introduction to electron microscopy andres kaech preparation › static › winterschool ›...

Introduction to

Electron Microscopy

Andres Kaech

Preparation

Center for Microscopy and Image Analysis

Biology Electron microscope

High vacuum

Electron beam

Sensitive to vibration/motion

(High magnifications)

Biological samples need to be

transferred into a solid state...

...keeping the sample close to the

native state

Not suitable for EM

Resistant to high vacuum

Resistant in electron beam

Thin – permeable for electrons

(for TEM)

Contrast

Physical demands of electron microscopy

Aqueous/hydrated

Soft

Light elements

(C, O, H, N, S, P etc.)

“Large”

Biology Electron microscope

High vacuum

Electron beam

Sensitive to vibration/motion

(High magnifications)

Any treatment changes the

specimen!

Not suitable for EM

Resistant to high vacuum

Resistant in electron beam

Thin – permeable for electrons

(for TEM)

Contrast

Physical demands of electron microscopy

Aqueous/hydrated

Soft

Light elements

(C, O, H, N, S, P etc.)

“Large”

Physical demands of electron microscopy

2 cm

What is (was) this?

Critical Point Drying

Coating

RT-SEM

WARM SPECIMEN

Main preparation pathways for TEM

Dehydration

Chemical fixation

Freeze-fractured/etched specimenFreeze-dried

specimen

Freeze-fracturing/Freeze-drying/Coating

RT-SEM

Low temperature processing

Cryo-Ultramicrotomy

Cryo-TEM

Cryo thin section

Cryo-SEM

RT specimen processing

High pressure

freezing

Plunge freezing

thawing

Immunolabeling

Replica

RT-TEM

Bare grid technique

Staining

Ultramicrotomy

Embedding

Low-temperature embedding

RT-embedding

FROZEN SPECIMEN

Freeze-substitution

Propane jet

freezing

RT-TEM, FIB-SEM

Main preparation pathways for TEM

Embedding

Fixation

Dehydration

Staining

Thin sectioning

TEM Requires thin specimen: 70 nm

Requires solid specimen (embedding in plastic)

Plastic only soluble in solvents (e.g. acetone)

Solvents dissolve biological matter

Embedding

Fixation

Dehydration

Staining

Room temperature processing for TEM

Thin sectioning

TEM

Stabilization of biological material

Chemical fixation (cross-linking) with Aldehydes, OsO4, Ur2+…

Glutaraldehyde CH2 CH2 CH2 C

O

H

C

O

H

1 mm

1 mm

1 mm

1 mm3: penetration within 30-60 min at 20-37°C

Maximum size for good preservation

Room temperature processing for TEM

Osmiumtetroxide

• Cross linker mainly of unsaturated lipids

some proteins & phenolic compounds

• Provides contrast

• Can solubilise some proteins

Os

OO

O O

Embedding

Fixation

Dehydration

Staining

Thin sectioning

TEM

Post-fixation with OsO4

Chemical fixation

The specimen must be small to enable

quick fixation with chemicals

Chemical fixatives

penetrate small specimen

at sufficient speed

Chemical fixatives block

their penetration if specimen

is too large...slow or no

fixation

1 mm

1 mm

1 mm

G. Griffith, Fine structure immunocytochemistry, Springer 1993

1 mm3: penetration within 30-60 min at 20-37°C

Room temperature processing for TEM

Embedding

Fixation

Dehydration

Staining

Thin sectioning

TEM

Room temperature processing for TEM

Well preserved Not well preserved

Liver tissue

Embedding

Fixation

Dehydration

Staining

Thin sectioning

TEM

Room temperature processing for TEM

Substitution of water with solvent (ethanol, acetone)

Usually performed with gradient of different concentrations.

Embedding

Fixation

Dehydration

Staining

Thin sectioning

TEM

Room temperature processing for TEM

Infusion with “plastic” formulation followed by polymerisation

Specimen embedded in Epon

• Plastic formulations consist of monomers, hardener, accelerator

• Polymerization by heat or UV light

• Epoxy resins, acrylic resins

• Note: Resins are toxic and allergenic

Embedding

Fixation

Dehydration

Staining

Thin sectioning

TEM

Room temperature processing for TEM

Cutting sections of ca. 70 nm -> electron transparent

Ultramicrotomy

Embedding

Fixation

Dehydration

Staining

Thin sectioning

TEM

Room temperature processing for TEM

Cutting sections of ca. 70 nm -> electron transparent

Room temperature processing for TEM

30 nm

70 nm

100 nm

150 nm

200 nm

300 nm

Embedding

Fixation

Dehydration

Staining

Thin sectioning

TEM

Cutting sections of ca. 70 nm -> electron transparent

Embedding

Fixation

Dehydration

Staining

Thin sectioning

TEM

Contrast enhancement with heavy metals

Room temperature processing for TEM

UAc H2O Pb-citrate H2O5 min 30 sec 5 min 30 sec

Parafilm

Droplet with staining solution

Grid with sections facing down

• Uranium ions: phosphate groups of lipids (membrane contrast)

