Live-cell imaging microscope

• Subject Area: Basic Research in Biology and Medicine
• Term: Funded in 2022
• Project identifier: Deutsche Forschungsgemeinschaft (DFG) - Project number 506524377

Here we apply for a live cell imaging system that can visually record the proliferation and death of living cells over an extended time. This will allow a much more precise characterization of both processes relative to the currently used assays that provide a single snapshot at a fixed time point. In recent years, it has become apparent that this increased precision is required for an accurate description of how cells respond to external stimuli or to genetic manipulation. All applicants work in tumour and/or cell biology and the precise characterization of cell proliferation, cell cycle progression and cell death is central to virtually every project in the applicants’ laboratories. To do so, the applicants use a well-established infrastructure: for example, we use FACS-analyses of labelled cells to monitor cell number, DNA content, S-phase progression and apoptosis. For long-term observations in cell number, we use a combination of cell counting devices and crystal violet staining of plates. Here, we apply for a live cell imaging system that allows the recording of time-lapse videos and constant monitoring of cell number and several optical parameters over a period of multiple days in a non-invasive manner. This is possible since the imaging instrument itself will be housed in a tissue culture incubator.The requirements to precisely document proliferation and death are constantly increasing and many laboratories by now use live cell imaging systems. It is apparent to us after testing these instruments that the analysis of cell behaviour with these instruments is much more precise than any method we have used before. This has several central reasons:• For each condition, the constant monitoring yields multiple data points, hence the error in describing for example growth rates or other quantitative parameters is much smaller in comparison to, for example, crystal violet staining. As consequence, all statements about differences between two experimental conditions are much more precise.• Heterogeneity among cells becomes immediately apparent during the experiment. For example, we use lentiviral-based pool infections of shRNA- or sgRNA-expressing vectors to deplete proteins. In these vectors, the expression of the sh/sgRNA is linked to a GFP marker. Long-term monitoring of GFP expression in parallel with cell growth allows us to determine whether cells that grow out over time still retain full or partial expression of shRNA/GFP, allowing us to draw conclusions about selection pressure by sg/shRNA.• The parallel monitoring of cell death enables clear statements whether depletion exerts a pro-apoptotic phenotype.• Finally, the better documentation greatly facilitates publication of the data, for example by fulfilling requirements for statistical documentation.

• DFG Programme: Major Research Instrumentation
• Major Instrumentation: Live-Cell-Bildgebungssystem
• Instrumentation Group: 5040 Spezielle Mikroskope (außer 500-503)
• Applicant Institution: Julius-Maximilians-Universität Würzburg
• Leader: Professor Dr. Martin Eilers