4D imaging of the dynamic molecular, cellular and tissue organization in living systems

Microscopy is an essential tool for revealing how living systems are composed and how they function. Recent advances have enabled researchers to see deep inside tissues and at high resolution within cells. A limiting factor, however, has been that the powerful illumination required causes damage to living samples, which reduces how long they can be observed. A new technology, called the inverted lattice lightsheet microscope, overcomes this problem and allows for the first time gentle long-term imaging of live cells and tissues. This new microscope is a game changer in our ability to observe living cells, the basic units of life, and examine their full range of behaviours in health and disease. In addition to low phototoxicity and high resolution into 3D multicellular structures, the Zeiss lattice lightsheet 7 microscope has a unique inverted design, which is ideal for imaging cell and tissue cultures. This configuration also allows multiple samples to be tracked in parallel in multiwell plates, which controls for sample bias and generates statistically powerful datasets from a single long-term imaging experiment. The multiwell option can greatly increase experimental throughput and enable small scale chemical and genetic screens.
This proposal is to purchase and install an inverted lattice lightsheet microscope in the University of Exeter Bioimaging Center. The microscope will be available to life sciences researchers across Exeter and in our partner Universities in the SouthWest, Bath, Bristol and Cardiff. Using this instrument, we will be able to see in great detail how cells change identity and build tissues during development, how different cell types communicate and cooperate to provide complex functions, and how multicellular tissues are disrupted by infection and disease.
The Exeter Bioimaging Centre has a range of high-resolution microscopes but lacks an instrument capable of tracking cell behaviour in multicellular structures or small organisms. Our team of applicants brings together world-leading senior researchers, microscope technology specialists, and highly talented junior independent investigators. The cutting-edge research that will benefit from the microscope includes studies on fungi, plants, marine invertebrates, zebrafish and human. Example projects span investigations into molecular signalling, stem cell fate, embryo formation, organogenesis, drug safety, cell-cell communication, plant pathogen interactions, and the biophysics of ciliary motion. The high volume of quantitative data obtained with the new microscope will foster interdisciplinary collaborations between biologists and mathematicians and physicists aimed at elucidating the rules of life using rich, complex and exciting long-term imaging information.

The capability to capture fast cellular changes is key to studying dynamic biological processes. Imaging of a complex biological specimen over a long period of time requires the combination of high speed, very low phototoxicity and photobleaching and high resolution. The Nobel prize-winning innovation of the lattice light sheet technology delivers these tasks. The lattice light sheet microscope has little phototoxicity and photobleaching compared to the conventional confocal microscope. It illuminates a single thin section of the sample at a time and collects the signal only from the in-focus plane through a second orthogonal objective lens, thereby minimizing the effects of photo-damage. The lattice structured light beam projects a thin (less than 1 micron) layer of light sheet to achieve the high-resolution imaging. The Zeiss Lattice Lightsheet 7(LLS7) we request in this application is a recent addition to the lattice light sheet microscope family. Zeiss brings in a unique inverted configuration, which images the samples from beneath. This was not previously possible with a light sheet microscope because of the refractive index mismatch between the excitation objective and the detection objective. Zeiss's design of the detection objective compensates the refractive index mismatch. The inverted configuration brings in the capability of imaging samples on a regular microscopy slide, a glass-bottom cell culture dish or even a multi-well plate for parallelized high throughput assays. The accessible inverted configuration allows the exchange of culture media and easy delivery of chemical compounds to the sample. These features together make the Zeiss LLS7 a powerful and easy- to- use live imaging system for cells and 3D structures. In this application we exemplify how the Zeiss LLS7 would empower researchers in multiple disciplines including cell signaling, cell fate transition, embryo and tissue morphogenesis, pathogen immunity and organ development and function.