• Lead ions preferably bind to proteins

Embedding

Fixation

Dehydration

Staining

Thin sectioning

TEM

Interpretation/orientation

Room temperature processing for TEM

HEP2 cells infected with Chlamydia pneumoniae

Embedding

Fixation

Dehydration

Staining

Thin sectioning

TEM

Room temperature processing for TEM

Aldehydes: Slow (seconds to minutes), lots of artefacts like shrinkage…

OsO4: Depolimerisation of proteins

Shrinkage

Conformational changes of proteins

Loss of lipids

Mechanical effects

Loss of Lipids

Shrinkage during polymerisation

Compression, knife marks

Staining artefacts (precipitation of heavy metals)

Interpretation mistakes

Embedding

Fixation

Dehydration

Staining

Thin sectioning

TEM

Cryo preparation for TEM

Cryo-Immobilization

Stabilization of biological material by freezing

Liquid water and vitrified water

Frozen water with ice crystals

Embedding

Fixation

Dehydration

Staining

Thin sectioning

TEM

Cryo preparation for TEM

Well frozen mouse cerebellum

Not well frozen mouse cerebellum

Embedding

Fixation

Dehydration

Staining

Thin sectioning

TEM

Cryo preparation for TEM

High pressure freezing (HPM)

Freezing under high pressure (2100 bar)

Adequate freezing of samples up to 200 µm thickness without anti-freeze

Plunge freezing in liquid ethane/propane:

Only suspensions (< 1 µm) or thin tissues containing anti-freeze

Propane jet freezing (JFD):

Adequate freezing of suspensions not thicker than 15 µm

Thicker specimen require anti-freeze

Slam freezing:

Suspensions and thin tissues (few µm, only front well frozen ca. 1 µm)

Embedding

Fixation

Dehydration

Staining

Thin sectioning

TEM

Cryo preparation for TEM

Relative sizes

Plunge/slam freezer Propane jet freezer

High-pressure freezer

Embedding

Fixation

Dehydration

Staining

Thin sectioning

TEM

Freeze-substitution

Substitution of water/ice with solvent (ethanol, acetone)

Usually combined with simultaneous fixation with chemicals

(OsO4, Uranyl-acetate…)

Cryo preparation for TEM

-100

-90

-80

-70

-60

-50

-40

-30

-20

-10

0

0 5 10 15 20 25 30 35

Time (h)

Te

mp

era

ture

(°C

)

-90°Cacetone-90°Cacetone

Embedding

Fixation

Dehydration

Staining

Thin sectioning

TEM

Infusion with “plastic” formulation followed by polymerisation at

low or room temperature

Cryo preparation for TEM

Embedding

Fixation

Dehydration

Staining

Thin sectioning

TEM

Same procedure as RT

Cryo preparation for TEM

Embedding

Fixation

Dehydration

Staining

Thin sectioning

TEM

Reduced extraction of cell constituents

Reduced shrinkage

Mechanical effects

Loss of Lipids

Shrinkage during polymerisation

Compression, knife marks

Interaction of heavy metals with biology provides electron

density

Interpretation/orientation

Cryo preparation for TEM

No RT fixation artefacts

Ice crystal damage possible

Specimen courtesy of Bettina Sobottka, Neurologische Klinik, University of Zurich

Room temperature vs. cryo preparation

500 nm

Conventionally fixed (glutaraldehyde) High pressure frozen

Mouse cerebellum

Specimen courtesy of Bettina Sobottka, Neurologische Klinik, University of Zurich

