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Locations: Irchel building 42, Level H and F

Locations: Irchel building 42, Level H and F

Self-study sessions:

Monday: Y42 G53

Tuesday: Y42 G53

Wednesday: Y42 J11

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Introduction

to

Biological Electron Microscopy

Andres Kaech

Center for Microscopy and Image Analysis

Scanning electron microscope (SEM)Transmission electron microscope (TEM)

The types of electron microscopes

Electron beam

Specimen ~100 nm

Electron beam

Specimen

Projection Surface

Hela Cells

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Examples TEM

Mouse cerebellum

500 nm

Mitochondrium

Nucleus

GolgiRibosomes

SynapseDendrite

Microtubule

Examples TEM

Mouse cerebellum

100 nm

Mitochondrium

Nucleus

Golgi

Lipid bilayer

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Examples SEM

Mouse kidney

500 µm

Mouse kidney (glomerulus)

10 µm

Examples SEM

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Pseudomonas aeruginosa

500 nm

Examples SEM

Wave-particle duality

Resolution depends on aperture and wavelength (Diffraction limited resolution)

Optical properties(Diffraction, chromatic abberation, spherical abberation, astigmatism etc.)

Abbe’s equation d = 0.61 λ/NA sin nNA

e-

Properties of electrons

Very similar to photons:

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Resolution of biological objects limited by specimen preparation: Practical resolution: > 1 nm

TEM: 40 – 300 kV

Effective instrument resolution TEM: 0.5 nm (120 kV)

Effective instrument resolution SEM: 1 nm

Resolution of electron microscopes

The higher the energy of the electrons, the lower the wavelength, the higher the resolution

SEM: 0.5 – 30 kV

Widefield light microscopeTransmission electron microscope

Condenser lens

Objective lens

Projector lens

Specimen

Illumination

Final image

Transmission electron microscope vs. Widefield light microscope

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Confocal laser scanning microscopeScanning electron microscope

Beam scanner

Detector

Lens system

Lens system

Specimen

Illumination

Scanning electron microscope vs. Confocal laser scanning microscope

Specimen holder

Specimen on a TEM grid

Specimen holders and stages - TEM

3 mm3 mm

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Transmission electron microscope

Goniometer: x, y, z, r

Specimen size:

• 3 mm in diameter!

• Ca. 100 nm in thickness(electron transparent)

Specimen holders and stages - TEM

Specimen holders and stages

Scanning electron microscope

Specimen stage (x, y, z, r, tilt)

Objective lens

Stage

Specimen stub

Stub holder

Specimen size:

• 100 mm in diameter

• 2 cm in z-direction (not electron transparent)

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Biology Electron microscope

Aqueous/hydrated

Soft

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

“Large”

High vacuum

Electron beam

Sensitive to vibration/motion (High magnifications)

Not suitable for EM

Physical demands of electron microscopy

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”

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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

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?

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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, FIB-SEM

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

Bare grid technique

Room temperature preparation for TEM

Embedding

Fixation

Dehydration

Staining

Thin sectioning

TEM

Ultramicrotomy -> 70 nm section

Resin like Epon, Acrylic resins

In a series of increasing concentration of acetone, ethanol

Glutaraldehyde, OsO4, UAc…

Contrast enhancement with heavy metals

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Room temperature processing for TEM

Room temperature preparation for TEM

Embedding

Fixation

Dehydration

Staining

Thin sectioning

TEM

Ultramicrotomy -> 70 nm section

Resin like Epon, Acrylic resins

In a series of increasing concentration of acetone, ethanol

Glutaraldehyde, OsO4, UAc…

Contrast enhancement with heavy metals

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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

Cryo preparation for TEM

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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 freezerEmbedding

Fixation

Dehydration

Staining

Thin sectioning

TEM

Cryo preparation for TEM

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

Room temperature vs. cryo preparation

500 nm

Conventionally fixed (glutaraldehyde) High pressure frozen

Thin sections of plastic embedded mouse cerebellum

Embedding

Dehydration

Staining

Fixation

Thin sectioning

TEM

Cryo-Immobilization

Cryo preparation for TEM

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Images of frozen hydrated sections in a cryo-TEM

Green algae

A. Al-Amoudi et al. / Journal of Structural Biology 150 (2005) 109–121

Cryo preparation for TEM

3D

From: J.C.Russ, 1998

Ultrastructural details, interaction of organelles

Are these objects related to each other?

