Subproject Z02: Light and electron microscopy

Principle Investigator:

Dr. York-Dieter Stierhof
Universität Tübingen

ZMBP, Zentrale Einheit Mikroskopie

Auf der Morgenstelle 5
Tel 07071-2976662/3

Fax 07071-293287

york.stierhofspam prevention@zmbp.uni-tuebingen.de

Summary:

The focus of the light (LM) and electron microscopy (EM) service/research unit of the ZMBP is on cryoimmobilisation-based specimen preparation techniques, detection of low copy number antigens, correlative LM and EM (CLEM), and high resolution/superresolution LM. The LM equipment includes a confocal laser scanning microscope (CLSM) Leica SP8 (resonant scanner, HyD hybrid detector, fluorescence lifetime imaging microscopy (FLIM), fluorescence correlation spectroscopy (FCS)) for fluorescence resonance energy transfer fluorescence (FRET)-FLIM, fluorescence intensity decay shape analysis microscopy (FIDSAM) and single molecule spectroscopy, a CLSM Zeiss LSM 880 (high-resolution (fast) Airyscan, GaAsP detectors), a custom-built direct stochastic optical reconstruction/photoactivated localization (dSTORM/PALM) superresolution LM (also FRET-FLIM, spectral mapping, single particle tracking (spt)PALM, highly adaptable to additional applications), and an epifluorescence microscope Imager M2 (Hamamatsu ORCA-flash 4.0 sCMOS camera, apotome). The EM equipment includes a Jeol TEM 1400plus (4K CMOS camera (tiling/stitch­ing/tomo­graphy)) and a Hitachi S-800 SEM. In addition a cryo-field emission gun- (FEG)-SEM in the Geoscience Institute (scanning transmission EM (STEM), (cryo-)FIB (focussed ion beam technology for serial block-face 3D reconstructions)) and an analytical double-corrected 200 kV (S)TEM (high-resolution 3D reconstructions) in the Natural and Medical Sciences Institute will be installed in 2018/2019. This year, a helium ion microscope was installed at the Institute for Applied Physics; core facility LISA⁺). Extensive EM projects are included in TPs A01, A02, A04, A06, B06, C03, C05, C06, C07, D06, D08, and D09, superresolution LM projects in TPs A04, A06, B06, C03, D02, D03, and D09. Preparation techniques required include: Cryofixation/high-pressure freezing (at MPI/Tübingen), conventional dehydration, low temperature dehydration and embedding, freeze-substitution (FS), embedding in epoxy resins and methacrylates for immunolabelling, serial sectioning for 3D reconstruction, cryosectioning (cryo-ultramicrotome Leica UC7/FC7), immunogold labelling of thawed cryosections (Tokuyasu technique) and of resin sections (& quantitative analysis), CLEM techniques (including superresolution LM), and fast sample processing (microwave). 3D reconstruction techniques using serial block-face imaging (FIB-SEM) has to be established.