Introduction to Electron Microscopy Andres Kaech Preparation · Introduction to Electron Microscopy...

1

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...

...which preserves the structures as a function of the living state…

…and not as a function of specimen preparation

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”

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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


Aqueous/hydrated

Soft

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

“Large”


2 cm

What is (was) this?

3

Dehydration

Critical Point Drying

Freeze-fractured/etched specimenFreeze-dried

specimen

Freeze-fracturing/Freeze-drying/Coating

RT-SEM

Low temperature processing

Low-temperature embeddingRT-embedding

RT-TEM

Cryo-Ultramicrotomy

Cryo-TEM

Cryo thin section

FROZEN SPECIMEN

Freeze-substitution

Cryo-SEM

RT specimen processing

Coating

RT-SEM

Ultramicrotomy

Staining

WARM SPECIMEN

High pressure freezing

Propane jet freezing

Plunge freezing

Embedding

Chemical fixation

thawing

Immunolabeling

Replica

RT-TEM

Preparation pathways overview

Dehydration



specimen


RT-SEM



RT-TEM

Cryo-Ultramicrotomy

Cryo-TEM

Cryo thin section

FROZEN SPECIMEN

Freeze-substitution

Cryo-SEM


Coating

RT-SEM

Ultramicrotomy

Staining

WARM SPECIMEN



Plunge freezing

Embedding

Chemical fixation

thawing

Immunolabeling

Replica

RT-TEM

Main preparation pathways for TEM

4

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

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Osmiumtetroxide

• Cross linker mainly of unsaturated lipidssome proteins & phenolic compounds

• Provides contrast

• Can solubilise some proteins

Os

OO

O O

Embedding

Fixation

Dehydration

Staining

Thin sectioning

TEM

Post-fixation with OsO4

Embedding

Fixation

Dehydration

Staining

Thin sectioning

TEM


Well preserved Not well preserved

Liver tissue

6

Embedding

Fixation

Dehydration

Staining

Thin sectioning

TEM


Substitution of water with solvent (ethanol, acetone)Usually performed with gradient of different concentrations.

Embedding

Fixation

Dehydration

Staining

Thin sectioning

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

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Embedding

Fixation

Dehydration

Staining

Thin sectioning

TEM


Cutting sections of ca. 70 nm -> electron transparent

Ultramicrotomy

Embedding

Fixation

Dehydration

Staining

Thin sectioning

TEM



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30 nm

70 nm

100 nm

150 nm

200 nm300 nm

Embedding

Fixation

Dehydration

Staining

Thin sectioning

TEM


Embedding

Fixation

Dehydration

Staining

Thin sectioning

TEM

Contrast enhancement with heavy metals


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

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Embedding

Fixation

Dehydration

Staining

Thin sectioning

TEM

Interpretation/orientation


HEP2 cells infected with Chlamydia pneumoniae

Embedding

Fixation

Dehydration

Staining

Thin sectioning

TEM


Aldehydes: Slow (seconds to minutes), lots of artefacts like shrinkage, osmotic effects, conformational changes of proteins, loss of ions and small molecules. OsO4: Depolimerisation of proteins

ShrinkageConformational changes of proteinsLoss of lipids

Mechanical effectsLoss of LipidsShrinkage during polymerisation

Compression, knife marks

Staining artefacts (precipitation of heavy metals)

Interpretation mistakes

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Embedding

Fixation

Dehydration

Staining

Thin sectioning

TEM

Cryo preparation for TEM

Cryo-ImmobilizationStabilization of biological material by freezing

Liquid water and vitrified water

Frozen water with ice crystals

Embedding

Fixation

Dehydration

Staining

Thin sectioning

TEM


Well frozen mouse cerebellum

Not well frozen mouse cerebellum

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Embedding

Fixation

Dehydration

Staining

Thin sectioning

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


Relative sizes

Plunge/slam freezer Propane jet freezer

High-pressure freezer

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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…)


