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Cryogenic Transmission Electron Microscopy for Environmental Microbiology

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.

Research application

Supporting the Biogeochemical Transformations, Cell Signaling and Communication 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.

Available Instruments

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.

Contacts