A new correlative super-resolution protocol to visualise cellular function and structure

Super-resolution microscopies are commonly utilised to obtain nanometre-scale information regarding the structures within a cell. This information is rarely accompanied by any functional detail, a key requirement when observing a cellular system. The most recent publication from the applied biophotonics group describes the development of a correlative super-resolution protocol. This protocol enables the fast second-messenger signalling of primary cell types to be visualised in regard to a cell’s underlying structure.

The protocols focuses on the principle of examining calcium sparks in living cells (main panel, left), and then aligning the image frames to the corresponding DNA-PAINT super-resolution images of the underlying clustering patterns of the Calcium release channels, the ryanodine receptors (magnified inset, right)

Application of the correlative super-resolution protocol has been undertaken across various cell types and disease models throughout Miriam Hurley’s PhD studies. Specifically, the group has applied the protocol to study the movement of calcium (Ca2+) against the nanoscale patterns of the ryanodine receptor, a Ca2+ release channel which gives rise to these Ca2+ signals within wildtype primary cells.

The protocol relies upon the combination of total internal reflection fluorescence (TIRF) imaging of the elementary Ca2+ signals with subsequent DNA-PAINT imaging of the RyRs. The publication outlines a straightforward image analysis protocol of feature extraction and image alignment between correlative datasets to demonstrate how such data can be used to visually identify the ensembles of Ca2+ channels which are locally activated during the genesis of cytoplasmic Ca2+ signals.

This work has formed the basis of Miriam’s PhD studies, funded by the Leeds Anniversary Research Scholarship and the Royal Society Research Grant (first research grant awarded to PI, Izzy Jayasinghe).

Access the full article at: https://doi.org/10.1016/j.ymeth.2020.10.005.

Nanoscale Microscopy Group celebrate Physiology Friday

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On the 12 October, Miriam ran an event funded by The Physiological Society to promote Physiology Friday at The University of Leeds. The aim of this outreach event was to raise awareness of how technological advancements in microscopy are improving our understanding of how human physiology can change within health and disease at the cellular level, specifically related to the heart. 

Within research, the knowledge of our own physiology in health and disease has developed in part from the advancement in microscopy, which has enabled the visualisation of single proteins. This advancement can be likened to the difference between an individual who is legally blind compared to someone who has 20/20 vision. Our event utilised visual impairment glasses which distorted an individual’s vision. Participants were asked how well they could resolve the outline of a structure in the absence and presence of the glasses to convey the significance of the development which has occurred within microscopy. Supplementary posters led individuals through the physiological relevance of this technological advancement, with images detailing the single protein structure within the heart. With the use of an anatomical heart model, the function and structure of the heart within health and disease was explained. We communicated how current research is using a super-resolution version of microscopy to characterise how a change in function during heart failure can be due in part to a change in the protein structure within a heart cell. To help convey the scale at which the cellular remodelling within heart failure is occurring, real-life examples were used. For example, if a person was tall enough to reach a plane flying at 30,000 feet then the proteins that we are imaging would still be smaller than the width of that person’s hair. Smartphone microscopes were available to allow individuals to experience first-hand the ability that a microscope has to study an object at a more detailed scale compared to what can be seen by the human eye alone. 

Over the course of the day a dialogue was created with students from all disciplines. This sparked a variety of questions on the topic of physiology; what is a heart attack? What happens to the proteins within your heart during heart failure? The event even led to one student exclaiming that “microscopes are so cool”, with another stating “I learned how much I need to take care of my body”. 

Many thanks to The Physiological Society who funded and supported this event – now to start planning the next one! 

Miriam Hurley writes for Physiology News

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In June 2018 Miriam attended a Public Engagement Training Day which was run by The Physiological Society. Building upon the skills learnt at this event Miriam gained funding from the Society to organise and run an event at The University of Leeds to promote Physiology Friday. Her reflections upon this training day can be read on Page 47 in the Winter 2018 Issue of Physiology News which can be accessed here.

Three Minute Thesis Competition held at The University of Leeds

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Miriam recently entered the ‘Three Minute Thesis’ competition held at The University of Leeds. A video of the presentation has since been uploaded to the conference YouTube channel which you can access here. A particular highlight of this competition for Miriam was hearing fellow researchers from a variety of disciplines communicate the wide-ranging impact of their research. The other presentations on the channel are a wonderful demonstration of this.