1

Preparation for SEM

RT-SEM

Low-temperature embeddingRT-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 SEM

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-driedspecimen

Freeze-fracturing/Freeze-drying/Coating

Cryo-SEM

FROZEN SPECIMEN

Freeze-fractured/etched specimen

2

Room temperature processing for SEM

Critical point drying

Fixation

Dehydration

Coating

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

3

Critical point drying

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

Room temperature processing for SEM

Critical point drying

Fixation

Dehydration

Coating

SEM

Air drying

Room temperature processing for SEM

4

Critical point drying

Fixation

Dehydration

Coating

SEM

Room temperature processing for SEM

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

5

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

Critical point drying

Fixation

Dehydration

Coating

SEM Interpretation/orientation

Room temperature processing for SEM

6

Critical point dried, fractured liver tissue

Center for microscopy and image analysis, University of Zurich

Room temperature processing for SEM

Cryo processing for SEM

Sublimation(partial freeze-

drying)

Fixation

Freeze-fracturing

Coating

Cryo-SEM

Cryo-Immobilization (same as for TEM)

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Cryo processing for SEM

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 SEM

Sublimation(partial freeze-

drying)

Fixation

Freeze-fracturing

Coating

Cryo-SEM

…under high vacuum and at low temperature

2 cm

-120°C…-150°C

8

Cryo processing for SEM

Sublimation(partial freeze-

drying)

Fixation

Freeze-fracturing

Coating

Cryo-SEM

EF…Exoplasmatic fracture facePF…Plasmatic fracture face

Ice

Cytoplasm

Cryo processing for SEM

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)

9

Cryo processing for SEM

Sublimation(partial freeze-

drying)

Fixation

Freeze-fracturing

Coating

Cryo-SEMElectron microscopy ETH Zurich

Freeze-fractured Vero cell: NO sublimation

Cryo processing for SEM

Sublimation(partial freeze-

drying)

Fixation

Freeze-fracturing

Coating

Cryo-SEMElectron microscopy ETH Zurich

Freeze-fractured mouse intestine: with sublimation

10

Cryo processing for SEM

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 SEM

Sublimation(partial freeze-

drying)

Fixation

Freeze-fracturing

Coating

Cryo-SEM Interpretation/orientation

11

Vero cells

Cryo processing for SEM

Nucleus

Outer nuclear membrane

Inner nuclear membrane

NP

Vero cells HSV infected

Cryo processing for SEM

Virus particle in perinuclear space

NP

NP

NP

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High-pressure frozen, freeze-fractured brain tissue

Electron microscopy ETH Zurich

Cryo processing for SEM

Cryo processing for SEM

Critical point dried, dry-fractured brain tissue

13

Cryo processing for SEM

Critical point dried, dry-fractured brain tissue

Special preparation methods

Tokuyasu techniqueNegative staining

14

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

Tokuyasu technique for immunolabeling

Localisation and identification of molecules, proteins

Cultured human lymphoblastoid cells: Intracellular cholesterol was frequently detected on highly curved membranes such astubulovesicular elements associated with the Golgi apparatus (A, arrowheads). (C) Internal vesicles of MVB appeared to be enriched incholesterol, Bars 200 nm.

Moebius, W., Y. Ohno-Iwashita, et al. (2002). "Immunoelectron microscopic localization of cholesterol using biotinylated non-cytolyticperfringolysin O." The Journal of Histochemistry and Cytochemistry 50(1): 43-55.

15

Low temperature processing

RT-TEM

Cryo-Ultramicrotomy

Cryo thin section

FROZEN SPECIMEN

RT specimen processing

Staining

WARM SPECIMEN

Plunge freezing

Chemical fixation

thawing

Immunolabeling

Cryo-protection

Tokuyasu technique for immunolabeling

Cryo-sectioning

Chemical fixation

Plunge-freezing

Immunolabeling

Thawing of section

TEM

Tokuyasu technique for immunolabeling

Sucrose infiltration

Contrast enhancement

Drying of section

Formaldehyde (low amounts of glutaraldehyde, uranylacetate)

2.3 M Sucrose solution as cryo-protectant prevents ice-crystal formation during freezing

Vitrified, frozen specimen

Cryo-ultramicrotome: Sections of ca. 150 nm

Interpretation

Picking up section with a droplet of frozen sucrose (2.3 M). Thawing of section at RT

Antibodies connected to gold particle (primary, secondary antibodies)

Uranyl-acetate in methyl-cellulose (staining and embedding). Embedding prevents drying artefacts.

16

Tokuyasu technique for immunolabeling

Cryo-ultramicrotome

Ultramicrotome

Cryo-chamber (-180°C to 60°C)

LN2 supply (Dewar)

RAT brown adipocyte

Protein A-gold triple labeling

Albumin 5 nm

GLUT4 10 nm

Cath. D 15 nm

George Posthuma, University Medical Center Utrecht

200 nm

Tokuyasu section

Tokuyasu technique for immunolabeling

17

Negative staining for TEM

Contrast based on heavy metals surrounding the topography of sample:Uranyl-acetate or Phosphotungstic acid

Negative staining for TEM

Only small particles like bacteria, viruses, proteins

No dehydration required

Contrast based on heavy metals surrounding the topography of sample

Very fast preparation (minutes)

Very quick method to image small particles at high resolution in the transmission electron microscope:

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Negative staining

500 nm500 nm

Human rotaviruses

T4 phages on bacterium

100 nm 100 nm 100 nm

Bacterium with flagella