Light microscopy

Our service group hosts a broad range of light microscopes which are operated as shared equipment and are open for all members of the institute. This includes conventional widefield epifluorescence microscopes (Z1 Observer, Z1 Imager), confocal laser scanning microscopes (LSM700, 1x LSM880 with Fast-Airyscan detector, 1x LSM800 with Fast-Airyscan detector, 2-photon laser and NDD Big2 detector), screening microscopes (Cellomics ArrayScan, Celldiscoverer 7) and a 3D light sheet microscope. Furthermore, we also support instruments located in individual departments (Z1 Observer, Dept. Meissner; Z1 Observer and V16 Stereo-Zoom, Dept. Herrmann) and other equipment such as basic cell culture and stereo microscopes, which can be accessed by users upon request.

Besides technical support, the focus of our group is on user training and application support. We assist all users in performing their imaging experiments, might it be a simple routine task or the implementation of a complex imaging workflow including a data analysis pipeline (Fig. 1). Prior to using a microscope, an instrument specific introduction given by a member of the service group is mandatory independent of the user’s background or the type of instrument used. We thus encourage all users to contact us in time to discuss and thoroughly plan before starting the actual experiment. Initial project planning should also support the user to select suitable dyes, suitable well-plate formats and proper instrument hardware as well as selecting proper imaging parameters according to downstream data analysis.

Figure 1: Imaging biological samples is often an iterative process. Our service group supports users at all levels including project planning, data acquisition and analysis as well as workflow optimization.

Frankly speaking, there is no universal imaging procedure available. A simple widefield experiment might be fast and sufficient to detect a certain signal, but not provide enough resolution to localize it unambiguously. Confocal microscopy might provide higher resolution, but the high laser intensity might bleach the sample and prevent long-term live cell imaging. In particular, imaging large three-dimensional samples requires careful balancing sensitivity, light intensity, resolution and speed, but also optimization of sample preparation including sample staining and clearing. Once experiments are running, we are open to discuss the results to further optimize individual steps including sample preparation, data acquisition, image processing as well as data analysis and interpretation.

Within recent projects, we implemented a broad range of imaging techniques including automated live cell and multiwell plate-based high-throughput screening, Airyscan super-resolution microscopy as well as 3D imaging approaches using 2-photon imaging, Fast-Airyscan confocal imaging and light sheet microscopy. Furthermore, we provide access to image processing workstations and various software packages such as Image J, FIJI, Zen Intellesis, Arivis Vision 4D, Cell Profiler, Columbus, HCS Studio and Voreen amongst others. Combining image acquisition and data analysis tools, we setup various imaging pipelines which are now available for all users. For example, in collaboration with Melissa Bothe (AG Meijsing), we established an automated pipeline for RNA and DNA-Fish imaging including acquisition of z-stacks, image segmentation, quantification and statistical analysis which is now used by several research groups throughout the institute. In a similar approach, established a workflow to quantify phase separation events inside the nucleus accompanying gene regulation (AG Hnisz). In collaboration with the Yaspo lab and Zeiss, we established semi-automated workflows to screen and analyze cancer spheroids. These involve targeted acquisition schemes to identify and characterize spheroid formation in 3D cell culture systems as well as drug response and end point assays (Buschow et al., 2019, Application note; see http://go2.zeiss.com/FsaMq00PPbX0J3mp000SLiC). Similar approaches are now used by other groups to study embryogenesis using gastruloids (Dept. Herrmann) or stem cell differentiation (Dept. Meissner).

Our own research focus hereby lies on implementation of correlative workflows to image samples on different imaging platform combining live cell imaging, high-resolution 3D imaging and ultimately ultrastructural analysis using TEM or SEM (Fig. 2; for more information please also visit our TEM pages). Another focus is the implementation of super-resolution light microscopy, which are not yet available at our institute.

 

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