The Cryogenic Transmission Electron Microscopy (TEM) suite supports EMSL’s mission to improve our fundamental understanding of environmental and biological systems through
- High-resolution imaging and analyses of bacterial-mineral interactions
- Characterization of complex biosystems in normal and environmentally stressed conditions, including microbial communities, biofilms, plants, fungi, and host-viral interactions
- Morphological studies of cellular components, such as photosynthetic units, plant organelles, and microbial extracellular polymeric substances
- 3-dimensional (3-D) reconstruction by TEM tomography of cellular and environmental features
- Immunogold labeling of frozen-hydrated or plastic sections for localization of immune response in cells.
Supporting the Biogeochemical Transformations, Cell Signaling, and Communications and the Rhizosphere Function integrated research platforms, Tecnai cryoTEM can characterize a wide variety of microbial-mineral interactions. Across spatial scales, the structures of interest can be imaged on tens of microns scale down to sub-nanometer resolution.
Tecnai and TEM sample prep suite includes the following instrumentation:
- Tecnai T-12 TEM (FEI)
- LaB6 filament
- Operation voltage 120 kV
- Tomography stage +/- 70 deg
- Cryostage (liquid nitrogen)
- 1.6A maximum spatial resolution
- 2 x 2K Ultrascan 1000 CCD
- High Pressure Freezer (Leica)
- Cryo-immobilization of aqueous samples under high pressure
- Cryo-fixation of cellular constituents without introducing significant structural alterations
- Automatic Freeze Substitution and Low Temperature Embedding System Leica EM AFS2
- Freeze substitution and progressive lowering of temperature for embedding and polymerization of resins
- Leica UC7 Ultramicrotome
- Precise cryo sectioning
- 50- to 120-nm frozen-hydrated sections of cellular material
- 120-nm sections of soft materials such as polymers
- Ultracut UCT Ultramicrotome (Leica)
- Precise room temperature sectioning
- 50- to 120-nm for ultrastructural TEM imaging
- 150- to 300-nm for TEM tomography
- 300- to 1,500-nm for material analyses
- Immuno-gold Processing Platform
- for immunocytochemistry incubations
- Dissecting Microscope (Leica).
Tips for success
- Compatible research samples include
- Biosystems in normal and environmentally stressed conditions
- Unicellular and filamentous organisms, cell cultures, and biofilms
- Plant-microbe-fungi interactions under different environmental conditions
- Host-viral/phage interactions
- Micro- and nanocrystals of natural minerals
- Manufactured particles and polymers.
- Sample preparation plays a critical role in successful imaging.
- The best ultrastructural detail is obtained by following the cryopreparation protocol when cell environments in culture conditions are preserved until the moment of high-pressure freezing, which occurs on a millisecond timescale.
- Traditional chemical processing also provides valuable information. We can customize the processing scheme according to the sample nature.
- Advanced sample preparation methodologies are also available, such as high-pressure freezing followed by automatic freeze substitution for material embedding, and ultramicrotomy for preparation of ultrathin sections.
- 3-D resolution for electron tomography is highly dependent on sample thickness, sample tolerance to electron dose, and desired field of view. Most tomography datasets will be limited to 4-nm resolution.
- Samples need to be shipped to the TEM lab either unfixed on blue ice or in a fixative.
- Upon request, we can perform the cell culture in our laboratory from microbial inoculum or plates, including temperature-controlled incubation and light conditions.
- Anaerobic processing is also available for redox systems sensitive to oxygen. Samples can be processed in an anaerobic glovebag.
- Low-dose image acquisition is available and recommended for samples sensitive to radiation damage and, generally, for cryo imaging.
- Access to cryo-TEM, along with other capabilities at EMSL, is gained through the standard EMSL user proposal call submission process.