Preparation for SEM - ZMB UZH · …under high vacuum and at low temperature 2 cm-120°C…-150°C...
Transcript of Preparation for SEM - ZMB UZH · …under high vacuum and at low temperature 2 cm-120°C…-150°C...
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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
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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
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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
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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
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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
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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
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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)
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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
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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
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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
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Cryo processing for SEM
Critical point dried, dry-fractured brain tissue
Special preparation methods
Tokuyasu techniqueNegative staining
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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.
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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.
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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
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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