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Raluca Niesner, Deutsches Rheuma-Forschungszentrum, Berlin, Germany
Martin Fuhrmann, German Center for Neuredegenerative Diseases, Bonn, Germany
Techniques for imaging and image analysis made substantial progress due to major improvements in microscope techniques during the recent years. The mere documentation of a specimens’ status quo was replaced by in-vivo imaging, single cell and 4D analysis, spatio-temporal analysis of sub-cellular components, and the analysis of cells in the context of their environment to name just a few. Not least these techniques to some extent account for the dawn of new fields of research such as tissomics, toponomics, and cytomics.
Advances in dynamic intravital two-photon microscopy - focus on optical performance and molecular specificity
1Deutsches Rheuma-Forschungszentrum Berlin, Germany; 2Neuroimmunology in ECRC, Charité – University Hospital, Berlin; 3Institute for Neuropathology, Charité – University Hospital, Berlin; 4LaVision Biotec GmbH, Bielefeld; 5Dermatology, Eppendorf University of Medicine, Hamburg; 6BSRT and Laboratory for Molecular Psychiatry, CC15, Charité – University Hospital, Berlin
Understanding physiological and pathological mechanisms in hardly accessible organs necessary, for instance, to develop novel selective therapy strategies, implies the investigation of cellular dynamics, cell-cell interactions and cellular function in genuine environment: the organism. Two-photon laser-scanning microscopy best enables highly-resolved visualization of cellular motility in the living organism, i.e. intravitally. However, limitations of standard TPLSM based on Ti:Sa laser excitation and photomultiplier detection, which are related to still reduced penetration depth, depth-dependent deterioration of the 3D spatial resolution, photobleaching of the cromophores, photodamage of the tissue and unspecific autofluorescence, prevents us from unequivocally monitoring and quantifying cellular communication intravitally. The benefits of two technological improvements of TPLSM for dynamic intravital imaging are discussed here: an infrared (IR) excitation source, i.e. the optical parametric oscillator, and a structured-illumination-like laser-scanning technique, i.e. striped-illumination multi-beam TPLSM. Using these technical advancements, the communication between antigen-carrying units on the surface of follicular dendritic cells and B-cells within the germinal center of the popliteal lymph node in living mice could be imaged. Moreover, the cellular function, which is the key of tissue and organ function, is still a hardly to access information for the most interesting studies, i.e. dynamic, highly-resolved studies in the living organism. Concretely referring to the neuronal function, it is reliably given by the intracellular Ca-level, which can be measured in CerTN L15 mice expressing a FRET-based Ca-biosensor in neurons. Here we present for the first time truly quantitative and dynamic Ca-level monitoring with diffraction-limited resolution in the brain of living mice by means of FRET-Fluorescence Lifetime Imaging (FLIM). The FLIM technique is based on a synchronized multichannel multi-detector TCSPC device. Using this method we are able to monitor the changes in the neuronal function, which build the roots of neurodegeneration.
Two photon in vivo imaging in neurodegenerative diseases
German Center for Neurodegenerative Diseases, Germany
Due to increasing age and lifespan the incidence of dementia is rising. Neurodegenerative diseases like Alzheimer's and Prion disease are characterized by the deposition of malfolded aggregated proteins. Hallmarks of these diseases represent neuron and synapse loss leading ultimately to cognitive decline. Moreover, neurodegenerative diseases exhibit a strong neuroinflammatory component that influences disease progression. Repetitive two photon in vivo imaging over the progression of disease allows to analyze kinetics of disease specific details like synapse and neuron loss. Moreover, the use of transgenic and knockout animal models enables to recapitulate and to interfere with neurodegenerative diseases.
Optogenetics in cardiovascular research: a new tool for light-induced depolarization of cardiomyocytes and vascular smooth muscle cells in vitro and in vivo
University Bonn, Germany
Purpose: Electrical stimulation is commonly used for activation of myocytes in clinics as well as in cardiac research. However, this has considerable side effects, lacks cell specificity and longer lasting or sub-threshold depolarization are not possible. In addition, electrical stimulation is problematic in loosely coupled cells such as vascular smooth muscle cells. We aimed to overcome these limitations by expression of channelrhodopsin 2 (ChR2), a light-gated cation channel, in muscle cells in order to stimulate these using light.
Methods: Embryonic stem cells were transfected with a plasmid encoding a ChR2-EYFP fusion protein under control of the chicken β-actin promoter and were used for generation of transgenic mice by diploid aggregation. Functional expression of ChR2 was analyzed in cardiomyocytes and transfected A7R5 VSMCs using patch clamp experiments, in the heart in vivo using ECG-recordings and in the aorta using isometric force measurements of aortic rings with a wire-myograph.
Results: Membrane-bound ChR2-EYFP was detected in cardiomyocytes of the whole heart and in smooth muscle cells of the abdominal aorta. Isolated ventricular cardiomyocytes from the adult heart and ChR2-expressing A7R5 vascular smooth muscle cells showed large inward currents upon illumination with blue light, and brief pulses of light evoked free running action potentials. Light pulses on the atrium or ventricle of the ChR2 mouse in vivo induced atrial or ventricular pacing in the ECG. Efficient stimulation was observed even using illumination areas as small as 0.05mm² (corresponding to ~50 cardiomyocytes). Continuous illumination of ventricular areas led to spontaneous arrhythmic extrabeats. Aortic rings from the ChR2 mouse contracted upon illumination and the force was similar to noradrenalin-induced contractions with fast on- (< 800 ms) and off-kinetics (< 2.5 s). Contractions could be maintained up to 10 min by constant illumination.
Conclusions: Light induced depolarization of cardiomyocytes and smooth muscle cells expressing ChR2 allows non-contact stimulation of the heart and the aorta.
High-Content Cell-Based Phenotypic Screening: Applications of Automated Confocal Microscopy in Cell Biology
German Center for Neurodegenerative Diseases, Germany
With the advent of automated microscopy and sophisticated automated image analysis softwares the high-throughput screening world is facing an exciting challenge. A new cell-based perspective for high-throughput assay has become possible in alternative to classic homogeneous assays.
The possibility to quantitatively relate multiple parameters to each other, on a single cell based level, within a large cell population is opening new pathways for target discovery and the understanding of complex intracellular mechanisms. Here we show the results of the application of automated microscopy to an high content screening in a genome wide loss of function analysis for endocytosis function.
Finally we will discuss the future development of automated microscopy and the use in cell-based phenotypic assays