A digital light-sheet microscope for the analysis of biological processes under almost natural conditions

Biological imaging has been moving consistently towards experimentation employing increasingly physiologically relevant systems for many years, and new technologies are making such experimental approaches progressively feasible. Such systems, comprising whole model organisms, tissue explants, and three-dimensional (3D) cell cultures, must be imaged in a way that minimises perturbations in order to maintain their physiological integrity as experimental models. Light-induced photodamage and phototoxicity have long been challenging in the field of biological imaging and can have quite a dramatic effect on health and function at all biological levels of organisation. Imaging such large and sensitive specimens thus demands an efficient approach to 3D imaging that lessens exposure of the sample to light.

The light-sheet microscope stands for a planar illumination technique that has revolutionised how optical imaging of biological specimens can be performed. Basically, the light-sheet technique is made possible by decoupling the illumination and detection optical pathways, allowing for novel illumination strategies that optimise the light exposure and signal collection efficiency. With light-sheet microscopy, lower illumination intensities can be used in combination with planar illumination to provide improved signal with minimal sample light exposure, enabling imaging with high frame rates and over long periods of time. Fields that have already benefited considerably from such an imaging modality include developmental biology, neurobiology, drug discovery, plant biology, and beyond.

However, custom-made light-sheet microscopes can be very difficult to use, usually requiring two or more objectives, non-standard sample preparation protocols, difficult alignment procedures, and a well-planned workflow for dealing with the massive amounts of data generated by such techniques. Our aim is to purchase a commercial light-sheet microscope that is the simplest, most intuitive and most reliable system available that in combination with a straightforward data handling and analysis platform can empower researchers to investigate biological processes under almost natural conditions.

Recording physiological function in multiple dimensions and across scales often defies the speed performance of conventional microscopes. Point scanning microscopes such as standard confocal or two-photon microscopes can only record one pixel at a time, resulting in pixel rates below 1 MHz. Yet, much higher pixel rates are required to capture fast processes, including beating heart development. In addition, high-speed imaging is required for the multiscale investigation of slower processes, such as cell migration. The past decade has seen the emergence of the light-sheet microscope as the most potent technique for fast, volumetric imaging of live tissues and whole organisms. The ability to carry out fast, 3D optical sectioning with an extended field of view and limited photodamage is a unique advantage of light-sheet imaging.

We propose to purchase the digital light-sheet microscope LEICA TCS SP8 DLS and establish a multiuser facility for volumetric imaging and analysis open to all researchers at Imperial College London and beyond. The system is uniquely suited for high-resolution volumetric imaging of large and sensitive specimens such as embryos, organoids or plants, as the light-sheet module is fully implemented into the well-established LEICA confocal platform. Uniquely, analysed specimens can be readily retrieved from the imaging chamber and subjected to further analysis to model essential dynamics and causal mechanisms of biological and homeostatic function. Currently, Imperial College London lacks an open access commercial light-sheet microscope, restricting the scientific community to using confocal microscopes even though they suffer from higher phototoxicity and slower acquisition speeds. The high speed, low phototoxicity and high tissue penetration of the light-sheet microscope will drive scientific discovery and advance knowledge into mechanisms that maintain health and wellness under stress and biological or environmental challenge.

The installation of the LEICA TCS SP8 DLS in the open access FILM at the South Kensington Campus of Imperial College London complemented with the appropriate IT platform, bioimage analysis software and technical support will be of immediate benefit to BBSRC-funded researchers at Imperial College London recording physiological and homeostatic function in multiple dimensions and scales. The high speed, low phototoxicity and high tissue penetration that can be achieved with the LEICA TCS SP8 DLS will drive scientific discovery and advance knowledge into essential mechanisms that maintain health and wellness under stress and biological or environmental challenge. The multiuser facility will enhance the research output of 13 highlighted current BBSRC-funded projects based at Imperial College that encompass Projects, Fellowships and Training Programmes totalling >£15M of BBSRC investment, extending to late 2023. As the LEICA TCS SP8 DLS is uniquely suited for high-resolution volumetric imaging of large and sensitive specimens such as embryos, organoids or plants, it will enable a wide range of transformative bioscience, addressing multiple challenge areas of the BBSRC Strategic Priority Areas including "Agriculture and food security (Sustainable agriculture systems, Food safety and nutrition and Crop and farmed animal health)", "Bioscience for Health (Lifelong Health, Nutrition for Health, One Health)" and "World-class and Underpinning Bioscience (Talented and productive workforce, Transformative new business opportunities)". Overall, the multiuser facility will enhance knowledge and advance innovative research in the BBSRC remit.

By providing 1:1 microscopy training to all new users both internal and external, FILM will train highly skilled multidisciplinary researchers to take full advantage of the unique imaging abilities of the LEICA TCS SP8 DLS. Over 200 postgraduate students enrolled into Imperial College London Doctoral Training Programmes (e.g. the BBSRC DTP for Multidisciplinary training for the Biosciences (BB/M011178/1)) the EPSRC Centre for Doctoral Training in Chemical Biology - Innovation for the Life Sciences (EP/S023518/1) and the EPSRC Centre for Doctoral Training in Neurotechnology for Life and Health (EP/L016737/1)) will also directly benefit from access to the LEICA TCS SP8 DLS at FILM with specific projects. Importantly, the proficient training provided by FILM will greatly benefit students and research staff outside the field of microscopy, enabling the wider application in state-of-the-art microscopy. In conclusion, the multiuser facility will support new training initiatives.

The installation of the LEICA TCS SP8 DLS in FILM will also enhance Imperial College London's strategic priority to support academic-industrial collaborations and support entrepreneurial activities. As FILM has a second facility located at the Hammersmith Campus adjacent to the new White City Campus with its growing community of start-ups, SMEs and major corporations, it is uniquely positioned to advertise and hold showcase events to introduce the research capabilities of the LEICA TCS SP8 DLS once in operation. Initiatives to enable sharing of the LEICA TCS SP8 DLS will include the promotion on Clustermarket, an equipment sharing platform that will drive collaborations between academia and industry. In summary, the multiuser facility will promote multidisciplinary research within academia and industrial collaborations.