EM Préparation .pdf



Nom original: EM Préparation.pdfTitre: Microsoft PowerPoint - EM_321.pptAuteur: ziegler

Ce document au format PDF 1.5 a été généré par PScript5.dll Version 5.2.2 / Acrobat Distiller 9.0.0 (Windows), et a été envoyé sur fichier-pdf.fr le 10/05/2012 à 16:05, depuis l'adresse IP 138.102.x.x. La présente page de téléchargement du fichier a été vue 2092 fois.
Taille du document: 12.5 Mo (86 pages).
Confidentialité: fichier public


Aperçu du document


University of Zurich

Center for Microscopy and Image Analysis

Introduction to
Electron Microscopy

Preparation

Courtesy: Andres Kaech

Physical demands of electron microscopy

Biology
Aqueous/hydrated
Soft
Light elements
(C, O, H, N, S, P etc.)
“Large”

Electron microscope
Not suitable for EM



High vacuum
Electron beam
Sensitive to vibration
(High magnifications)

Physical demands of electron microscopy

Biology
Aqueous/hydrated

Electron microscope
Not suitable for EM

High vacuum

Soft

Electron beam

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

Sensitive to vibration
(High magnifications)

“Large”

Biological samples need to be
transferred into a solid state...

Resistant to high vacuum

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

Thin – permeable for electrons
(for TEM)

…and not as a function of
specimen preparation

Resistant in electron beam

Contrast

Physical demands of electron microscopy

Fungi porcini fresh

Fungi porcini air dried

Physical demands of electron microscopy

Biology
Aqueous/hydrated

Electron microscope
Not suitable for EM

High vacuum

Soft

Electron beam

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

Sensitive to vibration
(High magnifications)

“Large”

Resistant to high vacuum
Resistant in electron beam
Any treatment changes the
specimen!

Thin – permeable for electrons
(for TEM)
Contrast

The goal of biological electron microscopy

Provide the structural basis for the correlation of structure and function

Time resolution

Preparation pathways overview
Low temperature processing

RT specimen processing
Plunge freezing
Propane jet
freezing

WARM SPECIMEN

FROZEN SPECIMEN

High pressure
freezing

Chemical fixation

Dehydration
Freeze-substitution

Freeze-fracturing/Freeze-drying/Coating

Cryo-Ultramicrotomy

Embedding

Low-temperature embedding
RT-embedding

Critical Point Drying

Ultramicrotomy
thawing

Cryo thin section

Coating
Immunolabeling

Freeze-dried
specimen
Staining

RT-SEM

RT-TEM

Freeze-fractured/etched specimen

Replica

RT-TEM

RT-SEM

Cryo-SEM

Cryo-TEM

Preparation pathways overview
Low temperature processing

RT specimen processing
Plunge freezing
Propane jet
freezing

WARM SPECIMEN

FROZEN SPECIMEN

High pressure
freezing

Chemical fixation

Dehydration
Freeze-substitution

Freeze-fracturing/Freeze-drying/Coating

Cryo-Ultramicrotomy

Embedding

Low-temperature embedding
RT-embedding

Critical Point Drying

Ultramicrotomy
thawing

Cryo thin section

Coating
Immunolabeling

Freeze-dried
specimen
Staining

RT-SEM

RT-TEM

Freeze-fractured/etched specimen

Replica

RT-TEM

RT-SEM

Cryo-SEM

Cryo-TEM

Main preparation pathways for TEM
RT specimen processing

Low temperature processing

FROZEN SPECIMEN

WARM SPECIMEN

Chemical fixation

Dehydration
Freeze-substitution

Embedding

Low-temperature embedding
RT-embedding

Ultramicrotomy

Staining

RT-TEM

Main preparation pathways for TEM
Fixation
Solvents dissolve biological matter

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

Embedding
Requires solid specimen
(embedding in plastic)

Thin sectioning

Requires thin specimen: 70 nm

Staining

TEM

Main preparation pathways for TEM
Classical preparation
(chemical fixation at RT)

Fixation

Dehydration

Embedding

Thin sectioning

Staining

TEM

Cryo-preparation
(cryo-fixation)

Room temperature processing for TEM

Fixation

Stabilization of biological material
Chemical fixation (cross-linking) with Aldehydes,
OsO4,…

