Live Cell Imaging

Scientific Focus

a) The instructive extracellular matrix

Figure 1: Alterations and cues within the extracellular matrix of the lung can instruct cells to modify their cellular behaviour. (Burgstaller G, et al. The instructive extracellular matrix of the lung: basic composition and alterations in chronic lung disease. Eur Respir J 2017.)

The main scientific focus of the platform “Live Cell Imaging” lies on identifying novel mechanisms of cell invasion and cell migration, but also on the instructive function of the extracellular matrix (ECM) and how this influences the behavior of cells. Aberrant migration behavior of cells, their invasion and deposition of ECM, are inherent features of many lung diseases (cancer, metastasis, interstitial lung diseases including IPF).

b) High-throughput screening for novel inhibitors of aberrant ECM deposition

Figure 2: High-throughput screening of inhibitors of ECM deposition and their validation with precision cut lung slices

The current approved pharmacological therapies mostly slow down, but do not stop disease progression. Therefore, the identification of novel therapeutic targets and potential pharmacotherapeutics is a major clinical need. Together with a solid knowledge of cellular mechanisms, we seek to develop and establish several medium- and high-throughput assays (96- and 384 well plates) for the screening of large compound libraries to discover novel therapeutically exploitable small molecules, which specifically block pathological functions in cell invasion and ECM deposition.

c) Advanced lab and imaging techniques

Figure 3: Advanced 4D imaging of precision cut lung slices. (Source: Gerald Burgstaller, HMGU // Adapted according to Burgstaller G, et al. Multidimensional immunolabeling and 4D time-lapse imaging of vital ex vivo lung tissue. Am J Physiol Lung Cell Mol Physiol 2015).

We apply a combination of standard laboratory techniques (Immunohisto(cyto)logy, Western-Blotting, qPCR, primary cells), various mouse disease and gene knock-out models, human and mouse ex-vivo lung tissue slices (PCLS), all together with advanced (live) staining and 4D imaging techniques (Lightsheet-, Laserscanning-, 2-Photon-, Superresolutionmicroscopy). Ongoing collaborations include state-of-the-art know-how such as CripsR/Cas, proteomics, transcriptomics (microarray technology), and single-cell RNAseq. Furthermore, we are in constant touch and information interchange with clinicians to enhance efforts in translational science.

Imaging Core Unit:

The CPC’s imaging core unit currently manages several state-of-the art microscopy systems, including widefield/brightfield microscopy (Leica M205FA, Zeiss AxioImager2 equipped with two AxioCams and an Apotome System), confocal laser scanning microscopy (Zeiss LSM 710), a stand-alone confocal spinning disk system for high-throughput fluorescent applications (BD Pathway), an automated slide scanner (Zeiss Mirax Scan), a Zeiss PALM MicroBeam microdissection system for contamination-free specimen capture and  a high-speed live cell imaging system (Zeiss AxioObserver Z1 including an incubation system for long-time experiments under physiological conditions, autofocus and a high-sensitive EMCCD camera). Furhermore the imaging core unit is equipped with a state-of-the-art Lightsheet fluorescence microscopy (LSFM, SPIM, Zeiss Z1) for the fast and gentle live 4D imaging of vital ex-vivo tissue, 3D organoids/spheres, and whole organs. Most of our systems are motorized and thus provide the necessary automation for high-throughput applications for fixed samples and living cells or tissues. Especially the live cell imaging system is apt for the observation of dynamic subcellular structures (molecules and organelles) over time (2D), in space (3D), in various colours (4D) and at different positions (5D). Two highly powerful computer workstations combined with a server for data storage provide a good deal of essential software tools for digital image analysis, 3D reconstruction of Z-stacks, deconvolution, cell/molecule/object tracking and tracing, morphometry and colocalization (ZEN (Zeiss) including a deconvolution package, Imaris (Bitplane), Vision4D (Arivis)). The imaging core unit trains microscope users, provides expertise in all microscope related issues, helps in experimental planning and assists in image analysis and processing.