Good Vibrations: Visualising life with multiphoton/Coherent Raman Scattering microscopy

In the past 15 years, there have been significant technological advances in the instrumentation available for imaging cells and tissues without the addition of fluorescent labels. This is important as the addition of these labels can change the behaviour of the system itself, obfuscating any biological results. These advances have led to increases in the speed of imaging, the level of detail obtained and the information content of these so-called “label-free” technologies. Advances in image interpretation driven by artificial intelligence enable the analysis of ever larger and more detailed experiments. Hence, we are entering an era where label-free imaging can be used to analyse detailed changes in complex, live cell and tissue models. Of all the label-free techniques, Coherent Raman Scattering (CRS) microscopy has been identified as a “Method to Watch” (Nature Methods, 2022) for imaging cells and tissues. CRS confocal microscopy captures quantitative data about the strength of discrete Raman vibrations corresponding to individual bonds across each pixel in an image. The morphology of a cell can be readily visualised in 3D by tuning to specific carbon-hydrogen (C-H) bond vibrations that reflect the environment of these bonds in proteins or lipids. In its hyperspectral mode, CRS microscopy generates pseudo-Raman spectra for every pixel within the desired field of view with unrivalled spatial resolution, providing an information-rich approach to microscopy and a powerful tool for chemical analysis. The proposed commercial multiphoton/CRS confocal microscope will allow world-leading scientists in the Institute for Regeneration and Repair (IRR) at the University of Edinburgh to map the distribution of different molecular species, probe changes in molecular composition, directly image small molecules and apply a range of image analysis techniques in live cell and tissue models - label-free - using an accessible, reliable platform for the first time. Currently there is only one equivalent commercial multiphoton/CRS microscope in the UK, which is not in a biology-led facility. The University of Edinburgh is world-leading in its use of CRS microscopy in the biosciences and has received funding for its development from UKRI, industry and charities. The IRR core Imaging Facility has recently expanded its label free imaging resources and with our CRS expertise we are exceptionally well placed to build on and expand our bioscience capabilities through investment in a commercial multiphoton/CRS microscope. The microscope will be a new tool that will enable us to address fundamental bioscience questions including: identifying hallmarks of cellular ageing; determining mechanisms of lineage commitment in tissue engineering; sensing intracellular conditions during tissue repair; and predicting causality in bioimage analysis. The acquisition of a commercial multiphoton/CRS microscope will immediately benefit researchers across the University of Edinburgh and Scotland, but will also be advertised to UK users through the UK Technology Specialists Network and imaging forums such as the Royal Microscopical Society, and the Society of Photo-Optical Instrumentation Engineers. By embedding the microscope in the IRR Imaging Facility, we will ensure that its continuity of operation and ease of access to all users is underpinned by trained professional staff. The impact of the research it enables will include an enhanced understanding of fundamental process which govern life; a reduction of animal use in research (through the development of more meaningful human cell and tissue models); and the development of cell and tissue models of healthy, aged and diseased tissue for biomedical research.