Light sheet microscopy for live cell imaging of photosensitive systems

Lead Research Organisation: Marine Biological Association
Department Name: Marine Biology

Abstract

Researchers at the Marine Biological Association (MBA) have a long standing reputation for excellence in the use of microscopy to study marine organisms. The ability to observe the unusual and novel physiology of these organisms has led to many significant discoveries. The new microscopy facility requested in this proposal will allow MBA researchers to examine cellular processes at unprecedented levels of resolution, with minimal stress on the cells or organisms under study, providing a world-class facility for future research into cell biology of marine organisms.

Research at the MBA relies on advanced fluorescence microscopy techniques to study marine model organisms, using fluorescent dyes and proteins to view where specific proteins are found in a cell or to directly image cellular processes such as changes in intracellular pH or calcium, which are central to many signalling pathways. Other aspects of MBA research use model organisms to study how cellular processes have evolved or may operate in more complex multicellular organisms. For example, we use algae to study the structure and function of flagella, as many aspects of flagella are highly conserved with the cilia found in the human body. Marine phytoplankton are tiny algae that represent the base of the food chain in our oceans, supporting fishing and seafood industries, but despite their importance to life on our planet we still understand very little about them. Many phytoplankton surround themselves with intricate extracellular coverings made from silica or calcium carbonate. We aim to understand how and why these algae produce such intricate and ornate cell coverings to aid the design of similar structures for use in biotechnology and biomedical applications. Other research uses marine organisms from lineages that diverged at the very base of the animal tree of life, such as sea anemones, to examine how complex regulatory processes involved in embryo development evolved in multicellular animals. The marine fungi represent another group of organisms that are poorly understood. Recent research has highlighted that fungi are widespread in marine environments, suggesting that they are likely to play similar roles in nutrient recycling and as pathogens as the terrestrial fungi. We are developing model species of marine fungi for laboratory studies, so that we can understand much more about their physiology and their potential roles in the environment.
Fluorescence microscopy of very small cells or structures requires that the subject is illuminated with a bright excitation light, which for many samples can be damaging. This is particularly relevant for photosynthetic organisms (such as marine phytoplankton) or for long term studies (such as time lapse imaging of biomineralisation or embryo development). MBA researchers are therefore currently limited in their ability to study important aspects of their model organisms. This proposal aims to address these limitations through the acquisition of a light sheet microscope. Light sheet microscopes are very effective at reducing damage from excitation light because they illuminate the sample with a very thin layer of light from the side, rather than passing light directly through the sample from above or below. Illumination from the side also has the effect of increasing the resolution of the microscope as there is no interference from out of focus areas of the sample. Light sheet microscopy has been shown to be particularly effective for long term time lapse imaging of photosensitive organisms for extended periods and therefore it is an ideal solution to the problems currently faced by MBA researchers.

The new facility will be used to support the ongoing research of the current research teams, with interests from marine fungi and algae through to marine animals, and further afield. The resource will therefore underpin many diverse avenues of research and therefore represents excellent value for money.

Technical Summary

Our research relies on live cell fluorescence microscopy of marine model organisms to view the movement of proteins or organelles, or to examine fluorescent biosensors for intracellular pH and calcium. An inherent issue with live cell microscopy is phototoxicity from the excitation light. This is particularly the case for photosensitive organisms (e.g. photosynthetic marine phytoplankton) or for long term studies (e.g. time lapse imaging of biomineralisation and embryo development). The existing widefield and confocal imaging facilities at the MBA are therefore sub-optimal for many applications, limiting the ability of MBA researchers to study important aspects of their marine model organisms.
We therefore need to supplement our imaging facilities with a new state-of-the-art digital light sheet microscope facility that provide unprecedented research opportunities for many years to come and will allow significant technical advances to be made in cellular imaging of marine organisms.

The requested combined confocal and digital light sheet microscope will allow long term, low photoxicity live cell imaging of samples. However, the light sheet illumination also offers many advantages over widefield and confocal microscopy for fast imaging at high-resolution, such as the ability to image sub-cellular Ca2+ elevations. Light sheet illumination will therefore allow MBA researchers to apply new approaches that are not currently possible. The Leica SP8 DLS also provides confocal illumination with super-resolution capability, which greatly enhances our existing confocal facility. The ability to combine light sheet illumination with confocal on a single instrument opens the way for further novel approaches, such as photoactivation or FRAP applications. While the microscope will be used extensively by MBA researchers, we aim to make the facility broadly available to the wider scientific community studying the cell biology of marine and other BBSRC-relevant model organisms.

Planned Impact

The primary beneficiary of the research supported by this equipment will be the scientific community. The research has wide-ranging implications for multiple research fields that address human health (for example the research will be of direct interest to end-users associated with human genetic disorders resulting from ciliary dysfunction). The work also addresses issues around food security since several aspects of the research described are relevant to understanding biological processes that underpin marine primary production that supports global fisheries and the biology of toxic algae. Scientific beneficiaries also include those directly involved in cellular signalling processes through to those engaged in broader research areas through the development of novel imaging tools and techniques. The scientific community will also benefit through access to the equipment to BBSRC-funded researchers and those engaged in BBSRC-relevant research funded by other organisations.
The general public will benefit from the increased dissemination of knowledge and information about the microbes living in the sea. These are fundamental to life on Earth though the perception of their importance is often overlooked by the public, particularly in comparison with the larger more charismatic marine animals. The microscopic visualisation of marine protists, many of which display stunning morphologies, provides an ideal mechanism for conveying information and theories around evolution, diversity, cellular function and biotechnology.

We will ensure that the impact of this research is delivered through a variety of mechanisms and activities:

Firstly, through engagement with scientific groups and networks, such as The Ciliary Interest Group, the BBSRC Phyconet Network, The European Marine Biological Resource Centre (EMBRC) and The British Beekeepers Association

Secondly, through running advanced research workshops that will fully utilise the proposed equipment. These include:
Microelectrode Techniques for Cell Physiology, a 2-week BBSRC-funded workshop (BB/P003923/1) that has run annually for 34 years and continues to be substantially oversubscribed. The Workshop provides intensive hands-on and theoretical training to postgraduate and postdoctoral researchers in state of the art single cell physiological approaches and live cell imaging. The Workshop would benefit tremendously from the high-resolution live cell imaging facility requested in this proposal. The Workshop is jointly organised by Dr. D. Ogden (ENS Paris and Crick Institute) and the PI (CB).
The Plymouth Microscopy Course. This annual 12-day workshop was established in 2003 and provides intensive practical and theoretical training in cutting-edge optical microscopy applications. The Workshop is jointly organised by Drs. Brad Amos and Alex Sossick (LMB, Cambridge), Prof Gail McConnel, University of Strathclyde) and the PI (CB). This Workshop has run as an EMBO Practical Workshop from 2006 to 2018 and is funded for 2019. Aplications for ongoing funding are pending.

Thirdly through active public engagement, including through our very active knowledge exchange and public outreach programme. For example, In addition to our normal frequent outreach activities, we will establish a freely accessible web-based interactive image database of "marine microbes and their cellular structures". The MBA is also a Learned Society with a Royal Charter and a membership of more than 1500. Members regularly receive newsletters and updates on knowledge exchange activities.

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