

The conference was organised by Dr Ann Wheeler and colleagues of the ESRIC and showcased their world class line up of microscopy platforms including a state-of-the-art Nikon STORM and SIM instrument and a Leica STED system.

Our research featured on the news of local TV station: ‘Made in Leeds’

On the back of our recent publication in Cell Reports, our research has enjoyed a wide ranging body of TV coverage. This included 20+ online newspapers and science & technology websites. Among this coverage, was a brief recording for the local television station Made in Leeds which was featured on the 6:30pm news on 11/1/2018. 

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Here is the link to a clip where Isuru is explaining the context and the value of the super-resolution microscopy technology in studying both healthy and disease physiology of the heart. Featured in the video, was Miriam during one of her imaging experiments.