Central Laboratory for Microscopy (CINIMA)
From cell to organism
Due to the successful combination of intrinsic molecular specificity with high spatial resolution, fluorescence microscopy is a versatile technique largely employed in both routine and high-end research applications of biosciences and biomedicine. Currently, these applications reach from the investigation of fixed biopsies, living cell cultures, vital organ models or even living organisms on a molecular basis and down to electron-microscope-like spatial resolution, i.e. supra-resolution techniques (like STED microscopy, PALM, STORM or SSIM/SPEM). The expertise at the Core facility for inovative imaging und analysis (CINIMA) at the DRFZ covers standard fluorescence techniques like wide-field and confocal fluorescence microscopy to the newest developments in vital organ explants and intravital multi-photon microscopy.
Confocal laser-scanning microscopy and wide-field fluorescence microscopy
For fast screening of histology samples of different organs, the DRFZ research groups employ the fully automated wide-field fluorescence microscope (Keyence). In order to achieve three-dimensional images at high resolution the Zeiss LSM710 confocal microscope is used. Both whole field images reaching to a few mm² (when tile scans are made) and images depicting cellular interaction/co-localization at sub-cellular level using the 63x oil-immersion lens (NA = 1.3, i.e. resolution at
488 nm excitation wavelength of 229 nm laterally and 577 nm axially) can be acquired. A central feature of the confocal microscope at the DRFZ is the possibility of live cell visualization and monitoring in a temperature- and CO2-regulated incubation chamber. With this system, spectral resolution of up up to five colors can be acquired. In order to achieve multiplexing in histology, we a Toponome imaging cycler is used. This system allows to detect more than 100 markers in one histological section, by sequentially staining and bleaching the sample, a process termed Multieptiope Ligand Cartography (MELC). We are developing customized algorithms to analyze the data generated using this system, in the sense of multiplexed histocytometry.
Multi-photon laser-scanning microscopy for deep-tissue and intravital microscopy
While the standard wide-field and confocal microscopy based on single-photon excitation intrinsically allows for high resolution due to UV/visible illumination, they do not allow for high imaging depths in intact, living organs. Due to the benefits of ultra-short pulsed near infrared (NIR) and infrared (IR) illumination, multi-photon microscopy counteracts exactly this shortcoming of standard fluorescence microscopy. The intrinsic 3D resolution and the excitation within the tissue optical window, i.e. at wavelengths where neither water nor hemoglobin absorb radiation, makes multiphoton microscopy an ideal tool for the visualization of cellular motility and communication deep in vital organ models or in organs within living organisms at low endogenous fluorescence and sub-cellular resolution.
The DRFZ and its partners at the Charité already have a wide expertise concerning multi-photon imaging within explanted organs, e.g. hippocampal brain slices (Dr. Jan Leo Rinnenthal, Dr. Helena Radbruch, Prof. Frank Heppner, Institute for Neuropathology, Charité) and spleen slices (Prof. A.E. Hauser, Dr. R. Niesner), and even more, within living organisms in intestine (group of Prof. A.E. Hauser), lymph node (group of Prof. A.E. Hauser), bone-marrow (group of Prof. A.E. Hauser) and organs of the central nervous system, i.e. brain stem (Dr. Helena Radbruch), brain cortex (Dr. Jan Leo Rinnenthal, Prof. Frank Heppner, Simon Bayerl, Prof. Vajcozy) and neuronal retina (Dr. R. Niesner, Prof. Paul). Currently, we focus our attention in extending this expertise to intravital spleen and kidney imaging, to longitudinal bone marrow endoscopy and to the use of optical coherence tomography in the mouse eye.
While extending the expertise in the field of deep-tissue and intravital multi-photon imaging with applications especially in immunology plays a central part in the work at the DRFZ imaging core-facility CINIMA, tight collaborations with other imaging facilities in Berlin as well as with systems biology groups ensure the access of DRFZ scientists to a broad palette of fluorescence imaging techniques and data evaluation. In 2011 JIMI (Joint network for Intravital MIcroscopy) was founded in order to bundle the expertise of Multi-Photon intravital imaging present at the DRFZ, Berlin (Dr. R. Niesner und Prof. Dr. A. Hauser) with the expert knowledge of microscopy at the MDC (Dr. A. Sporbert) and image analysis present at the HKI in Jena (Prof. M.T. Figge) . The goal of JIMI is to make intravital microscopy accessible for researchers from different fields in life science. We provide help in planning and performing intravital microscopy experiments as well as in the analysis of the data. This is completed by consulting and assistance through regular seminars of the JIMI members and of invited experts of the field of optical imaging in biosciences and biomedicine. Between 2012 and 2016, JIMI has been granted funding by the DFG. In this time period, JIMI supported 38 projects out of 8 national and international institutions and published 27 scientific articles (www.jimi-network.de). Furthermore, JIMI is majorly involved in leading national and international imaging networks like CTLS (headed by Prof. Spencer Shorte, Imagopole, Institute Pasteur, Paris) or German BioImaging (led by Prof. Elisa May, University of Konstanz) and closely cooperates with leading microscopy core-faclities as well as microscopy companies in Germany, France and UK. To further increase the access of the JIMI users to information and to enhance scientific exchange, we organized yearly work-shops dedicated to live and intravital microscopy: the Imaging cytometry meetings at the annual meeting of the DGfZ (German Society for Cytometry), in October 2015 and October 2016, both in Berlin.
Fluorescence Life Imaging
Prof. Dr. Anja E. Hauser
Dr. rer. nat. Raluca Niesner
Fluorescence Life Imaging