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High Speed super-resolution confocal laser scanning microscope for sub-diffraction analysis at the multi-user Leicester Advanced Imaging Facility

Abstract

We aim to install a high speed super-resolution confocal laser scanning microscope (CLSM) for sub-diffraction analysis at the multi-user Leicester Advanced Imaging Facility (AIF) for both specialist and non-specialist users. At Leicester AIF, there are currently three CLSMs, which are heavily used. These systems are >10 years old and lack an option to be upgraded for sub-diffraction imaging analysis, which has quickly become a standard in cell biology. There is a strong demand from the current CLSM users at Leicester and their collaborators, including industrial partners to obtain a super-resolution CLSM. A Zeiss 880 CLSM with Airyscan Fast (subject to tender process) will fulfil these requirements, combining super-resolution confocal imaging with increased speed and sensitivity. The key feature of the Airyscan Fast technology is that a 32-pixel sensor is used instead of pinhole found in traditional confocal microscopes. The 32 pixels are arranged in circular symmetry coaxial with the point spread function (airy disk), and each pixel, in essence, serves as an independent pinhole probing its region of the point-spread function (airy disk) of the image. As a result, the system becomes more photon-efficient (as is the case with a fully open pinhole in a traditional confocal microscope), without sacrificing the spatial resolution. The system is packaged in a compact, plug-and-play, user-friendly manner. The system allows imaging of both fixed samples and live cell imaging. The improved resolution in this system is obtained at any magnification, resulting in a general improvement of the images for all the users. The system is designed for multicolor samples of standard chromophores, thus, no special requirement is needed for sample preparations. All together, these features of the system fulfill requirements for the Leicester AIF to provide up-to-date high quality and user-friendly imaging capability to a wide range of users who conduct research within the BBSRC remit.

Summary

Optical imaging of molecules, cells, tissues and whole organisms brings invaluable information when we address biological questions. It is literally "seeing is believing", if we are interested in finding out about microscopic events in a cell/tissue as well as their quantities and dynamic behaviour that will help us to understand their biological roles. For example, if they are found on the cell surface membrane, their role may be relevant to cell membrane function. If two proteins are found at the same site in a cell, they may work together to deliver a cellular role. If these proteins are missing in disease cells/tissues, restoration of these proteins may be an effective therapeutic strategy. To highlight the locations of the molecules of interest, we need to "mark" the molecules. Fluorescent markers that associate with the target molecules have revolutionized our ability to study cellular and tissue developmental and physiological processes. They can be used in both fixed samples and in live-cells. These markers are detected by optical microscopes, where a specialised system called Confocal Laser Scanning Microscope (CLSM) has been playing a vital role. CLSMs allow us to collect signals only from the focal plane, excluding out-of-focus light using a small aperture in front of the detector. Therefore, we can look inside cells and tissues without physically cutting them into sections and look at specific molecules floating in solution without getting masked by surrounding excess of other molecules. CLSMs have a flexibility to change the pixel resolution of the image by modulating the pixel setting or zooming into a smaller area of the cell. This means that both large tissue samples and tiny bacterial samples can be analysed by CLSMs. The quality of images are dependent on their "resolution", which is defined as the minimal distance between two points in the sample that can still be distinguished by the detector (our eyes or a camera) as separate points. Resolutionof conventional fluorescence microscopy is limited to about 200 nm, which is termed the "diffraction limit". In the last two decades, novel methodologies have brought substantial improvements to the resolution, which can become 50-120 nm, dependent on methodologies. This major breakthrough in cell biology was awarded the Nobel Prize in Chemistry in 2014 "for the development of super-resolved fluorescence microscopy". These microscopes can be called super resolution microscopes (or nanoscopes as they offer nanometre resolution). The information provided by super-resolution microscopy is unique and cannot be obtained by any other means. For example, high resolution imaging by electron microscopy (EM), where resolution can be now down to a few Ånstrongs cannot replace super-resolution microscopy as EM cannot use fluorescent markers and alternative methodologies to localise a protein of interest in relation to the observed structures are limited. Researchers are aware of this issue and actively working on it but methodology development to precisely "mark" proteins of interest with specialised tags is still ongoing. Therefore, a question such as whether protein two proteins are co-localising at a particular cellular structure or not, needs to be addressed by super-resolution microscopy. Unsurprisingly, super-resolution microscopy has quickly become very popular in the cell biology research field. An improved resolution by a factor of 1.7 to 2 (about 100-120 nm) has become the new standard in cell biology. CLSMs with super-resolution capability are commercially available. These super-resolution CLSMs can come with a detector for improved sensitivity and speed, allowing imaging of live cells where the target molecules may make dynamic movement. By installing one of these super-resolution CLSMs at the multi-user Leicester Advanced Imaging Facility, we aim to promote world-class cell biology.

