HC 10: Visualising Cells Electron Microscopy (EM)
Why use electrons?
𝜆
• Resolution set by wavelength: Abbe for light: 𝑑 = → visible light limits lateral resolution to ~200–250 nm.
2𝑁𝐴
• Electrons accelerated to high voltages have very short wavelengths, enabling atomic / molecular scale imaging.
o Example: at 200 kV an electron’s wavelength ≈ 2.5 pm (≈0.0025 nm) — orders of magnitude shorter than visible light → enables sub-
nanometre/atomic resolution in principle.
• Electrons interact strongly with matter (charge + mass), so:
o Pros: high scattering → high resolving power, lenses (electromagnetic) can steer beams
o Cons: beam damages biological samples, requires vacuum, complex sample prep
Basic EM instrument essentials:
1. Vacuum column (avoid electron scattering in air; keep sample clean).
2. Electron source (gun):
o Thermionic (heated filament — e.g., LaB₆)
o Field emission (FEG) — higher brightness, coherence, better resolution
3. Electromagnetic lenses & apertures (change currents to focus/zoom; imperfect — spherical & chromatic aberration)
4. Specimen stage & manipulators (incl. cryo-stages)
5. Detectors (phosphor screen, CCD, CMOS, direct electron detectors/DDD, EDX for composition)
Key practical: lens strengths are changed by current; apertures limit aberrant electrons (tradeoff resolution vs contrast).
Major EM types & when to use them:
• Transmission EM (TEM) — “look through” thin samples.
o Use for: cell ultrastructure (thin sections), viruses, macromolecules, crystals, high-res single particle analysis (SPA), cryoEM.
o Requires sections or thin film (≤ ~1000 nm practical; typical resin sections 50–70 nm; cryo thin films ~100–300 nm or thinned by FIB).
• Scanning EM (SEM) — “look at surface” by scanning beam and detecting secondary/ backscatter electrons.
o Use for: surface topology, 3D impression, volume imaging with serial block face (SBF-SEM) or FIB-SEM, large volumes, correlative
workflows.
o Strengths: large depth-of-field, compositional signals (backscatter, EDX).
• Cryo-EM (vitrified samples) — preserves native hydrated state; no chemical fixation/staining. Methods:
o Single Particle Analysis (SPA) — purified macromolecules (particle averaging → high resolution structures).
o Cryo-Electron Tomography (cryoET) — 3D of intact cells/organelles (thicker samples; often combined with cryoFIB milling).
o CEMOVIS (cryosectioning) — thin vitreous sections for TEM.
o cryoFIB-milling — thin lamellae from thick cells/tissues for cryoET.
• Serial Block-Face SEM (SBF-SEM) / FIB-SEM — large 3D volumes; use when you want ultrastructural 3D context over many µm.
Sample preparation — two broad strategies:
• Conventional (room-temperature) EM — fixation / dehydration / embedding / staining
o Workflow (typical): chemical fixation (aldehydes) → post-fixation (OsO₄) → dehydration (ethanol/acetone) → resin embed → ultrathin
section (50–70 nm) → heavy-metal stains (uranyl acetate, lead citrate).
o Immuno-gold: antibody + gold probes on sections for localization (requires special fixation/embedding for antigenicity).
o Pros: contrast, stability, thin sections for TEM, routine for cell ultrastructure.
o Cons: dehydration/chemicals can distort native structure; staining produces “contrast” but not native density.
• CryoEM (vitrified hydrated samples) — keep water, no staining
o Workflow (typical small samples): apply sample to holey grid → blot thin film → plunge-freeze into liquid ethane (vitrification) →
transfer under cryo conditions to microscope (liquid nitrogen temperatures).
o Advantages: preserves native conformation; densities correspond to biomolecular mass; enables SPA and cryoET; can combine with
direct detectors & movie correction.
o Challenges: low contrast / low SNR, radiation sensitivity → requires low-dose methods, motion correction, and computational
averaging.
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, Contrast mechanisms & detectors:
• TEM contrast: mass-thickness & scattering; biological samples (H, C, N, O) scatter weakly → need heavy-atom stains (conventional EM) or
computational averaging (cryoEM).
• SEM signals: secondary electrons (surface topography), backscattered electrons (atomic number contrast), EDX (elemental composition).
• Detectors:
o Photographic film (historical)
o Indirect digital: scintillator + CCD/CMOS — decent but limited detective quantum efficiency (DQE)
o EDX detectors for elemental analysis (e.g., Ca deposits)
o Direct electron detectors (DDD) — much higher DQE, fast frame rates → enable movie correction (beam-induced motion), critical
advance for high-res cryoEM)
Thickness, sectioning & sample limits:
• TEM requires thin samples that electrons can transmit:
o Typical resin ultrathin sections: 50–70 nm (conventional EM).
o Cryo thin films across holes (SPA): tens to a few hundred nm; thick cells/tissues require cryoFIB lamellae (~100–300 nm).
