Seeing is believing - Methods of observing vital processes in cells and organisms

At the molecular level, cellular structures and processes were originally described mainly by biochemical and genetic approaches or indirectly using functional assays and chemical drugs. Rapid development of imaging techniques and specific probes enabled verification and, in the most cases, also highly detailed and direct molecular characterisation in cells and organisms. In this course, problem-oriented lectures will guide students through the exhibition of tools - mainly fluorescent probes – and methodology for studies of biological structures and processes at the molecular level and with focus on quantitation. At the beginning of a lecture, a biological question to solve will be defined. Afterwards, we will describe the available tools and methods used to answer these questions by top scientists. Alternatively, we will suggest different options and discuss advantages and limitations of these diverse approaches. To mention just a few, the topics will cover determination of components of signalling and metabolic pathways and timing of individual steps in cells or small animal (plant) models, quantitative imaging of ions, investigation of cytoskeletal dynamics, membrane trafficking and relocalisation/structural changes in nucleoprotein assemblies or monitoring of cellular responses to oxidative stress. The technology required for the appropriate use of mentioned probes will be explained, mainly by providing examples where clever combination of the right imaging techniques and smartly-designed probes led to break-through findings. Advanced fluorescence techniques such as super-resolution imaging and variants of correlation spectroscopy will be elucidated in more detail to highlight their advantages but also to indicate their limitations and, often, improper use. We will also open the room with label-free techniques (e.g. second and third harmonic imaging) to indicate current state of these methods which fulfil the dream of a biologist to avoid probes and labels.