Fluorescence Light Sheet Microscopy for Live 3D and 4D imaging

.Individual cells in a plant or an animal are exposed to changes in their environment (biochemical signals, temperature, light variations...). Cells have to interpret this information to adapt and respond appropriately. To understand the molecular mechanisms leading to a particular response (e.g., cell death, cell growth, cell migration, etc), biologists have to measure the levels and localisation of proteins in the cells. Each individual cell might respond differently from its neighbour and at a different time so it is crucial to follow the events in real time and in each individual cell. This can be achieved using live cell imaging coupled with the use of fluorescent labels. However, technological limitations have restricted the measurements to flat samples, such as cells attached to a glass coverslip.
In 2004, a German group invented a new microscope called light sheet microscopy, which illuminates the sample using, as the name indicates, a sheet of light. This technology also includes rotation of the sample to acquire different views. Images can be acquired very quickly through the depth of a sample (up to 1 mm thick, which is far more than with conventional microscopes). Because of the illumination geometry, the sample does not suffer from the toxicity induced by the laser light, enabling longer term imaging of living organisms. For example, the development of a zebrafish or drosophila embryo, from a few cells up to a whole organism, with single cell resolution can be observed in real-time. The first commercial light sheet microscope was released at the end of 2012. We propose to purchase one of the first commercial light sheet instruments in the UK and to install it in the Liverpool Centre for Cell Imaging (CCI). The CCI is an open access facility, so the microscope will be accessible to groups from several universities and companies. To illustrate the breadth of the science that will be served by this equipment, we briefly present below three exemplar projects:
1. Repair of muscles during ageing.
As people age, their skeletal muscles starts to deteriorate. Skeletal muscle cells do not divide and hence need to have a robust mechanism in place in case of damage or loss. Repair requires a unique population of specific muscle stem cells called satellite cells. Failure in satellite cell function can lead to delayed, impaired or failed recovery after injury and such failures increase in old age. With the new microscope, we will follow for the first time, the movement, division and differentiation into muscle cells of the satellite cells, in real time on damaged muscle fibres.
2. 3D cell culture models for drug testing
The reduction, replacement, and refinement of animal experiments is one of the BBSRC priorities. It requires better in vitro models to improve drug discovery and drug testing. Three dimensional multi-cellular spheroid models allow faster and less expensive screening in a 3D cellular organisation. To develop drug delivery and toxicity testing using 3D cell culture system, we need to understand how the cells survive, divide and move at different positions within the spheroid. Using Light sheet microscopy, we will image in real time cell survival, proliferation and migration of individual cells in the whole spheroid. This has attracted the attention of biotech companies developing biomaterials for 3D culture and pharmaceutical companies for drug testing.
3. Uncovering cell specific changes in the plant circadian clock
This project is about understanding the regulation of intracellular signals in plants triggered by the day and light changes (circadian clock). Using 3D analysis of seedlings with the light sheet microscope, we will elucidate each cell type specific regulation of the circadian clock intracellular network. This research has important implications, because the circadian clock regulates many agronomic important processes including yield, water use efficiency, disease resistance and flowering time

Cell biology has undergone a revolution from largely non-quantitative observations in fixed cells to high-throughput quantitative data in live cells. Although confocal microscopy is extremely powerful for following events in single living cells, information from thicker samples is more challenging. Two photon microscopy allows deeper penetration but is limited by phototoxicity and photobleaching. Light Sheet Fluorescence Microscopy (LSFM) is a revolutionary development in microscopy. LSFM reduces photobleaching and phototoxic effects in living samples allowing long-term imaging in real time in three and four dimensions. The first description of LSFM was over a century ago, yet it was only in 2004 that Stelzer's group applied it to a developmental biology project and made the technology adequate for imaging living 3D biological samples. The imaging community has adopted LSFM and it is now commercially available (Nov. 2012 from Zeiss). At the time of writing there are no commercial LSFM systems in the UK. We propose to install a Z.1 LSFM in Liverpool Centre for Cell Imaging to serve a range of projects within the BBSRC remit for users from the University of Liverpool, as well as external academic and industrial partners. The proposed projects include imaging of circadian clock proteins in Arabidopsis, neuron mapping in zebrafish brain, cell migration in drosophila, 3D cell culture system characterisation for their use in drug delivery and toxicity, repair and maintenance of skeletal muscle during ageing, stem cell potential by re-implantation in mouse kidneys, etc. Liverpool is uniquely placed to implement this step-changing technology, by having a facility manager who was previously trained in the Stelzer's laboratory and a multidisciplinary team of developmental, cell and plant biologists as well as physicists and mathematicians for optimal exploitation of the quantitative data generated.

Where and who is our user pool?
A key beneficiary of this investment in Light sheet microscopy will be our user pool. The CCI is setup specifically for live cell imaging in control environmental conditions with capacity to image live cell cultures, plants (Arabidopsis), Chick Embryo and Drosophila. Therefore our primary user base will be academics interested in the quantitative measurement of real-time biological events in a variety of 3D systems. We current host an average of 35 users from across the University of Liverpool, UK and the rest of the world. With recent investment from the Optical Microscopy across council initiative (MR/K015931/1) we aim to extend this. Moreover, we currently have an expression of interested with Euro-Bioimaging to become a Euro-Bioimaging Node. While our facility is well established we currently do not have any LSFM capacity.
1. The Biotechnology Industry will have specific interests in 3D structures of biological materials, cultures and biofilms. The agro-biotechnology industry will also be interested in 3D imaging of plants for example to understand plant development, target sites within a plant for herbicide resistance and tracking pathogen invasion in 3D.
2. The pharmaceutical industry will be interested in imaging cell organisation within model systems of tissues and to use those for drug testing by monitoring cell fate in real time in a 3D environment.
3. Scientific Software Industry- The 3D imaging data generated by the light sheet microscopes is generating a whole new set of challenges.
4. Scientific community. The major impact will come through access to the light sheet microscope to a wide user base. We will ensure the academic impact of this work through timely seminars, workshops and publications.
5. Outreach. The PIs and CoPIs have active collaborations with the Liverpool World Museum and local schools. The team will use these links to host events showcasing the 3D imaging technique and develop teaching resources.
6. A next generation of Scientists. The Centre for Cell Imaging will provide strong training of young scientists using the facility. The Centre for Cell Imaging train annually ~6 undergraduate and 10 postgraduate scientists, who will have access to the new light sheet microscope. The Centre for Cell Imaging is also active in with working with schools. For instance, sixth form projects are run within the CCI.
LSFM is set to become an important microscopy technique, access to this machine will be accompanied by training and workshops. On top of individual hand-on training, we plan a series of events and workshops on LSFM explaining its application as well as specific and specialist training on imaging techniques from Zeiss and external speakers. We will also include training on image analysis. Workshops will be available to new and existing CCI users in the UK.