• SEM for volume imaging: remove surface layer repeatedly (SBF-SEM with diamond knife) or mill with focused ion beam (FIB-SEM).
Radiation damage & imaging strategies:
• Electrons damage biomolecules (ionization, heating). To minimize:
o Low-dose imaging strategies (minimize total electron dose).
o Use cryo conditions (liquid N₂) — reduces damage and preserves structure.
o Dose fractionation + motion correction (DDD cameras) is standard for SPA.
o For tomography, distribute dose across tilt series carefully (dose per tilt, dose weighting).
From 2D to 3D: when & how
TEM produces 2D projections of a 3D object; overlap and projection ambiguity arise.
• If you need 3D:
o Electron tomography (tilt series → reconstruct tomogram) — best for cellular contexts (organelles, viruses, inclusions) at ~3–5 nm (or
better with subtomogram averaging).
o Subtomogram averaging — extract repeated motifs from tomograms and average to improve resolution (used for spikes on virus,
axoneme motors, MIPs).
o Single Particle Analysis (SPA) — for many identical particles in random orientations (purified complexes), can reach near-atomic
resolution (~2–4 Å).
o Electron crystallography — 2D crystals (membrane proteins) → high resolution.
Common practical numbers:
• Electron wavelength (example): 200 kV → λ ≈ 2.5 pm (tiny).
• Resin ultrathin sections: 50–70 nm.
• Negative staining resolution: ~2 nm (quick screening of particles).
• CryoEM SPA: can reach ~2–3 Å (atomic models).
• cryoET subtomogram averages: often ~3–10 Å depending on particle count & alignment.
• SEM resolution (high-end): ~1–5 nm at high vacuum; great depth of field.
Comparison: what technique for what biological question?
• Need macromolecular structure (isolated protein/virus)? → cryoEM SPA (purified particles, aim for atomic detail).
• Need structure of proteins in situ or large complexes in cells? → cryoET + subtomogram averaging (or CEMOVIS + cryoET after FIB
lamella).
• Need large 3D ultrastructure across cells/tissue (connectivity, organelle relationships)? → SBF-SEM or FIB-SEM (volume EM).
• Need surface topology (membrane morphology, extracellular matrix)? → SEM.
• Need localization of specific protein / gold labelling? → immunogold TEM (conventional EM with careful fixation/embedding).
• Need native hydrated/functional states? → cryoEM methods (avoid chemical fixation).
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Why use electrons?
𝜆
• Resolution set by wavelength: Abbe for light: 𝑑 = → visible light limits lateral resolution to ~200–250 nm.
2𝑁𝐴
• Electrons accelerated to high voltages have very short wavelengths, enabling atomic / molecular scale imaging.
o Example: at 200 kV an electron’s wavelength ≈ 2.5 pm (≈0.0025 nm) — orders of magnitude shorter than visible light → enables sub-
nanometre/atomic resolution in principle.
• Electrons interact strongly with matter (charge + mass), so:
o Pros: high scattering → high resolving power, lenses (electromagnetic) can steer beams
o Cons: beam damages biological samples, requires vacuum, complex sample prep
Basic EM instrument essentials:
1. Vacuum column (avoid electron scattering in air; keep sample clean).
2. Electron source (gun):
o Thermionic (heated filament — e.g., LaB₆)
o Field emission (FEG) — higher brightness, coherence, better resolution
3. Electromagnetic lenses & apertures (change currents to focus/zoom; imperfect — spherical & chromatic aberration)
4. Specimen stage & manipulators (incl. cryo-stages)
5. Detectors (phosphor screen, CCD, CMOS, direct electron detectors/DDD, EDX for composition)
Key practical: lens strengths are changed by current; apertures limit aberrant electrons (tradeoff resolution vs contrast).
Major EM types & when to use them:
• Transmission EM (TEM) — “look through” thin samples.
o Use for: cell ultrastructure (thin sections), viruses, macromolecules, crystals, high-res single particle analysis (SPA), cryoEM.
o Requires sections or thin film (≤ ~1000 nm practical; typical resin sections 50–70 nm; cryo thin films ~100–300 nm or thinned by FIB).
• Scanning EM (SEM) — “look at surface” by scanning beam and detecting secondary/ backscatter electrons.
o Use for: surface topology, 3D impression, volume imaging with serial block face (SBF-SEM) or FIB-SEM, large volumes, correlative
workflows.
o Strengths: large depth-of-field, compositional signals (backscatter, EDX).
• Cryo-EM (vitrified samples) — preserves native hydrated state; no chemical fixation/staining. Methods:
o Single Particle Analysis (SPA) — purified macromolecules (particle averaging → high resolution structures).
o Cryo-Electron Tomography (cryoET) — 3D of intact cells/organelles (thicker samples; often combined with cryoFIB milling).
o CEMOVIS (cryosectioning) — thin vitreous sections for TEM.
o cryoFIB-milling — thin lamellae from thick cells/tissues for cryoET.
