2D & 3D Electron Microscopy

• Provide reliable and equitable access to EM instrumentation and expert support.
• Deliver high-quality imaging and specimen preparation services.
• Provide consultation and technical guidance on experimental and analysis strategies to identify ultrastructural phenotypes, with particular expertise in those involving mitochondria and membranous organelles.
• Develop user competence through structured training and certification.
• Encourage internal and external collaborations.
• Maintain a sustainable shared-resource model that maximizes scientific impact, publications, grant competitiveness, and institutional return on investment.

• TEM (unrestricted service accessibility): Thermo/FEI Tecnai 12 low-voltage (Max. 120 keV acceleration voltage) digital TEM fitted with a bottom-mounted AMT BioSprint12 12Mpx CCD camera. 
• COMING SOON: The 20+ year-old Tecnai 12 is to be replaced soon with a new Thermo-Fisher/FEI Talos 12 120 kV TEM fitted with an embedded Ceta 16 Mpx CMOS camera and configured for automated montage imaging and tilt-angle tomography workflows. 
• LIMITED SEM : Only for select samples, with conditional accessibility! The Elstar electron column of the Helios 5 FIB-SEM can perform standard and ultra-high resolution (down to ~1 nm) SEM imaging. For topographical and heavy metal stain-contrast images back-scattered and secondary electrons can be captured using conventional Everhart-Thornley detector or via through-the-lens and in-column detectors as well as a directional backscattered electron detector (DBS). *The SYSTEM IS PRIORITIZED for 3D FIB-SEM PROJECTS, which requires extra clean specimen chamber and lengthy (24+ hour) recordings. We DO NOT ACCEPT hydrated samples, bulky organic samples (prone to outgassing under high vacuum), magnetic materials or powders.
  SEE SERVICES for ACCESSIBILITY LIMITATIONS

*NOTE: The Helios system is prioritized for FIB-SEM (dual-beam) workflows. Single-beam SEM imaging requests are only accepted on a case-by-case base, after consultation, without hard commitment to completion time. 

• FIB-SEM. Thermo-Fisher/FEI Helios 5 CX focused ion beam scanning EM (FIB-SEM) with a high-resolution Tomahawk ion column with Ga+ liquid metal ion source. From resin-embedded biological specimen, using the Auto Slice-and-View 4 (ASV 4.x) automated acquisition application software, the system is capable to deliver image stacks with isotropic (xyz) resolution down to ~2 nm. The maximum stack volume at each resolution is limited by the total acquisition time, which, for practical reasons, is capped at 48 hours (approximately 2000 image slices). Individual exemptions from the 48 hour cap will be considered based on usage load. The stack dimensions below reflect these limitations at 4096 x 3536 pixels scanning array.

            Voxels    x(um)   y (um)   z(um)

            2 nm         8.2       7           4

            3 nm        12.3     10.6       6

            4 nm        16.4     14.1       8

            5 nm        20.5     17.7      10

            6 nm        24.6     21.2      12

Larger scanning array (6144 x 4096 pixels) is available, but the acquisition speed will be slower, thus limiting z progression to <2000 slices in 48 hours. 

• COMING SOON: TEM electron tomography (tilt-angle series). The new Talos 12 will be capable of tilt-angle tomography of 120-150 nm-thick sections.

• FIXATION: Chemical fixation consultations. To maintain a biosafety level 1 (BSL1), chemical fixation that renders the sample inanimate (kills living organisms) must be done in the user’s laboratory. We offer consultation in fixation strategies/approaches.
• COMING SOON: High-pressure freezing (HPF) and freeze-substitution (FS). We have a Leica EM ICE HPF instrument capable of instantaneous vitrification of small-volume cellular or subcellular samples providing better preservation of the native ultrastructure than chemical fixation. This is followed by the FS procedure using a Leica EM AFS2 automated FS and low-temperature embedding system. Please check back later when this service goes live.   
•  EMBEDDING SERVICES: processing the chemically fixed sample (via steps of post-fixation w/ or w/o en bloc heavy-metal contrast staining, dehydration, resin infiltration and resin curing) to become embedded in a hard-plastic block suitable for sectioning by ultramicrotome and for imaging in the ultra-high vacuum inside the EM. Protocols are categorized by the type of the fixed sample (tissue, cell pellet or cell monolayer) and the intended imaging modality (TEM, FIB-SEM). See details in the Price Table & Calculator Excel document.
  
  
• STANDARD SECTIONING SERVICES for TEM include the cutting of (ultra)thin (50-100 nm) sections from the resin-embedded blocks and picking them up on standard copper mesh grids (or other optionally available EM grid type). Two Leica UCT ultramicrotomes are used for this by our expert technical personnel.
  • Thick (aka semi-thin) sections (0.5-1 um thick) mounted on glass slides and stained with toluidine blue can be requested to prescreen for quality (e.g. even fixation) orientation and gross content of the block.
  • Post-staining of the thin sections with heavy metals (uranyl acetate, lead citrate) to further enhance structure contrast is also offered.

• NON-STANDARD SECTIONING:
  • Sectioning of embedded monolayers with the ultramicrotome requires fragmenting the disc-like block and mounting fragments to empty ‘dummy’ blocks with acrylic glue.
  • Leveling and quality-checking of blocks for FIB-SEM (pre)require cutting out of the biological sample-containing piece of the resin block and mounting it on an SEM pin stub using a conductive silver paste.

• NEGATIVE STAINING: On-the-grid “drop-stain-dry” method to outline particles of interest with heavy metal stain(s). Used for fast prescreening of purified protein preparations before single-particle cryo-EM or for the crude visualization of membrane vesicle preparations or molecular aggregates. It requires a glow-discharged support film grid (supplied at market price). A glow-discharge device is readily available in a neighboring cryo-EM prep lab. WARNING: this service is not available for staining or imaging materials known to be infectious to vertebrates (e.g. certain bacteria or viruses)!
• SPUTTER COATING: Non-conductive resin-embedded biological samples require a conductive film coat to prevent charge build-up during SEM imaging. Routinely, we coat the sample with a 4 nm gold/palladium film using a Leica ACE600 high-vacuum single-sputter coater with Au/Pd, 80/20, 17.9 g/cm3, 0.17 mm Target.

• Current TEM: Thermo/FEI Tecnai 12, 120 kV digital TEM with a bottom-mounted AMT BioSprint12 12Mpx CCD camera. Location: 552 JAH
• Successor TEM: Thermo-Fisher/FEI Talos 12 120 kV TEM with an embedded Ceta 16 Mpx CMOS camera. Configured for automated montage imaging and tilt-angle tomography. Location: TBD
• FIB-SEM: Thermo-Fisher/FEI Helios 5 CX Gallium FIB-SEM with MAPS correlative and montaging application and Auto Slice & View (ASV 4) volume EM auto-acquisition software. Location: 443 JAH
• Au/Pd Sputter Coater. Leica ACE600 high-vacuum single-sputter coater with Au/Pd, 80/20, 17.9 g/cm3, 0.17 mm Target. Location: 443 JAH
• HPF. Leica EM ICE for HPF and Leica EM AFS2 for FS. Location: 552 JAH