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Correlation Light and transmission or scanning Electron Microscopy for low expression proteins.

Coverslip options:

  1. Photoetch gridded coverslip for chemical fixation (ibidi, matsunami)
  2. Gridded glass-bottom dish by photo-etching for chemical fixation (ibidi, matsunami)
  3. Gridded sapphire coverslip by Quorum K950X Turbo Evaporator with Leica CLEM coating mount


Workflow for RT-CLEM (LM before the plastic embed):

  1. Culture tissue cells on the gridded coverslip, (in which the fluorescent proteins are expressed)
  2. Observe living cells by confocal fluorescence microscopy with gridded patterns
  3. Chemical fixation with half-strength Karnovsky’s solution (2% paraformaldehyde - 2.5% glutaraldehyde, EM-grade) in sodium cacodylate buffer including 2mM CaCl2 or NaHCa buffer
  4. Rapid freeze tissues or cultured cells by Leica EM-HPM100 Sapphire disc system or Variant CryoPress (as required)
  5. Freeze substitution by Leica EM AFS2 with FSP (as required)
  6. Infiltration and resin embedding in epoxy resin by Leica EM-TP
  7. Polymerization at 70 °C for a few days by (vacuum) oven
  8. Remove the coverslip to leave the gridded pattern
  9. Trim the plastic block to identify the correlated same field
  10. Semi-thin section and stain with 1% toluidine blue solution
  11. Ultrathin section by Leica EM UC7 or ARTOS-3D
  12. Grid stain with uranyl acetate (or alternative) and lead citrate
  13. Correlation imaging by TEM or SEM


Workflow for In-resin CLEM (LM after the plastic embed):

  1. General procedures for high-pressure freezing and freeze substitution
  2. Confocal fluorescence microscopy fluorescent molecule-tagged protein expressed cells in Lowicryl HM20 ultra-sections
  3. Grid stain with uranyl acetate (or alternative) and lead citrate
  4. Correlation imaging by TEM or SEM


Workflow for Cryo-CLEM:

  1. Confocal fluorescence microscopy living GFP-tagged protein expressed cells on gold EM grids
  2. Cryo-Confocal fluorescence microscopy (as required) [under construction]
  3. Plunge freeze in liquid ethane or ethane/propane mixture by the TFS Vitrobot
  4. Cryo-EM imaging