Inverted confocal live cell imaging microscope

• Subject Area: Microbiology, Virology and Immunology
• Term: Funded in 2020
• Project identifier: Deutsche Forschungsgemeinschaft (DFG) - Project number 439819241

Understanding infectious diseases at single cell level unravels molecular mechanistic insights that could lead to disease treatment and prevention. To this end, our groups have developed new cell biological markers that allow for visualization of previously unknown mechanism within the life cycle of viruses and parasites. Those dynamic mechanisms of infection can only be fully understood when investigated using live cell imaging. Time scales, however, can vary drastically: from seconds and minutes, for viral particle trafficking and parasite motility, to hours and days, for parasite development, viral spread within tissue or cellular stress responses. At the same time these events can unfold at a scale beyond the resolution of classical fluorescence microscopes. Within our proposed projects we aim to fill critical knowledge gaps about long term viral and parasitic infectious mechanisms. This comprises the challenge to image infected cells over a long time with sub-diffraction resolution, while mitigating the cytotoxic effects of light. For this we require a new confocal imaging system to fill this important gap in imaging technologies situated between super-resolution and confocal microscopy.Most of the proposed projects require continuous live cell imaging for 24 hours or more. Currently these time frames are not readily available on our live cell capable microscopes due to extensive usage. Further, malaria parasites and viral host cell, which are studied within the proposed projects, naturally grow under low oxygen pressure. Towards creating imaging conditions with more physiological relevance we require hypoxic incubation conditions to e.g. prevent loss of polarity in hepatocytes or parasite death. A key aspect of physiological long term live cell imaging is reducing phototoxicity but at the same time achieve resolutions slightly below the diffraction limit. Specifically, malaria parasites have been shown to be very susceptible to fluorescent illumination. Therefore, highly sensitive detectors must be used for image acquisition. This however needs to be reconciled with increased spatial resolution to reveal significantly more cellular detail in three dimensions. Particularly, for the small sized malaria parasites the ideal technology would enable us to detect events such as formation of polar rings, chromosome segregation, centrosome duplication, and more. All this has to be achieved while maintaining an imaging speed that allows significant quantitative cell biological analyses.Hence, we want to implement a versatile and sustainable imaging solution that can open new dimension in our understanding of infection biology of those globally relevant pathogens.

• DFG Programme: Major Research Instrumentation
• Major Instrumentation: Invertiertes konfokales Mikroskop für Lebendzellbildgebung
• Instrumentation Group: 5090 Spezialmikroskope
• Applicant Institution: Ruprecht-Karls-Universität Heidelberg
• Leader: Dr. Julien Guizetti