We would like to acquire two complementary microscopic systems to perform observations on living cells, allowing for physiological examinations. The confocal laser scanning (CLSM) system provides excellent opportunities for fluorescent manipulations and measurements (photoactivation, photoconversion, photobleaching, Förster resonant energy transfer (FRET)); the specificity of the signal recording is increased by spectral detection. In this system, we can focus the tests on the smaller regions within the field of vision, optionally designated. With the spinning disc (SD) microscopic system, we can record molecular processes in live cells even faster than the CLSM system, even at a millisecond image recording rate, in the entire field of vision. An important aspect is that living cells are subject to less light exposure during the recordings, so we can continue the observations much longer. The SD system to be procured is also suitable for displaying molecular processes in the immediate vicinity of the cell surface (within a distance of 100-150 nm) with increased resolution and high speed compared to CLSM tests (TIRF, i.e. full internal reflection microscopy). Both systems are capable of recording two fluorescent signals simultaneously with 2 detectors (CLSM) and 2 cameras (SDs), so that molecular interactions, concentration changes and transport processes in live cells are specifically tracked in real time. The 14 research teams participating in the tender are investigating the molecular and cell physiological effects of inflammatory and autoimmune diseases affecting tumour formation and metastasis, cellular processes of nervous plasticity and the self-digestion of cells (see attached biographies). The objectives are broad, but their common feature is that they aim to explore dynamic cell biological changes in living cells. The microscopes to be procured provide the necessary infrastructural conditions for the studies, and the outstanding scientific background of the participants provides the necessary knowledge platform for implementation. Below, the microscopic techniques to be used are presented through a few main themes. 1. In various immune cells (e.g. granulocytes, macrophages, dendritic cells, lymphocytes) processes following ligand binding of receptors, “dialogue” between receptors play a crucial role in many physiological and pathological immune processes. For example, cooperation between complement receptors and pattern recognition receptors for granulocytes important in inflammation, leading to the formation of neutrophil extracellular traps (so-called NETs). Changes in the intracellular Ca2+ signal accompanying neutrophil activation can be tested using fluorescent Ca-indicator inks (e.g. Fluo3/Fluo4) and genetically encoded Ca2+ indicators (e.g. GCaMP6) on both CLSM and SD systems for cell lines capable of forming NET. TIRF microscopy integrated in SD is essential to monitor the adhesion and spreading of various immune cells and processes near the cell surface (e.g. NET release). By using CLSM and SD systems, by detecting two fluorescent signals simultaneously, we can test the molecular effects that control the functioning of immune cells (e.g. interaction between receptors, intracellular transport processes) in real time, in close proximity to the cell surface and in parallel with the measurement of activation. 2. Homologous recombination is one of the most effective ways of correcting errors in the DNA of organisms, preventing cancer transformation and creating new gene variants. In addition to the in vitro approach, we test in whole organisms (worm and zebrafish) DNA-helicas in which sub-processes of cell division, through which molecular activities and with which protein partners they promote controlled recombination, accurate chromosomal segregation. Microscopes that allow simultaneous and rapid display of the two fluorescent signals are essential for detecting processes in organisms producing proteins that are involved in error correction mechanisms with different fluorescent labels. Analysis with CLSM and SD systems therefore complements the measurements carried out with the already existing two-photon microscopic system. 3. In recent years, by developing molecular tattoo technology, we have achieved groundbreaking results in the field of optofarmacology. With the development of light-activable pharmaceutical derivatives, we have the opportunity to regulate certain cell biological processes in the CLSM system by localising certain micrometres. The processes that control the dynamic transformation of the actin skeletons of nerve cells (e.g. growth and search for paths of axons) can be explored with high spatial and temporal resolution. MyosinII axon growth and actin dynamics flow