Dehydration

Embedding

Thin sectioning
Staining

TEM

Glutaraldehyde

Glutaraldehyde polymerises

Glutaraldehyde reacts with proteins (Crosslinking)

Room temperature processing for TEM
Osmiumtetroxide



Cross linker mainly of unsaturated lipids, some proteins &
phenolic compounds



Main used as secondary fixative



Causes elastic electron scattering



Can solubilise some proteins

Room temperature processing for TEM

Fixation

Aldehydes:
Slow (seconds to minutes)
Conformational changes of proteins
Change of membrane permeability
Osmotic effects lead to dimensional alterations
Loss of diffusible ions and small molecules
Masking of antigens
OsO4: Depolimerisation of proteins

Dehydration

Embedding

Thin sectioning
Staining

TEM

Room temperature processing for TEM

Fixation

Aldehydes:
Slow (seconds to minutes)
Conformational changes of proteins
Change of membrane permeability
Osmotic effects lead to dimensional alterations
Loss of diffusible ions and small molecules
Masking of antigens
OsO4: Depolimerisation of proteins

Dehydration

Embedding

Thin sectioning
Staining

TEM

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

Room temperature processing for TEM

Fixation

Aldehydes:
Slow (seconds to minutes)
Conformational changes of proteins
Change of membrane permeability
Osmotic effects lead to dimensional alterations
Loss of diffusible ions and small molecules
Masking of antigens
OsO4: Depolimerisation of proteins

Dehydration

Embedding

Thin sectioning
Staining

TEM

Shrinkage
Conformational changes of proteins
Loss of lipids

Room temperature processing for TEM

Fixation

Aldehydes:
Slow (seconds to minutes)
Conformational changes of proteins
Change of membrane permeability
Osmotic effects lead to dimensional alterations
Loss of diffusible ions and small molecules
Masking of antigens
OsO4: Depolimerisation of proteins

Dehydration

Shrinkage
Conformational changes of proteins
Loss of lipids

Embedding

Infusion with “plastic” formulation followed by polymerisation

Thin sectioning
Staining

TEM

Room temperature processing for TEM

Plastic formulations consist of monomers, hardener, accelerator
Polymerization by heat or UV light
Epoxy resins, acrylic resins
Note: Resins are toxic and allergenic
Specimen embedded
in plastic (Epon)
Embedding molds

Room temperature processing for TEM

Fixation

Aldehydes:
Slow (seconds to minutes)
Conformational changes of proteins
Change of membrane permeability
Osmotic effects lead to dimensional alterations
Loss of diffusible ions and small molecules
Masking of antigens
OsO4: Depolimerisation of proteins

Dehydration

Embedding

Thin sectioning
Staining

TEM

Shrinkage
Conformational changes of proteins
Loss of lipids
Mechanical effects
Loss of Lipids
Shrinkage during polymerisation

Room temperature processing for TEM

Fixation

Aldehydes:
Slow (seconds to minutes)
Conformational changes of proteins
Change of membrane permeability
Osmotic effects lead to dimensional alterations
Loss of diffusible ions and small molecules
Masking of antigens
OsO4: Depolimerisation of proteins

Dehydration

Embedding

Thin sectioning
Staining

TEM

Shrinkage
Conformational changes of proteins
Loss of lipids
Mechanical effects
Loss of Lipids
Shrinkage during polymerisation

Cutting sections of ca. 100 nm (electron transparent)

Room temperature processing for TEM

Room temperature processing for TEM

30 nm
70 nm
100 nm
150 nm
200 nm
300 nm

3 mm

Thickness of section: ~100 nm

Room temperature processing for TEM

Carbon

Collodion

Copper grid
Dimensions of ultrathin sections:
Area:
0,5 x 0,5 mm
Thickness: 30 – 100 nm
Volume:
0,00001 mm3.

