Central Laboratory for Microscopy (CINIMA)

By using established and novel technologies of optical imaging, we are able to monitor the orchestration of cells and molecules in tissue which are relevant for a better understanding of chronic inflammatory mechanisms.

The expertise of the Core facility for INnovative IMaging und Analysis (CINIMA) at the DRFZ encompasses standard fluorescence microscopy methods, such as wide-field and confocal microscopy. We extended our expertise in the field of immunofluorescence histology by applying and further developing automatized multiplexed microscopy as well as evaluation algorithms of these data. In addition, our unique customized and self-developed microscopy technologies allow the functional analysis of single cells in organ models and whole living organisms, with a special focus on non-linear optical multi-photon microscopy.

Confocal laser scanning microscopy, multiplexed wide-field microscopy and light-sheet microscopy: imaging living cells and fixed samples

Histological samples which are relevant for many research groups at the DRFZ are typically investigated in an automatized manner using the wide-field microscope (Keyence). For research questions which imply the visualisation of cellular and tissue structures of only few 100 nm, a confocal system (Zeiss LSM 880) is employed. It allows sequential investigation of five spectrally resolved parameters on the same sample. Additionally, this system is equipped with an incubation chamber in which both temperature and CO₂ are controlled and its optical focus stabilisation allows for the investigation of living cells over the time course of days. In order to achieve multiplexing in immunofluorescent histology, a Toponome Imaging Cycler based on a wide-field fluorescence microscope is used. In this way, more than hundred markers can be sequentially visualised on the same sample. The basic principle of this multi-epitope-ligand-cartography technique is based on the automatised sequence of sample staining, washing, imaging and photobleaching. Our major task is to develop adequate evaluation algorithms, which extract the full information contained in the multiplexed data. For instance, this technology is used in the frame of the DFG SPP1937, to analyse the heterogeneity of innate lymphoid cells in situ and to compare homeostasis and chronic inflammation. Since 2018, we offer researchers access and support to a state-of-the-art light-sheet microscope, which enables the visualization of single cells in the context of whole cleared organs, in a three-dimensional manner.

Optical coherence tomography and multi-photon microscopy for dynamic and functional imaging in living organisms

In order to visualise and quantify cellular dynamics and function deep in the organs of living mice, we develop and provide expertise in intravital multi-photon microscopy methods. Time-resolved multi-photon microscopy methods based on time-correlated single-photon counting (TCSPC) allow the quantification of metabolic enzymatic activity in living cells and tissues based on the endogenous fluorescence of the coenzymes NADH and NADPH. Additionally, by using Förster resonance energy transfer (FRET)-sensors, we enable real-time quantification of absolute intracellular calcium concentrations in hematopoietic cells and neurons. Moreover, we set a main focus of development on longitudinal intravital imaging in different organs of mice such as femoral bone marrow (LIMB), retina, and kidney – thereby providing unique insight into the evolution of chronic inflammatory mechanisms in vivo. Last but not least, we combine these cutting-edge technologies with imaging technologies of living organisms used in clinical setups such as optical coherence tomography (OCT), allowing direct correlation between mechanistic knowledge and clinically accessible information. The development is performed in the frame of a close collaboration between the groups “Biophysical analytics” on the physics and biophysics side (Niesner) and “Immune dynamics” on the immunology and animal welfare side (Hauser) and is provided to various DRFZ groups, Charité groups and groups at the Freie Universität Berlin and to researchers belonging to different consortia such as the transregional collaborative research center TRR130 or the research unit FOR2165.

Since 2013, CINIMA has been actively providing multi-photon microscopy expertise to the transregional consortium SFB TRR130 in the frame of a central project. It also leads a transregional network for intravital microscopy – JIMI (Joint network for Intravital MIcroscopy) – which was funded by the German Research Council (DFG) between 2012 and 2016. Our central lab for microscopy collaborates with national and international bioimaging networks such as the GermanBioImaging led by Prof. Elisa May in Konstanz and the CTLS (Core Technologies for Life Sciences led by the Pasteur Institute in Paris).

Keywords

Epifluorescence Microscopy
Confocal microscopy
MELC
Light-Sheet-Microscopy
Intravital microscopy
Fluorescence Life Imaging