High pressure freezer with a light and electrical stimulation

• Subject Area: Medicine
• Term: Funded in 2020
• Project identifier: Deutsche Forschungsgemeinschaft (DFG) - Project number 436509545

We hereby apply for a high pressure freezer with a light and electrical stimulation unit that is integrated into the freezer chamber. The requested device is a crucial research equipment that we would like to combine with our 3D electron tomographic imaging efforts to tackle the ‘dynamic ultrastructure’ of cardiomyocytes - cells whose function is to contract and relax, once every second of the human life. Our proposal builds on a solid track record of developing and utilising advanced imaging tools for monitoring cardiac ultrastructure and function in general, and on an actual test of the suitability of the here requested device for our studies.Our previous work was limited to chemically fixed preparations, where the limited temporal resolution (in the range of minutes rather than milliseconds) limited our ability to make statements on the functional relevance of observed 3D nanoscopic changes in cardiomyocyte subcellular domains during mechanical deformation. Millisecond-resolved 3D reconstructions of cardiac ultrastructure during the physiologically initiated (i.e. action-potential-triggered) contractile cycle will allow us to address crucial questions of fundamental relevance (e.g. length-dependent membrane integration of caveolae, convective T-tubular fluid transport, spatially-determined gain of Ca2+-induced Ca2+-release in excitation-contraction coupling). The requested device will allow us to conduct principally new research (contraction-cognisant nano-micro-macro integration) and drive insight into clinically relevant cardiovascular physiology (as – thanks to the built-in electrical stimulator – the system can be used with genetically non-modified tissue, including human samples, to explore disease-related remodelling). At the same time, we will create a pipeline for studying the ultrastructure of contracting cells within a true-to-nature temporal frame, and provide others with a unique reference source of 3D nm-resolution information on cardiac cell ultrastructure in different species, cardiac regions, contraction states, and patho-physiological conditions. These insights will be suitable for applications, from computational modelling of excitation-contraction coupling to the functional interpretation of molecular signalling pathways in the context of cell compartmentalization.The new research device will be operated at the Institute for Experimental Cardiovascular Medicine in Freiburg, in close proximity to the in-house cardiovascular tissue biobank, cell isolation and culture facilities, freeze-substitution equipment, molecular biology laboratory, and advanced multiphoton / confocal microscopy platform.We believe to be in a unique position with regard to track record, technical expertise, scientific abilities, and pilot data, to develop the tools and techniques required for millisecond-accurate 3D characterisation of cardiomyocyte nanostructure dynamics during the cycle of action-potential-induced contraction and relaxation.

• DFG Programme: Major Research Instrumentation
• Major Instrumentation: Hochdruck-Gefriergerät mit integrierter Licht- und Elektro-Stimulationseinheit
• Instrumentation Group: 3540 Gewebeeinbettungsgeräte, Fixier- und Färbegeräte
• Applicant Institution: Albert-Ludwigs-Universität Freiburg
• Leader: Professor Dr. Peter Kohl