Confcal microscopy-based cell sorting platform

• Subject Area: Basic Research in Biology and Medicine
• Term: Funded in 2025
• Project identifier: Deutsche Forschungsgemeinschaft (DFG) - Project number 563501122

Bottom-up synthetic biology pursues the remarkable vision of rebuilding a cell from molecular components such as lipids, DNA/RNA and proteins. This engineering approach to biology not only provides a fundamental understanding of cellular mechanisms but also innovative tools for biomedical intervention. We have demonstrated that functional cellular components can be artificially produced using an innovative DNA/RNA folding method ("DNA/RNA origami"). However, complex synthetic cells remain unattained as rational engineering approaches hit the limits of known structure-function relationships. To drive a significant leap in functional complexity, we propose a novel strategy combining rational engineering with the directed evo-lution of synthetic cells. The process begins with encoding functional RNA origami structures within lipid vesicles (GUVs). These RNA structures, transcribed from a DNA template (the "genotype"), will define key properties of the GUVs, such as permeability, division rate, or even a desired immune effector function (the "phenotype"). Building on these rationally engi-neered RNA structures as a foundation, we will initiate directed evolution to optimize the de-sired functionality. This involves iterative cycles of genetic diversification and selection, which requires the observation of the synthetic cells at high resolution over extended periods of time, and, subsequently, the selection of the ones that display the desired function from a large pool. For this purpose, we have recently developed an image-based sorting method, which enables the selection of desired vesicles by photopolymerization. Similar technologies are in demand when cells have to be isolated from a large pool. This par-ticularly concerns immune cells with high performance, which can then be studied to under-stand the determinants of their elite immunity. Here, we thus request a dedicated platform for the confocal microscopy-based sorting of syn-thetic and live cells consisting of three main components – a confocal microscope, a digital mirror device and a robotic arm for plate handling – offering high resolution (120nm), high throughput (up to 500 cells/sec) and a high degree of automation. In particular, the heart of this pipeline is a high-resolution state-of-the-art confocal laser scanning microscope equipped with a digital mirror device which enables targeted photopolymerization. The samples are placed on the sample holder by a robotic arm, connecting an incubation storage unit directly with the microscope allowing for high-throughput screening and optimal setup usage (e.g. overnight). Custom-written software integrates the hardware allowing for near-complete automation of the entire workflow.

• DFG Programme: Major Research Instrumentation
• Major Instrumentation: Konfokal-Mikroskopie-basierte Plattform zur Zellsortierung
• Instrumentation Group: 5090 Spezialmikroskope
• Applicant Institution: Ruprecht-Karls-Universität Heidelberg
• Leader: Professorin Dr. Kerstin Göpfrich