• Serial Block-Face SEM (SBF-SEM) / FIB-SEM — large 3D volumes; use when you want ultrastructural 3D context over many µm.
Sample preparation — two broad strategies:
• Conventional (room-temperature) EM — fixation / dehydration / embedding / staining
o Workflow (typical): chemical fixation (aldehydes) → post-fixation (OsO₄) → dehydration (ethanol/acetone) → resin embed → ultrathin
section (50–70 nm) → heavy-metal stains (uranyl acetate, lead citrate).
o Immuno-gold: antibody + gold probes on sections for localization (requires special fixation/embedding for antigenicity).
o Pros: contrast, stability, thin sections for TEM, routine for cell ultrastructure.
o Cons: dehydration/chemicals can distort native structure; staining produces “contrast” but not native density.
• CryoEM (vitrified hydrated samples) — keep water, no staining
o Workflow (typical small samples): apply sample to holey grid → blot thin film → plunge-freeze into liquid ethane (vitrification) →
transfer under cryo conditions to microscope (liquid nitrogen temperatures).
o Advantages: preserves native conformation; densities correspond to biomolecular mass; enables SPA and cryoET; can combine with
direct detectors & movie correction.
o Challenges: low contrast / low SNR, radiation sensitivity → requires low-dose methods, motion correction, and computational
averaging.
1|Page
, Contrast mechanisms & detectors:
• TEM contrast: mass-thickness & scattering; biological samples (H, C, N, O) scatter weakly → need heavy-atom stains (conventional EM) or
computational averaging (cryoEM).
• SEM signals: secondary electrons (surface topography), backscattered electrons (atomic number contrast), EDX (elemental composition).
• Detectors:
o Photographic film (historical)
o Indirect digital: scintillator + CCD/CMOS — decent but limited detective quantum efficiency (DQE)
o EDX detectors for elemental analysis (e.g., Ca deposits)
o Direct electron detectors (DDD) — much higher DQE, fast frame rates → enable movie correction (beam-induced motion), critical
advance for high-res cryoEM)
Thickness, sectioning & sample limits:
• TEM requires thin samples that electrons can transmit:
o Typical resin ultrathin sections: 50–70 nm (conventional EM).
o Cryo thin films across holes (SPA): tens to a few hundred nm; thick cells/tissues require cryoFIB lamellae (~100–300 nm).
• SEM for volume imaging: remove surface layer repeatedly (SBF-SEM with diamond knife) or mill with focused ion beam (FIB-SEM).
Radiation damage & imaging strategies:
• Electrons damage biomolecules (ionization, heating). To minimize:
o Low-dose imaging strategies (minimize total electron dose).
o Use cryo conditions (liquid N₂) — reduces damage and preserves structure.
o Dose fractionation + motion correction (DDD cameras) is standard for SPA.
o For tomography, distribute dose across tilt series carefully (dose per tilt, dose weighting).
From 2D to 3D: when & how
TEM produces 2D projections of a 3D object; overlap and projection ambiguity arise.
• If you need 3D:
o Electron tomography (tilt series → reconstruct tomogram) — best for cellular contexts (organelles, viruses, inclusions) at ~3–5 nm (or
better with subtomogram averaging).
o Subtomogram averaging — extract repeated motifs from tomograms and average to improve resolution (used for spikes on virus,
axoneme motors, MIPs).
o Single Particle Analysis (SPA) — for many identical particles in random orientations (purified complexes), can reach near-atomic
resolution (~2–4 Å).
o Electron crystallography — 2D crystals (membrane proteins) → high resolution.
Common practical numbers:
• Electron wavelength (example): 200 kV → λ ≈ 2.5 pm (tiny).
• Resin ultrathin sections: 50–70 nm.
• Negative staining resolution: ~2 nm (quick screening of particles).
• CryoEM SPA: can reach ~2–3 Å (atomic models).
• cryoET subtomogram averages: often ~3–10 Å depending on particle count & alignment.
• SEM resolution (high-end): ~1–5 nm at high vacuum; great depth of field.
Comparison: what technique for what biological question?
• Need macromolecular structure (isolated protein/virus)? → cryoEM SPA (purified particles, aim for atomic detail).
• Need structure of proteins in situ or large complexes in cells? → cryoET + subtomogram averaging (or CEMOVIS + cryoET after FIB
lamella).
• Need large 3D ultrastructure across cells/tissue (connectivity, organelle relationships)? → SBF-SEM or FIB-SEM (volume EM).
• Need surface topology (membrane morphology, extracellular matrix)? → SEM.
• Need localization of specific protein / gold labelling? → immunogold TEM (conventional EM with careful fixation/embedding).
• Need native hydrated/functional states? → cryoEM methods (avoid chemical fixation).
2|Page