Room temperature processing for TEM

Fixation

Aldehydes:
Slow (seconds to minutes)
Conformational changes of proteins
Change of membrane permeability
Osmotic effects lead to dimensional alterations
Loss of diffusible ions and small molecules
Masking of antigens
OsO4: Depolimerisation of proteins

Dehydration

Embedding

Thin sectioning
Staining

TEM

Shrinkage
Conformational changes of proteins
Loss of lipids
Mechanical effects
Loss of Lipids
Shrinkage during polymerisation
Compression, knife marks

Room temperature processing for TEM

Fixation

Aldehydes:
Slow (seconds to minutes)
Conformational changes of proteins
Change of membrane permeability
Osmotic effects lead to dimensional alterations
Loss of diffusible ions and small molecules
Masking of antigens
OsO4: Depolimerisation of proteins

Dehydration

Embedding

Thin sectioning
Staining

TEM

Shrinkage
Conformational changes of proteins
Loss of lipids
Mechanical effects
Loss of Lipids
Shrinkage during polymerisation
Compression, knife marks

Contrast enhancement with heavy metals

Room temperature processing for TEM
Contrast enhancement
Grid with sections facing down

Droplet with staining solution

Parafilm

Typical staining procedure:

UAc
5 min

H2O
30 sec each

Pb-citrate
5 min

H2O
30 sec each

Room temperature processing for TEM

Fixation

Aldehydes:
Slow (seconds to minutes)
Conformational changes of proteins
Change of membrane permeability
Osmotic effects lead to dimensional alterations
Loss of diffusible ions and small molecules
Masking of antigens
OsO4: Depolimerisation of proteins

Dehydration

Embedding

Thin sectioning
Staining

TEM

Shrinkage
Conformational changes of proteins
Loss of lipids
Mechanical effects
Loss of Lipids
Shrinkage during polymerisation
Compression, knife marks

Interaction of heavy metals with biology
provides electron density (contrast)

Room temperature processing for TEM

3 mm

Goniometer: x, y, z, r

Room temperature processing for TEM

Fixation

Aldehydes:
Slow (seconds to minutes)
Conformational changes of proteins
Change of membrane permeability
Osmotic effects lead to dimensional alterations
Loss of diffusible ions and small molecules
Masking of antigens
OsO4: Depolimerisation of proteins

Dehydration

Embedding

Thin sectioning
Staining

TEM

Shrinkage
Conformational changes of proteins
Loss of lipids
Mechanical effects
Loss of Lipids
Shrinkage during polymerisation
Compression, knife marks

Interaction of heavy metals with biology
provides electron density

Interpretation

Cryo preparation for TEM

Fixation

Dehydration

Embedding

Thin sectioning
Staining

TEM

Cryo-Immobilization
Stabilization of biological material by freezing

Cryo preparation for TEM

Freezing of soft condensed hydrated matter
Challenge

Ice-crystal formation during and after the freezing procedure
must be minimized or prevented

Vitrification/adequate freezing
(without visible ice crystal damage)

Cryo preparation for TEM
Liquid water and vitrified water

Well frozen golden delicious apple leaf

2 µm

Frozen water with ice crystals

Poorly frozen golden delicious apple leaf

1 µm

Electron Microscopy Center Zurich (EMEZ)

Cryo preparation for TEM
Effect of entry velocity in subcooled Freon 22
1.25 ms-1

5 µm

10 ms-1

5 µm
Handley et al. 1981

Cryo preparation for TEM
Plunge freezing:


Only suspensions (< 1 µm) or thin tissues containing anti-freeze
(anti-freeze -> osmotic effects!)

Slam freezing:


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

Propane jet freezing (JFD):


Adequate freezing of suspensions not thicker than 15 µm



Thicker specimen require anti-freeze

High pressure freezing (HPM)


Freezing under high pressure (2100 bar)



Adequate freezing of samples up to 200 µm thickness

Cryo preparation for TEM

Plunge/slam freezer

Relative sizes

Propane jet freezer

High-pressure freezer

Cryo preparation for TEM
Plunge freezing

Tweezers
TEM grid with specimen suspension

Cryogen (propane: -186°C, ethane: -180°C)

Metal container

LN2 bath for cooling of secondary cryogen: - 196°C

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

Cryo preparation for TEM
Slam freezing

Specimen holder
Specimen attached to holder

Polished metal block
LN2/liquid helium bath for cooling of metal block

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

Cryo preparation for TEM
Propane jet freezing
Specimen
carriers

TEM grid with specimen

Liquid propane jet

Specimen sandwich

 Adequate freezing of suspensions not thicker than 15 µm
 Thicker specimen require anti-freeze

Cryo preparation for TEM
High-pressure freezing

Temperature [°C]