Room temperature vs. cryo preparation

500 nm

Conventionally fixed (glutaraldehyde) High pressure frozen

Mouse cerebellum

RT-SEM

Low-temperature embedding

RT-embedding

RT-TEM, FIB-SEM

Cryo-Ultramicrotomy

Cryo-TEM

Cryo thin section

Freeze-substitution

Ultramicrotomy

Staining

WARM SPECIMEN

Embedding

thawing

Immunolabeling

Replica

RT-TEM

Main preparation pathways for SEM1 µm1 µm

Bare grid technique

Dehydration

Critical Point Drying

Coating

RT-SEM

Chemical fixation

Low temperature processingRT specimen processing

High pressure

freezing

Propane jet

freezing

Plunge freezing

Freeze-dried

specimen

Freeze-fracturing/Freeze-drying/Coating

Cryo-SEM

FROZEN SPECIMEN

Freeze-fractured/etched specimen

Room temperature processing for SEM1 µm1 µm

Critical point

drying

Fixation

Dehydration

Coating

SEM

1 µm1 µm

Critical point

drying

Fixation

Dehydration

Coating

SEM

Same as RT preparation for TEM

Room temperature processing for SEM

Sample finally in solvent like ethanol or acetone

1 µm1 µm

Critical point

drying

Fixation

Dehydration

Coating

SEM

SS Starting point

EE End point

CC Critical point

liquid

gas

solid

CC

SS

EE

Temperature

PressurePhase diagram of CO2

Critical point of CO2: 31°C, 74 bar

Critical point of H2O: 374°C and 221 bar

Room temperature processing for SEM

1 µm1 µm

Critical point

drying

Fixation

Dehydration

Coating

SEM

Air drying

Room temperature processing for SEM

1 µm1 µm

Critical point

drying

Fixation

Dehydration

Coating

SEM

Room temperature processing for SEM

1 µm1 µm

Critical point

drying

Fixation

Dehydration

Coating

SEM

Air dryingCritical point drying

Surface of rose blossom SPI

Electron Microscopy ETH ZurichSpider mite

Room temperature processing for SEM

1 µm1 µm

Critical point

drying

Fixation

Dehydration

Coating

SEM

Platinum/Gold (1-10 nm)

Primary electron beam

• Sputter coating

• Resistance evaporation

Thin heavy metal layer applied to the specimen surface

Room temperature processing for SEM

1 µm1 µm

Critical point

drying

Fixation

Dehydration

Coating

SEM Interpretation/orientation

Room temperature processing for SEM

Critical point dried, fractured liver tissue

Center for microscopy and image analysis, University of Zurich

Room temperature processing for SEM1 µm1 µm

Cryo processing for SEM1 µm1 µm

Sublimation

(partial freeze-

drying)

Fixation

Freeze-fracturing

Coating

Cryo-SEM

Cryo-Immobilization (same as for TEM)

Cryo processing for SEM1 µm1 µm

Sublimation

(partial freeze-

drying)

Fixation

Freeze-fracturing

Coating

Cryo-SEM

…under high vacuum and at low temperature

2 cm

-120°C…-150°C

Cryo processing for SEM1 µm1 µm

Sublimation

(partial freeze-

drying)

Fixation

Freeze-fracturing

Coating

Cryo-SEM

…under high vacuum and at low temperature

2 cm

-120°C…-150°C

Cryo processing for SEM1 µm1 µm

Sublimation

(partial freeze-

drying)

Fixation

Freeze-fracturing

Coating

Cryo-SEM

EF…Exoplasmatic fracture face

PF…Plasmatic fracture face

Ice

Cytoplasm

Cryo processing for SEM1 µm1 µm

Sublimation

(partial freeze-

drying)

Fixation

Freeze-fracturing

Coating

Cryo-SEM

Revealing the ultrastructure by removing the ice

embedding the biological material (under high vacuum)

Heating (for example: -100°C for 5 minutes)

Cryo processing for SEM1 µm1 µm

Sublimation

(partial freeze-

drying)

Fixation

Freeze-fracturing

Coating

Cryo-SEM

Electron microscopy ETH Zurich

Freeze-fractured mouse intestine: with sublimation

Cryo processing for SEM1 µm1 µm

Sublimation

(partial freeze-

drying)

Fixation

Freeze-fracturing

Coating

Cryo-SEM

Platinum/Gold (1-10 nm)

Primary electron beam

Thin heavy metal layer applied to the specimen surface

…at low temperature

Cryo processing for SEM1 µm1 µm

Sublimation

(partial freeze-

drying)

Fixation

Freeze-fracturing

Coating

Cryo-SEM Interpretation/orientation

Vero cells

Cryo processing for SEM1 µm1 µm

Nucleus

Outer nuclear membrane

Inner nuclear membrane

NP

Vero cells HSV infected

Cryo processing for SEM1 µm1 µm

Virus particle in perinuclear space

NP

NP

NP

High-pressure frozen, freeze-fractured brain tissue

Electron microscopy ETH Zurich

Cryo processing for SEM1 µm1 µm