An upright confocal microscope for multidisciplinary research

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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.