A Dual Camera Lattice Lightsheet Microscope To Deliver Transformative Multi-Channel Volume Imaging

The first studies of biological structures were by the early pioneers of microscopy, Robert Hooke and Antoni van Leeuwenhoek, in the 17th century. Robert Hooke, in 1665, was the first to introduce the term "cell" when he was viewing the "boxes" he saw in slices of cork using one of the earliest optical compound microscopes (two lenses: an objective lens and an eyepiece) that he developed. He probably didn't quite realise the significance of this discovery, as it was only when it became apparent that the great majority of organisms are composed of cells that Cell Theory was born. Cell Theory, first proposed by M.J. Schleiden and Theodore Schwann in 1839, states that cells are of universal occurrence and are the basic units of an organism. Over 300 years of microscope improvements have led to fascinating discoveries of how cells function and now fluorescence microscopy, a form of light microscopy where objects are tagged with light-emitting dyes, has become an essential tool to study the biology of the cell. Many technical developments have led to greatly improved image quality and resolution however when imaging cells a compromise between imaging speed, volume acquired and longevity of the sample is required which restricts the type of biological question that can be investigated. Dynamic observations in living cells are usually limited to a small subcellular area. Lattice Lightsheet (LLS) technology addresses these challenges providing a step change in the imaging datasets possible and the biological questions that can be asked. LLS microscopy is the first commercially available technology that will allow full volumetric imaging (3 cell volumes per second) at high resolution over long time periods without the damaging effects of laser illumination (phototoxicity). This opens up the possibility to study new cell biological questions about morphology, responses to stress, and in development. A lattice light-sheet uniquely creates a very thin (500nm) sheet of laser light which is passed across the sample to generate a series of images (optical sections) that can be reconstructed into a three-dimensional view of the sample. With LLS this sheet is thin enough to resolve the three-dimensional details within cells clearly. With other microscopy techniques, we are limited by the speed at which we can acquire these images and the length of time we can observe before the sample dies due to exposure to the laser light. Unlike other non-lightsheet microscopes, the illumination of the sample is restricted to the focal plane, one slice within the sample. Because the areas of the sample above and below this plane are not exposed to the light, the sample viability is improved allowing the gentle observation of important biological processes over longer periods of time without sacrificing the speed of image acquisition or image resolution. To be able to observe these processes in detail across the full volume of the cell is truly unique. This transformative technology will allow researchers in Durham and beyond to address fundamental questions in plant cell growth and development which will ultimately lead to a greater understanding of how plants adapt to different stresses (biotic and abiotic), and how animal cells divide and grow in normal and disease states. This new variant of LLS technology allows the simultaneous imaging of two channels at high speed fundamentally expanding the biological questions we can ask about where proteins and structures interact. The instrument will be supported by dedicated expert technical staff and shared with scientists in the N8 research partnership of Northern universities and nationally with the UK Plant Cell Biology Community.

When imaging live cells a compromise between speed, volume acquired and sample longevity is necessary, restricting the biological questions that can be investigated. Dynamic observations are often restricted to a small subcellular area or slice. Lattice Lightsheet (LLS) addresses these challenges providing a step change in the imaging possible and the biological questions that can be asked. LLS microscopy is the first commercially available technology that will allow full volumetric imaging at high resolution over long time periods without the damaging effects of phototoxicity. The Zeiss LLS 7 DualCam is a unique inverted commercial implementation of LLS first developed by Eric Betzig. Principle advantages of this system is that it is inverted, allowing users to use standard carriers such as dishes and can image two channels simultaneously. Automated optical alignment, adaptive optics, auto sample leveling, and auto immersion together make this system ideally suited to core facility use. Lattice Sheets generated range from 500nm thick (by 12um long) to 2um thick (by 180um long) giving a resolution of 290nm in XY and x 450m in Z following deconvolution. This gentle high-speed imaging can achieve 400fps in 2D and volumetric imaging of 3 3D cell volumes per second for several hours or even days. The instrument will enable Durham researchers to study new questions in plant and animal cell biology including cytoskeleton dynamics, cell division, membrane connections, cell migration, nucleo-cytoskeleton links, and more. The instrument will be shared with scientists in the N8 research partnership and nationally with the UK Plant Cell Biology Community. Durham is a centre for excellence in plant bioimaging and as such is ideally suited to develop LLS plant methodologies and share this LLS microscope with the community. The system will be supported by permanent dedicated facility staff, located within a core bioimaging facility, and be provided with DU large data storage.