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.