MitoCare Service Centers
Mission, Goals, Capabilities
The Research Electron Microscopy Laboratory in the Department of Pathology, Anatomy and Cell Biology aims to provide researchers with ultrastructure in specimen prepared from tissue, cellular or subcellular preparations. We are also open (on case by case base) to collaborative consultations to assist users in TEM related experimental design and analysis. Current capabilities allow 2D transmission electron microscopy. Tilt-angle tomography of ~150 nm thick sections of resin-embedded samples (or negative-stained particles) is currently suspended until further notice*.
The core instrument is an FEI Tecnai 12 120 keV digital TEM, equipped with an AMT XR111 CCD camera and upgraded with a tomography package that uses the FEI Xplore 3D Tomography software. Addition of a high field of view tomography holder and Xplore3D retrofit package allows acquisition of electron tomograms at high tilt angles. The sample preparation laboratory has two Leica UCT ultramicrotomes.
Services available include:
TEM Scoping Services
User levels: Full service (100% by facility personel).
Assisted use. May require training. User navigates over the specimen grid, sets magnifications, adjust focus, acquire and save images. Operator assists with (un)loading the specimen to(from) the scope and alignments. Scoping time is limited by the availability of the support/supervising personnel.
Self (unassisted) use. Requires previous experience in TEM operation AND specific experience with the FEI Tecnai 12 TEM. Self-user shall pass an initial skill assessment by the core director/manager. Scoping time can include afterhours/weekend.
TEM Sample Preparation Services
Cover preparation steps that are required to ready a specimen for TEM imaging. Tissue/cell sample acquisition and primary fixation (glutaraldehyde, paraformaldehyde or both) is the user/client responsibility. For consultation or training regarding primary fixation, please contact Dr. Csordas.
- Processing & Embedding services include secondary fixation (osmification), contrasting (usually uranylacetate), dehydration and embedding of the sample to resin blocks. Offered for chemically fixed (primary fixation) tissue or cell culture samples.
Negative staining of membrane particles, or molecules. Particle isolation and obtaining glow-discharged carbon-coated TEM grids is the user’s responsibility. We will advise as needed in obtaining glow-discharged grids. We do not offer staining of live infective particles such as virus particles on site: for that, only scoping of already negative-stained grids can be requested.
Embedding for Pre-embedding Immunogold.
Consultation must precede request! Samples are Adherent Cells plated on NUNC Thermanox plastic coverslips (15 mm round) in 12 well plates. First fixation, and immunolabeling is the user’s responsibility.
- Sectioning services offer the cutting of thick (toluidene blue-stained) sections for checking sample orientation, as well as (ultra)thin sections for TEM imaging. User can request post-staining (e.g. uranylacetate, lead citrate) of the thin sections for stronger contrast, for additional cost.
Special: thin sections from adherent cells (mostly for Pre-embedding Immunogold). The cells are embedded at the bottom of a platic (resin) disc. Small piece(s) with cells is cut out from the disc, mounted to a larger resin (dummy) block so it can be held in the ultramicrotome for sectioning. The monolayer of cells will be right under the cutting surface, which makes the sectioning more delicate and time-consuming than standard thin sectioning; thus higher-priced.
*Tilt-angle tomography requires a currently missing part, the tomography sample holder, for which a capital budget request is submitted.