mechanical constraints; pollen tube; plant hormones; plant reproduction; mycorrhiza; disease resistance; cell biology; deep tissue live imaging; chromatin; gametophyte; plant development
Orosa-Puente Beatriz, Leftley Nicola, von Wangenheim Daniel, Banda Jason, Srivastava Anjil K., Hill Kristine, Truskina Jekaterina, Bhosale Rahul, Morris Emily, Srivastava Moumita, Kümpers Britta, Goh Tatsuaki, Fukaki Hidehiro, Vermeer Joop E. M., Vernoux Teva, Dinneny José R., French Andrew P., Bishopp Anthony, Sadanandom Ari, Bennett Malcolm J. (2018), Root branching toward water involves posttranslational modification of transcription factor ARF7, in Science
, 362(6421), 1407-1410.
Andersen Tonni Grube, Naseer Sadaf, Ursache Robertas, Wybouw Brecht, Smet Wouter, De Rybel Bert, Vermeer Joop E. M., Geldner Niko (2018), Diffusible repression of cytokinin signalling produces endodermal symmetry and passage cells, in Nature
, 555(7697), 529-533.
Grossmann Guido, Krebs Melanie, Maizel Alexis, Stahl Yvonne, Vermeer Joop E. M., Ott Thomas (2018), Green light for quantitative live-cell imaging in plants, in Journal of Cell Science
, 131(2), jcs209270-jcs209270.
Vermeer Joop E.M., van Wijk Ringo, Goedhart Joachim, Geldner Niko, Chory Joanne, Gadella Theodorus W.J., Munnik Teun (2017), In Vivo Imaging of Diacylglycerol at the Cytoplasmic Leaflet of Plant Membranes, in Plant and Cell Physiology
, 58(7), 1196-1207.
In recent years, fluorescence-based microscopical imaging of living tissue has greatly advanced our understanding of plant developmental processes at the cellular level. This has largely been possible due to the use of Arabidopsis as a model species. The fast and straightforward generation of stable transgenic lines expressing fluorescent cellular markers allows for 3-dimensional (3D) imaging of developmental processes using confocal laser scanning microscopy (CSLM). The intimate relationship between the organization of tissues in all cell layers, their functional specialization, and the overall tissue morphology implies that a comprehensive understanding can be only be achieved using non-invasive imaging approaches throughout the depth of a tissue. Yet, 3D-microscopical imaging of living plant tissue is confronted by specific challenges including, (i) the inherent lack of optical clarity and strong light-scattering nature of plant cells [due to cell walls and numerous autofluorescent compounds] that prevents an efficient penetration/readout of the excitation/emitted light in deep tissue layers; (ii) the relative thickness of plant tissues exceeding the working distance of most objective lenses; and (iii) the moderate to low output signals from spatially and temporally restricted fluorescent markers are poorly captured with standard detectors. These technical limitations severely impede several research projects at the Institute of Plant Biology (IPB) that focus on cell-based mechanical constraints in organogenesis, hormone signaling in physiological responses, cellular dynamics in reproduction and pathogen responses in several plant species e.g. Arabidopsis, Marchantia, petunia, maize and wheat. All these research projects rely on the analysis of structural, physiological and signaling components in living, intact plant tissues throughout multiple cell layers and at subcellular resolution. Novel, non-invasive imaging techniques that offer fast imaging in deep tissue layers with enhanced resolution and sensitivity can overcome these limitations. To make a quantum leap in our capacity to analyze developmental processes at subcellular resolution and in order to remain competitive with top plant research groups in Europe we are applying for an all-in-one confocal/multiphoton laser scanning microscopy system that offers, in addition to the traditional confocal mode, (i) multiphoton excitation for imaging of deep-lying tissues, and (ii) an innovative Airyscan detector that combines high sensitivity with enhanced resolution properties. These requirements are currently met by a unique, commercially available system: the Zeiss LSM880. This confocal system can be equipped with near infrared lasers for multiphoton excitation and the revolutionary Airyscan detector (a 32 GaAsP-array detector offering the best currently available sensitivity], providing a 1.7x increase in resolution in all dimensions resulting in a 5x smaller confocal volume, without limitations regarding sample depth and acquisition speed. In addition, the multiphoton laser also allows to specifically ablate cells in deep-lying tissues. These ablation experiments are essential to our projects aiming at manipulating mechanical constraints and to assess the role of the positional information and interactions with surrounding cells during development. We, as well as Urs Ziegler (head of the Center for Microscopy and Image Analysis, ZMB) have tested the LSM880 system with Airyscan and multiphoton excitation (April 2015) and agree that this is currently the best system to solve the above problems. Yet, as other systems with similar properties are expected to become available, we will thoroughly and objectively assess them to choose the optimal system for our research projects.The high-end technology offered by the proposed all-in-one multiphoton confocal imaging system will provide a major impulse for our studies in multiple species and will help us to perform internationally competitive, top-level research at IPB. It will uniquely complement our existing high-end microscopy imaging platform comprising wide-field fluorescence microscopes and CSLM instruments, thereby considerably increasing the attractiveness and international competitiveness of IPB. Importantly, the ZMB of the University of Zürich does not possess the imaging instrument as requested here. In his continuous efforts to foster excellence in biological imaging, and with the shared vision that this instrument will meet interest beyond the plant science community, the Head of ZMB, Urs Ziegler, fully supports our proposal. Consequently, the requested equipment, while physically located at IPB, will be imbedded in the ZMB infrastructure thereby offering access to all ZMB users, thus guaranteeing optimal maintenance and user training. In the following part, we will describe several projects for which the requested equipment will be of central importance.