Biological Specimen Preparation for Scanning

Centre for Advanced 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 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 walls in wide sense. and air spaces

• 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 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 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

• , , 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.