Biological Specimen Preparation for Scanning Electron Microscope Centre for Advanced Microscopy Joanne Lee Specimen preparation for SEM Ideal sample for Biological sample SEM • Living organisms are • Rigid and still highly dynamic Fixing Drying • Contains no liquid • Contains high levels of Coating (stable in the vacuum water condition) • Mainly composed of • Conductive organic material Specimen preparation for SEM Dry sample Wet sample Vapour fixation Chemical fixation Dehydration Drying Mounting Coating SEM observation Fixation • Stop all cellular processes Thickness of liver tissue fixed at various times • Changes the dynamic systems within cells to an immobile and stable • To preserve cellular structures as close Diffusion through plant as possible to the living state tissue much slower Fixation assures a rapid stabilisation of because of the cell walls proteins in wide sense. and air spaces Glutaraldehyde • Reacts very quickly with proteins and amino acids. • Penetrates very slowly. • Forms irreversible crosslinks. • Crosslink proteins in the tissue as well as proteins in solution Paraformaldehyde • Penetrate very quickly but react slowly. • Cross-link proteins more slowly. • Fixation is reversible. • Suitable for immuno work because since antigenicity is maintained. Osmium tetroxide • Extremely toxic chemical • Very poor rate of penetration (pretreatment with aldehyde is generally recommended). • Crosslinks unsaturated lipids (membranes are well fixed and stained). • Osmic acid is produced during crosslinking with lipids and it improves specimen preservation (especially membranes) and contrast. Buffers Use buffers as a ‘vehicle’ for the chemical fixatives • Gentle liquid medium to bring the fixative to the cellular components. • Help maintain normal pH levels while the cells are being fixed. • Maintain osmotic conditions which help to avoid swelling or shrinkage. Buffers • Phosphate Buffer Inorganic, non-toxic, prone to growth of bacteria in storage → Need to make fresh solution • Sodium Cacodylate Buffer Inorganic, quite toxic • Pipes or Hepes Organic, can add ions to these buffers → often use in tissue culture work! Dehydration • Gradually replace the free water with an organic solvent • Ethanol, Acetone, Methanol Drying Chemical dry Air dry Critical point dry (HMDS) • Easy but harsh • Time consuming • Quick procedure • Insect • Plant tissue • Animal tissue and cells D.F. Bray, J. Bagu, and P. Koegler (1993) Microscopy research and technique 26:489-495 Critical Point Drying • Preserve the surface structure of delicate samples. • Can avoid damage due to surface tension when changing from the liquid to gaseous state. • Critical point is where the liquid and gas phases of CO2 are in equilibrium Mounting sample sample adhesive adhesive stub stub sample adhesive stub Coating • Increase conductivity of specimen • Prevention of charge-up by sampling • Reduce electron beam damage coating sample sample sample adhesive adhesive adhesive stub stub stub Coating Sputter coating HT Target atoms (-ve) •Au Argon •Au/Pd (Positive ions) •Pt Sample Artefacts Charging beam damage Contamination Cautionary notes • Every sample is different! • Understand your sample well. • To save your time, money and heartache, test sample preparation procedures on a small batch of your samples before processing all of them. • Artefacts may appear in micrographs..