Leeds integrated atomic force and confocal microscopy for life science applications

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Seeing is believing: imaging techniques have been instrumental for progress in biology since the inception of modern science. This project provides researchers at the University of Leeds with a unique instrument that integrates two types of advanced microscope for recording images and making mechanical and optical measurements at very small scales. With this capability, biological problems can be solved that have proven intractable with conventional techniques.Optical microscopes (OMs), continuously developed since the time of Galileo, are now extremely powerful and versatile. Their ability to magnify enabled a clearer view of biological structures and this capacity remains much appreciated today to visualize live cells and the structures inside them. However, fluorescent tags that can absorb and emit light, and methods to measure the time between absorption and emission with exquisite resolution, have given OM a new dimension. With time-resolved fluorescence, the dynamics of biomolecular motion, the chemical environment of biomolecules, and the interactions and distances between biomolecules can now be studied in great detail.Some fundamental limits remain, however. Physical properties such as mechanical forces and elasticity are also vital for the function of biological systems, from single biomolecules to cells and tissues, yet these are not accessible with optical microscopes. Since its invention three decades ago, atomic force microscopy (AFM) has emerged as a unique technique to directly measure mechanical properties at length scales down to individual molecules. Based on a tiny tip that scans across the sample, it is also able to produce height ('topographical') images with a resolution that is superior to that of optical microscopes, of less than one nanometer (1/1000 the size of a typical bacterium). AFM, as OM, can be applied in liquid environment and so biological samples can be probed alive. OM and AFM are complementary techniques, because they measure distinct physical parameters. Combining them into one device and characterizing the same sample at the same time with both techniques affords the ability to correlate data and gain new insight that cannot be obtained with either technique alone, or even with the two techniques applied separately one after the other.On a larger scale, cells and tissues can be studied in unique ways. For example, we will characterize the structure and mechanical properties of the perineuronal net, an insulation sheath that covers the surface of neurons and modulates how neuronal connections form. Such insight may help to develop ways to delay memory loss in dementia or to repair spinal cord injury. On an intermediate scale, molecular assemblies can be probed, for example how blood clots are structured on the nanometer scale and what the mechanical properties of the individual fibres are that make up the clot. This will help to better understand thrombosis and its resolution, key to the prevention and treatment of heart attack. On yet smaller scales, we will study biological membranes, fascinating structures made from many loosely interacting molecules (lipids and proteins) forming an ultrathin film that is highly dynamic and key to cellular communication yet stable enough to compartmentalize cells and tissues. With the combined instrument, we will be able to study the structure and dynamics of biological membranes, for example to understand lipid organization (important for cell signaling), crystallization of membrane proteins (to facilitate their structural analysis, an important step for drug development), and how light-harvesting plant membranes work, for 'next-generation' bio-inspired energy generation methods.