Research Report 2010 - MDC

Structure of the GroupGroup LeaderDr. Anje SporbertStart of group: March 2008Anje SporbertConfocal and 2-Photon MicroscopyIn March 2008, a microscopy core facility was established at the MDC focusing on confocal and2-Photon fluorescence microscopy. Starting with two confocal laser scanning microscopes inone room the facility provides now an instrumentation of seven different microscopes eachoptimised for specific applications in three rooms: four confocal laser scanning microscopes withdifferent configurations (one equipped with an 2-Photon laser), one wide-field fluorescencemicroscope with two lasers for TIRF-Illumination, one multifocal-2-Photon laser scanningmicroscope and one wide-field fluorescence microscope with UV-laser for laser-assisted microdissectionand catapulting. All microscopes are centrally located in rooms equipped with asterile bench, a CO 2 -cell incubator, a small lab bench and gas supplies for the microscope stageincubators offering an ideal environment also for live specimen experiments.The aim of the Microscopy Core Facility (MCF) is to provide researchers at the MDC a choice ofhigh-end fluorescence microscopes and the necessary support to enable them to performadvanced imaging experiments with different specimens ranging from fixed cells and tissuesections to live cells and organisms.Potential applicationsIn confocal microscopy lasers are scanned line-wiseover the sample to excite the fluorophores in the specimen.The emitted fluorescence is detected with a digitaldevice via a pinhole in front of it. This pinholerestricts the detected fluorescence to light originatingonly from the focal plane.The benefits of this techniqueare (i) a better separation of spectrally overlapping fluorophoresas often used in multi-colour fluorescencelabellings in many immuno-fluorescence techniquesand (ii) a higher axial resolution by the optical z- sectioningeffect which is especially prominent in thickerspecimen.The confocal microscopes in the MCF are equipped withdifferent sets of lasers and objectives to offer optimalconditions for multi-colour fluorescence imaging ofstructures ranging from the subcellular level to themorphology of small organisms (see Figure A/B).Stage incubators on three of the four confocal microscopesallow for live specimen imaging by controllingthe temperature and CO 2 -environment. This is a prerequisitefor short and long time-lapse experiments toobserve, i.e. the development of zebrafish/mouseembryos (Figure B), the migration of cells, the movementof organelles (Figure C) or the different stages ofthe cell cycle.The possibility to direct the laser beam to a definedposition and illuminate only this area in the sampletogether with the development of fluorescent proteinswith new properties opens up a range of exciting experimentaltools for live specimen at the confocal microscopes:a) Photobleaching a specified area in the specimenand observing how fast the fluorescence recoversinto that area (“FRAP”) gives indications on the224 Technology Platforms