2D & 3D Electron Microscopy
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Missions, Goals, Capabilities
MitoCare’s 2D & 3D EM Laboratories in Jefferson Alumni Hall aim to provide researchers with 2D and/or 3D ultrastructure of specimens prepared from tissue, cellular or subcellular preparations. We also offer service or collaborative consultations in ultrastructural experimental design and analysis. Current capabilities cover 2D TEM with image outputs of <1nm pixel resolution of positively or negatively stained samples. 3D focused ion beam scanning electron microscopy (FIB-SEM) uses the ‘auto slice & view’ workflow to generate image stacks of up to 20um x 20um x 20um volume at 3-to-10 nm isotropic voxel resolution of fixed, stained, and resin-embedded tissue or cell samples. Sample processing laboratory, ulramicrotomes for sectioning and Au-Pd sputter coating for FIB-SEM/SEM samples.
Electron Microscopes. TEM: FEI Tecnai 12 120 keV digital TEM, fitted with a bottom-mounted AMT BioSprint 12 MPx CCD camera. FIB-SEM: ThermoFisher Scientific/FEI Helios 5 CX DualBeam FIB-SEM with quick-loader and added STEM3 detector.
Sample Preparation Laboratory. Houses two Leica UCT ultramicrotomes and a Leica ACE600 high-vacuum sputter coater with Au-Pd target and film thickness sensor.
Service Consultation Fee: $50 for 30 minutes
For more details on services and pricing
Services available include:
I. SCOPING (EM IMAGING) SERVICES
One of our main contacts at ThermoFisher (manufacturer of the TEM and FIB-SEM) brought this nuance to my attention, saying that he felt this was a less commonly used word so it is better to define it.
TEM Scoping Services
Full service, 100% performed 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. Available 8:30am-3:30pm.
Self (unassisted) use. Requires previous experience in TEM operation AND specific experience with the FEI Tecnai 12 TEM. Self-user shall have passed an initial skill assessment by the core director/manager. Availability include afterhours/weekend.
FIB-SEM Scoping (Auto Slice&View, ASV) Service
ASV setup, currently full service only.
ASV is an automated workflow. Generally limited to 24h max. acquisition time.
II. EM SAMPLE PREPARATION SERVICES
Include preparation steps to ready a specimen for EM imaging. Pre-fixation tissue/cell sample acquisition and primary fixation (glutaraldehyde, paraformaldehyde or both) are the user’s/client’s responsibilities. For consultation or training regarding primary fixation, please contact Dr. Csordas.
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; separately for TEM vs. FIB-SEM.
Negative staining of membrane particles, or molecules. Particle isolation and obtaining carbon-coated TEM grids is the user’s responsibility. We will advise as needed in glow-discharging the grids. We do not handle live, potentially infective particles such as virus particles on site. For that, only the 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.
Post-Embedding processing and Sectioning services include specimen mounting procedures preceding the sectioning of cell monolayer samples or the finishing/leveling of FIB-SEM samples. Sectioning Services. Thick (toluidene blue-stained) sections for checking sample orientation with light microscope. (Ultra)thin sections for exploratory or quality-checking (from blocks for FIB-SEM) TEM imaging. User can request post-staining (e.g. uranylacetate, lead citrate) of the thin sections for stronger contrast, for additional cost. ASV Prepping includes the leveling and surface-finishing of FIB-SEM samples mounted on SEM stubs using the diamond knife of the ultramicrotome. Sputter Coating includes the layering of a conductive Au-Pd film over the non-conductive resin-embedded specimen.