0

1
Me
lting
Tem
per
atu
re

-20

3

2
liquid

-40
-60
-80
-100

Area of
supercooled water

ho
m
og
en
ou
sN
uc
lea
tio
n

Hexagonal

0

1

Te
Ice mper
at
ur
e

3

2

Pressure (x 103 bar)
-140

High pressure freezing at 2100bar

Redrawn from Kanno H, (1975) supercooling of water to -92°C under pressure Science 189: 880-881

Cryo preparation for TEM
High-pressure freezing
 Immediately after reaching 2100 bar, cooling should start and run as fast as possible
 Pressure built-up must be as rapide as possible (Dissociation constants)
Pressure [mbar], Temperature [°C]

30°C
2100 bar

1 bar

pressure
0°C

temperature

-160°C
100 ms

Time [ms]

560 ms

Cryo preparation for TEM
High-pressure freezing

Specimen carrier

Specimen
Extracellular fluid
Specimen carrier

 Freezing under high pressure (2100 bar)
 Adequate freezing of samples up to 200 µm thickness without cryo protectants

Cryo preparation for TEM

Cryo-Immobilization

Dehydration

Embedding

Thin sectioning
Staining

TEM

No RT fixation artefacts (aldehydes)
Fast…Ice crystal damage possible

Substitution of water with solvent (ethanol, acetone)
at low temperatures! Usually combined with simultaneous
fixation with chemicals! -> freeze-substitution

Cryo preparation for TEM
Freeze-substitution:
Substituting the frozen water in the specimen with a solvent


Starting at the lowest possible temperature



Simultaneous fixation (glutaraldehyde, OsO4, Uranyl-acetate…)



Temperature/time course to temperature for embedding
0
-10

Temperature (°C)

-20
-30
-40
-50

-90°C
acetone

-60
-70
-80
-90
-100
0

5

10

15

20

Time (h)

25

30

35

Cryo preparation for TEM

Important parameters of freeze-substitution



Choosing the solvent: Melting temp. versus dissolving power



Temperature/time course



Choosing the fixatives: Ultrastructure vs. immunolabeling

Cryo preparation for TEM

Cryo-Immobilization

Dehydration

Embedding

Thin sectioning
Staining

TEM

No RT fixation artefacts (aldehydes)
Fast…Ice crystal damage possible

Reduced extraction of cell constituents
Reduced shrinkage

Cryo preparation for TEM

Cryo-Immobilization

No RT fixation artefacts (aldehydes)
Fast…Ice crystal damage possible

Dehydration

Reduced extraction of cell constituents
Reduced shrinkage

Embedding

Infusion with “plastic” formulation followed by polymerisation
at low or room temperature!

Thin sectioning
Staining

TEM

Same procedure as RT

Cryo preparation for TEM

Cryo-Immobilization

No RT fixation artefacts (aldehydes)
Fast…Ice crystal damage possible

Dehydration

Reduced extraction of cell constituents
Reduced shrinkage

Embedding

Mechanical effects
Loss of Lipids
Shrinkage during polymerisation

Thin sectioning
Staining

TEM

Compression, knife marks

Interaction of heavy metals with biology
provides electron density

Interpretation

Room temperature vs. cryo preparation
Paramaecium (ciliate)
Conventionally fixed (glutaraldehyde)

High pressure frozen

1 µm
Elektronenmikroskopie ETH Zürich

Room temperature vs. cryo preparation
Light exposed retina, overview (rat)
Conventionally fixed (glutaraldehyde)

High pressure frozen

5 µm
Elektronenmikroskopie ETH Zürich


Aperçu du document EM Préparation.pdf - page 1/86
 
EM Préparation.pdf - page 2/86
EM Préparation.pdf - page 3/86
EM Préparation.pdf - page 4/86
EM Préparation.pdf - page 5/86
EM Préparation.pdf - page 6/86
 




Télécharger le fichier (PDF)


EM Préparation.pdf (PDF, 12.5 Mo)

Télécharger
Formats alternatifs: ZIP



Documents similaires


liver
armstrong 2006 nutritional strategies for football counteracting heat cold high altitude and jet lag
materials testing online
2011 acetonic extract of buxus sempervirens induces cell cycle arrest apoptosis and autophagy in breast cancer cells
art3 mitochondrial dynamics cancer oncogene 2013
1

Sur le même sujet..