1 1 SEM Specimen Preparation for Tissues and Biomaterials Iolo ap Gwynn The University of Wales Bioimaging Laboratory Aberystwyth Wales [email protected]RMS/ESB Workshop Sorrento 2005 2 SEM Imaging • Information required ? – Signal types – Resolution ? • Specimen preparation – Preservation ? – Dehydration ? – Coating • Microscope settings • Interpretation – Analysis 3 Specimen types (general) • Biomaterial – Surfaces – Particles – Matrices • Biological material – Cells – Tissues • Combined Biological/Material – Interfaces 4 Animal specimen Mount on stub Freeze Cryoprocessing Histochemical staining Immunolabel Autoradiography Chemical fixation Dehydration Embedding Sputter coating SEM TEM XRMA Section Critical point dry Air dry [Contrast stain] Histochemical staining Immunolabel Biological Material LM CryoSEM 5 Animal specimen Mount on stub Freeze Cryoprocessing Chemical fixation Dehydration Sputter coating SEM Critical point dry Biological Material 6 Freeze or Fix? Rabbit Articular Cartilage Chemical fix Freeze fracture
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SEM Specimen Preparation-iolo-printout - eCM Journal Preparation-SEM.… · Specimen Dehydration 2 Major Steps: ¾1. Water replaced by organic solvents: ¾Ethanol or acetone (time
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SEM Specimen Preparationfor
Tissues and BiomaterialsIolo ap Gwynn
The University of Wales Bioimaging LaboratoryAberystwyth
What do think you need to know?Search literature for methodsDiscuss with microscopist(s) !Choose possible approach(es)ExperimentChoose final approach(es)Interpret results
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Why Not Freeze Always?
Possible artefact formationRapid freezing not possibleComparison to published work
Not always correct!Access to fresh tissue not possible……etc.
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Chemical Fixation
The composition of a fixativeFixing agent(s)Vehicle (buffer, ions etc.)
Fixation conditionsTime; Temperature; pH
DehydrationDrying methods
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The Fixing Agent
Macromolecule cross-linkerKill cellsPossible provider of contrastWill create artefactsSeveral often used together
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The AldehydesGlutaraldehyde
Popular since 1960s – slow penetratingNeeds oxygen
FormaldehydeUsed in combination with glutaraldehydeFaster penetration BUT unstablePotentially least disruptive
Acrolein (acrylic aldehyde)Highly reactive and fast penetration
All crosslink proteinsAll remove basic groups
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Osmium Tetroxide
Cross linker mainly of unsaturated lipids, some proteins & phenolic compoundsMain use in secondary fixativesCauses elastic electron scattering (BSE) Can solubilise some proteins
Os
O
OO
O
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Effects of Fixing Agents
Main reaction = proteinsSome with lipids (fats)Rarely with carbohydrates
Reduction in pH (Buffer)Cell death = acidification
– Propane, Freons, Iso-pentane– Immersion or spraying
• ‘Slam’ freezing• High Pressure
– Special apparatus
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Conclusions
• Optimising freezing is possible• Smaller samples are easier• Many approaches possible• Experimentation necessary• Artefacts can form• Care with cryogens
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After Freezing
FrozenTissue
SEM with Cryo stage
Cryo Microtomy
Freeze Dry
Freeze Substitution
Freeze Fracture
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Freeze Substitution• Keep specimens @ < -90oC• Place in organic solvent for several days
Based on slide by M.Muller, Labor für EM I, ETH Zürich
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Surface coating structure
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High resolution coating for SE1 imaging (FESEM)
R: mainly in sampleSE Signal: SE1 + SE2 (small) from metal;little signal from specimen.Resolution: limited by coating thickness &diam. of e- beamSE produced beneath coating & containedCoating discontinuity common
SE2
SE1Coating: 1 nm; high Z (Cr, Ta, W) SE Escape depth: 1-3 nmBSE coefficient: HighR: Small
BSE
Beam – <1 KV
Based on slide by M.Muller, Labor für EM I, ETH Zürich
Sputter Coating for BSE Imaging: Double layer coating
Coating: 2 nm; High Z (Pt/Pd)BSE coefficient: HighR: Small
Beam 10-30 kV
BSE
R: irrelevantSE signal: NoneBSE signal: Depends on coatingResolution: High Beam and BSE penetrate C layer C layer improves stability & reduces charging
Based on slide by M.Muller, Labor für EM I, ETH Zürich