Spinning disk microscope for Wolfson Light Microscopy Facility

Lead Research Organisation: University of Sheffield
Department Name: Biomedical Science

Abstract

How components interact within cells is key to how cells behave and this in turn affects the organisation of tissues and ultimately the whole organism. Seeing how these interactions occur is key to our understanding of how a healthy organism forms and continues to stay healthy, and what happens when things go wrong in disease. Often visualisation of biological events causes us to formulate new hypotheses. Researchers thus use microscopy extensively to understand how cells, tissues and organisms function. Frequently, fluorescent labels are attached to components of interest which allows them to be observed in living cells. The time-scale of biological processes is highly variable: some processes happen very quickly, meaning that images need to be acquired speedily while others occur over a much longer time-scale of minutes to hours. There are a number of challenges to capturing fluorescence images because the exposure of cells to light can cause phototoxicity which damages cells and causes aberrant behaviour. Additionally, some fluorescent labels can be quite dim which makes the fluorescent signal difficult to detect.
The Wolfson Light Microscopy Facility (LMF) at the University of Sheffield houses a range of microscopes that are optimised for particular applications. Spinning disk confocal microscopy allows imaging of fast dynamic events in 3 dimensions. Our current spinning disk confocal microscope is extensively used to understand how normal physiology is regulated, and what changes as organisms age. It is also used in experiments with model organisms such as zebrafish and fruit flies which help us to understand how the immune system works. Our existing system is however now greater than 12 years old and is vulnerable because the original parts are no longer supported by the manufacturers. The aim of this proposal is to install a replacement spinning disk microscope. This will not only accommodate our existing needs but will open up a range of exciting new possibilities because the new generation spinning disk confocal microscopes offer significantly enhanced performance in terms of speed, sensitivity, and low phototoxicity. These advantages, coupled with the ability to capture wide fields-of-view, mean that this is a very powerful instrument which will have a wide range of uses within the LMF. We have identified projects ranging from cell and developmental biology, immunology and microbiology which will benefit from the acquisition of a state-of-the-art spinning disk confocal microscope.

Technical Summary

Spinning disk technology images fast, dynamic biological events in 3 dimensions. Based on a wide-field architecture, spinning disk (SD) confocal systems are relatively straightforward to operate, making them an ideal high-resolution live-imaging tool for a core imaging facility such as the Wolfson Light Microscopy Facility. We are seeking funds for a state-of the-art SD confocal microscope as a replacement for our existing system which is now over 12 years old. This system is extensively used and, because of its age, it is now vulnerable to breakdown and replacement parts are no longer supported by the original manufacturer.

This system will be used for fast, high resolution, 3-D imaging of live cells, tissues and organisms. The latest SD microscopes offer significant advantages over our existing system. The new systems are faster and more sensitive. SD microscopy also has many advantages over point scanning confocal imaging, including Airyscan. The cameras used in new SD systems have much higher quantum efficiencies (95%) than the PMTs (15%) or avalanche photodiodes (40%) of confocal systems, which ensures that the emitted signal is collected more efficiently. SD systems therefore have comparably shorter exposure times, can image at higher frame rates, with less photobleaching. Such advantages are specifically beneficial for live imaging, especially for the imaging of sensitive samples over extended periods of time. These advances, coupled with the flexibility of the new systems to image multiple samples and multiple different types of samples, will allow us to image biological processes in unprecedented spatial and temporal detail, allowing us to address a range of fundamental questions in cell and developmental biology, immunology and microbiology.

Publications

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