-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


13

Embedding

Fixation

Dehydration

Staining

Thin sectioning

TEM

Same procedure as RT


Embedding

Fixation

Dehydration

Staining

Thin sectioning

TEM

Reduced extraction of cell constituentsReduced shrinkage

Mechanical effectsLoss of LipidsShrinkage during polymerisation

Compression, knife marks

Interaction of heavy metals with biology provides electron density

Interpretation/orientation


No RT fixation artefactsIce crystal damage possible

14

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

High-pressure frozen, freeze-substituted mouse cerebellum

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Dehydration



specimen


RT-SEM



RT-TEM

Cryo-Ultramicrotomy

Cryo-TEM

Cryo thin section

FROZEN SPECIMEN

Freeze-substitution

Cryo-SEM


Coating

RT-SEM

Ultramicrotomy

Staining

WARM SPECIMEN



Plunge freezing

Embedding

Chemical fixation

thawing

Immunolabeling

Replica

RT-TEM

Main preparation pathways for SEM

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

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Fixation

Dehydration

Coating

SEM

SS Starting pointEE End pointCC 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



Fixation

Dehydration

Coating

SEM

Air drying


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Fixation

Dehydration

Coating

SEM



Fixation

Dehydration

Coating

SEM

Air dryingCritical point drying

Surface of rose blossom SPI

Electron Microscopy ETH ZurichSpider mite


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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



Fixation

Dehydration

Coating

SEM Interpretation/orientation


19

Critical point dried, fractured liver tissue

Center for microscopy and image analysis, University of Zurich


Cryo processing for SEM

Sublimation(partial freeze-

drying)

Fixation

Freeze-fracturing

Coating

Cryo-SEM

Cryo-Immobilization (same as for TEM)

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drying)

Fixation

Freeze-fracturing

Coating

Cryo-SEM

…under high vacuum and at low temperature

2 cm

-120°C…-150°C



drying)

Fixation

Freeze-fracturing

Coating

Cryo-SEM

…under high vacuum and at low temperature

2 cm

-120°C…-150°C

21



drying)

Fixation

Freeze-fracturing

Coating

Cryo-SEM

EF…Exoplasmatic fracture facePF…Plasmatic fracture face

Ice

Cytoplasm



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)

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drying)

Fixation

Freeze-fracturing

Coating

Cryo-SEMElectron microscopy ETH Zurich

Freeze-fractured Vero cell: NO sublimation



drying)

Fixation

Freeze-fracturing

Coating

Cryo-SEMElectron microscopy ETH Zurich

Freeze-fractured mouse intestine: with sublimation

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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



drying)

Fixation

Freeze-fracturing

Coating

Cryo-SEM Interpretation/orientation

24

High-pressure frozen, freeze-fractured brain tissue

Electron microscopy ETH Zurich



Critical point dried, dry-fractured brain tissue

Deh

ydra

tion

Crit

ical

Poi

nt D

ryin

g

Fre

eze-

frac

ture

d/et

ched

spe

cim

enF

reez

e-dr

ied

spe

cim

en

Fre

eze-

frac

turi

ng/F

reez

e-dr

ying

/Coa

ting

RT

-SE

M

Lo

w t

emp

erat

ure

pro

cess

ing

Low

-tem

pera

ture

em

bedd

ing

RT

-em

bedd

ing

RT

-TE

M

Cry

o-U

ltram

icro

tom

y

Cry

o-T

EM

Cry

o th

in s

ect

ion

FR

OZ

EN

SP

EC

IME

N

Fre

eze-

subs

titut

ion

Cry

o-S

EM

RT

sp

ecim

en p

roce

ssin

g

Coa

ting

RT

-SE

M

Ultr

amic

roto

my

Sta

inin

g

WA

RM

SP

EC

IME

N

Hig

h pr

essu

re

free

zing

Pro

pane

jet

fre

ezi

ng

Plu

nge

free

zing

Em

bedd

ing

Ch

em

ica

l fix

atio

n

tha

win

g

Imm

un

ola

belin

g

Re

plic

a

RT

-TE

M

Pre

para

tion

path

way

s ov

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ew

Introduction to Electron Microscopy Andres Kaech Preparation · Introduction to Electron Microscopy...

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Transcript of Introduction to Electron Microscopy Andres Kaech Preparation · Introduction to Electron Microscopy...