2D

Electron Tomography

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3D reconstruction of 2D projectionsof a 3-dimensional object

2D projections 3D rendered object

Reconstruction

Electron Tomography

3D reconstruction based on:

I) Projection of serial sections

Thickness of section for EM: 70 - 300 nm

Z resolution limited to section thickness

Imaging in TEM Imaging in SEM(new application)

Electron Tomography (I)

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II) Projection of a tilt series

Thickness of sections for tilt series: ~70 - 300 nm

Z resolution depending on number of different projections: 5 - 20 nm

NOTE: A combination of both methods is possible

3D high resolution of several successive sections

Imaging in TEM (RT or CRYO)

3D reconstruction based on:

Electron Tomography (II)

Marsh 2005

Aligned projections of aPancreatic beta cell line: Double-tilt series, ±60°, 1.5° increment, section 400 nm thick

Electron Tomography (II)

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Calculation of tomograms based on projections – virtual sections

Marsh 2005

Electron Tomography (II)

Segmentation of objects of interest

Marsh 2005

Electron Tomography (II)

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Rendering of segmented structures

Marsh 2005

Electron Tomography (II)

III) Projection of a random particle orientation (cryo-TEM)

3D reconstruction based on:

Single particle imaging

Imaging in a Cryo TEM

Electron Tomography (III)

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Plunge freezing

Tweezers

TEM grid or other carrier with specimen suspension

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

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

Metal container

Electron Tomography (III)

H. Stahlberg, Center for Cellular Imaging and Nanoanalytics (C-CINA), Biozentrum, University of Basel

3D reconstruction of the cyclic nucleotide gated potassium channel MloK1

Electron Tomography (III)

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IV) Serial “abrasion” in a focused ion beam scanning electron microscope

Slice and view

Z resolution depending milling capability/specimen and surface imaging: best resolution 5 nm (voxel)

Imaging in SEM

3D reconstruction based on:

Electron Tomography (IV)

Electron Tomography (IV)

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Electron Tomography (IV)

C. elegans embedded in Epon/Araldite

Electron Tomography (IV)

High-pressure frozen and embedded mouse brain (hippocampal slice culture)

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V) 3 view microtome in SEM

3D reconstruction based on:

Electron Tomography (V)

Large areas possible

Limited z-resolution: ca. 30 nm(limited by sectioning process)

Room temperature processing for SEM

Critical point drying

Fixation

Dehydration

Coating

SEMMouse kidney (glomerulus)

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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

Cryo processing for SEM

Sublimation(partial freeze-

drying)

Fixation

Freeze-fracturing

Coating

Cryo-SEM

Cryo-Immobilization (same as for TEM)

Fracturing at low temperature and under high vacuum

Sublime water from frozen sample

Enhance contrast

Image in the cold state (e.g. – 120°C)

< –

100°

C

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Cryo processing for SEM

Sublimation(partial freeze-

drying)

Fixation

Freeze-fracturing

Coating

Cryo-SEMElectron microscopy ETH Zurich

Freeze-fractured mouse intestine

< –

100°

C

Negative staining for TEM

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

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Negative staining

500 nm500 nm

Human rotaviruses

T4 phages on bacterium

100 nm 100 nm 100 nm

Bacterium with flagella

H/K -ATPase in cimetidine-treated resting gastric parietalcells (rabbit).

Immunolabelling on sections

Sawaguchi et al. 2004, Journal of Histochemistry & Cytochemistry

100 nm

Immuno electron microscopy