An upright confocal microscope for multidisciplinary research

The desire to visualise cellular structures and processes has been a central aim of biologists ever since the development of the light microscope and the advances in cell biology are intrinsically linked to the advances in microscope technology. The development of synthetic fluorescent probes made it possible to visualize the location of individual proteins and complexes within the cell and the development of different coloured probes allowed multiple proteins to be studied at the same time. This gives an insight not only into the localization of the proteins within the cell but also their interactions with other proteins. Along with this, the development of the laser scanning confocal microscope, using a detection pinhole to reject out of focus light, allowed researchers to see these fluorescent probes inside thick samples and obtain a "free from blur" optical section and three-dimensional model of the sample.

The ability to visualize protein dynamics in live cells was made possible with the discovery and subsequent sequencing of the green fluorescent protein from jelly fish. Using genetic engineering it is possible to form a protein chimera in which a protein of interest is fused to this fluorescent protein. The now fluorescently tagged protein of interests can then be expressed and observed in live cells. With the development of coloured variants of the jelly fish protein (and coral proteins), it has become possible to follow multiple different proteins inside living cells, tissues and even whole organism and has allowed researchers to use optical approaches to gain an understanding of how proteins interact, how cells communicate and how cells and tissues react to the their external environment.

Microscopes can come as essentially two models with respect to access to the sample to be imaged. An inverted model accesses the sample plated in dishes from the bottom and high resolution imaging needs imaging through thin transparent surfaces such as glass. An upright microscope accesses samples placed in dishes directly from the top with the use of lenses that can be "dipped" (hence dipping lenses) in the culture media. The use of this approach enables imaging of samples without additional interface which can be thick or opaque etc. Such approach allows imaging of samples that grow in a three-dimensional environment resembling their natural in vivo environment. Such environments can be original tissue or in-vivo like engineered biomaterials. These new developments provide a realistic insight into the role of how cells behave in their environment in health and disease, and an upright confocal microscope provides the ideal platform and critical for imaging cells under such modified environments.

Whist we currently have a Leica SP5 upright confocal microscope, it is at the end of its useful life and lacks sensitivity which is critical for imaging combined with new other technologies such as CRISPR, where single copies of fluorescently labelled protein genes are targeted to specific locations within the genome of cells and organisms. Although this targeted approach offers enormous potential for understanding the role of individual proteins in the cell, their level of expression is often so low that the resulting fluorescent signal is very weak. The latest generation of upright confocal microscopes provide the ability to perform these sophisticated multi-colour microscope experiments even on thick samples due to their improved light efficiency and detector sensitivity.

Here we propose to replace our old out-dated upright microscope with a new state-of-the-art Leica SP8 upright confocal microscope. This will allow improved delivery of a core service to a productive set of around 67 well-funded research groups who heavily use the current instrument and will provide them with access to a system with improved flexibility, improved sensitivity and improved resolution.

The existing Leica SP5 upright confocal in the Faculty of Biology Medicine and Health is heavily utilised but it is out-dated. Sample thickness, dimensionality or opaqueness often requires direct optical access to the sample and this is not possible on existing inverted microscopes. As the use of samples embedded in bio-materials and tissues increases, so does the demand for access to a state-of-the-art upright confocal system.

To meet this demand we propose the purchase of a new Leica SP8 upright confocal system equipped with a tunable white light laser, dipping objectives, spectral imaging, the latest hybrid (HyD) detectors and triggering of ancillary components. The tunable white light laser and spectral detection sliders allows the excitation and emission wavelengths to be matched exactly to that of any fluorophore, while the hybrid (HyD) detectors are twice as sensitive as traditional PMT detectors. These optical advances are increasingly important as we use technologies such as CRISPR to label single copies of endogenous genes, resulting in lower fluorescent signals, as well as the need for better multiplexing and spectral discrimination.
The dipping objectives provide direct optical access to cells in three-dimensional tissues and substrates and will also enable us to image cleared tissue, a technology that is gaining interest and already used with light sheet microscopy in Manchester. A significant group of users require the use of the upright confocal along with ancillary equipment such as fluidic devices and force measurement on custom-made stages.

This upright confocal will support a wide range of interdisciplinary science that lies within the BBSRC remit. Supporting this, the group of applicants and users of the microscope have more than 30 current BBSRC grants and >£50m current funding. Work on the microscope will span from bacteria, zebra fish, Caenorrhabditis and Drosophila model systems through to mouse and human cells and tissues.

This application requests an upright confocal microscope that will support the research of a large number of users whose projects address biological, physical, and medical questions. Thus, there are a wide range of direct and indirect beneficiaries of this research:

(1) Biotechnology. The data resulting from the use of the microscope contributes to the research that will reach a broad audience across disciplines including biomedical sciences, biophysics and tissue engineering. Such research automatically leads to the development of Biotechnological tools. Tools will range from cell lines stably expressing fluorophore-tagged proteins (that may become valuable for the screening of materials and drugs affecting cellular behaviour) to the development of new reagents and devices that promote imaging or other methodologies. We expect a high potential impact in the biotechnology area and will actively search for relevant systems/companies to share our knowledge. The impact will be direct and mid-term.

(2) Pharmaceutical industry. A number of projects in the Faculty relate to cell behaviour under changing mechanical and biochemical environments. Unravelling the mechanisms through imaging will provide a starting point for the development of pharmaceutical products influencing cellular responses to changing environments in healthy or diseased tissue. Modulating cell responses to changing properties is designed to promote regeneration. The new Faculty structure will support this undertaking. The impact will be direct and mid- to long-term.

(3) General public. Images generated from this project are colourful, intuitive, attractive and make science more accessible. They are useful for educating the public, and particularly children through school lectures, about science. As a nexus between different types of research the Bioimaging Facility will help focus on how disciplines can be integrated to deliver tangible benefits for society, in terms of finding new ways to understand and treat disease as well as to develop new material. Promoting health processes will improve life quality of thousands of people in the UK and beyond the borders. Furthermore, it will drastically reduce treatment costs, thus directly and indirectly impacting the healthcare system. The impact is indirect and can range from short- to long-term.

(4) Researchers of various backgrounds. The Bioimaging Facility with its imaging tools being key for a large variety of different projects across biology, medicine and physics promotes links between interdisciplinary research. The development of novel methods and experimental approaches is equally relevant for biosciences and engineering. Data analysis tools that have been, and will be, developed will be shared by all researches. Accordingly, scientists working in any of those areas might be highly interested in the state of the art imaging equipment. The impact will be direct and immediate.

(5) Staff working on the project. Researchers will work interdisciplinary, interact with many scientists of different backgrounds and companies and creatively solve problems. They will further develop communication, problem solving and entrepreneurial skills and acquire new technical and IT skills, which will be useful in any later profession.