Impact Summary

The proposed project aims to incorporate a state-of-the-art confocal laser scanning microscope (CLSM) with a resolution beyond the diffraction limit and improved sensitivity into the existing Advance Imaging Facility (AIF) at the University of Leicester. The proposed system not only improves the x and y resolution by a factor of 1.7 but also improves z-resolution making 3D localization more accurate. The introduction of nanoscopy has resulted in a stream of new discoveries and it is to be expected that this near-doubling of spatial resolution will also accelerate the generation of high-impact discoveries by the researchers using the AIF. It will support already BBSRC funded research within four BBSRC strategic priorities: Food Security, Fundamental Cell Biology, Bioscience for Health and Structural Biology. By understanding the underlying cellular mechanisms it is possible to design, synthesize and test biologically active compounds and specific tools to treat many diseases. Some of the diseases that are being investigated through BBSRC funded research at Leicester including cancer and neurological diseases such as Alzheimer's as well as Pain relief (Opioids). Also Agri-tech, understanding of animal and bacterial molecular mechanisms will feed knowledge into the farming industry, sustainable plant based product development, crop enhancement and animal vaccine development through existing collaborations with Leicester. The imaging undertaken on the requested CLSM technology and is expected to bring impact to three main areas, academic, commercial and societal (public engagement). Academic impact In addition to the 20 academic applicants in this proposal that have stated a need for the CLSM microscope system to enhance their research that is within the BBSRC remit, the facility will be open to all users at the university including PI's, post-docs, PhD students, technicians and undergraduates as well as the other Midlands Innovation universities (Birmingham, Aston,Cranfield, Keele, Loughborough, Nottingham and Warwick). As part of their ongoing research programs all applicants and users will present work at National and International meetings, publish academic papers and enter biological images competitions. Industrial Impact A number of applicants have BBSRC funded projects involving industry partners, covering both pharmaceutical and Agri-tech industries as well as bodies such as Public Health England, the Animal and Plant Health Agency and the Defence Science and Technology Laboratory. Through these existing partnerships, new projects will be forged that utilise the new CLSM imaging technology. When combined with existing technology available at UoL, such as the cryo-electron microscope facility, this creates an impressive offering to external partners. Thus further collaborative research projects will be developed and new partnerships explored. Societal Impact Informing the general public is a key aspect in engaging the public with the need for scientific research as well as informing them of new breakthroughs in both science and medicine. The CLSM will produce fascinating imaging that will inspire and interest the public. We intend to develop a series of posters showing the beauty of microscopy and potentially preclinical imaging for public engagement activities and art expositions. The college of Life Sciences has a number of ongoing activities to promote their research to the public, both adults and children, which will be utilised to share research and images obtained using the CLSM. These activities will be supported by the Public engagement manager, who will provide advice and guidance to researchers to develop public engagement programmes that support their research. In addition, press releases will be generated to increase public interest in the research images.
Committee Not funded via Committee
Research TopicsX – not assigned to a current Research Topic
Research PriorityX – Research Priority information not available
Research Initiative Advanced Life Sciences Research Technology Initiative (ALERT) [2013-2014]
Funding SchemeX – not Funded via a specific Funding Scheme
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