Electron Microscopy

Transmission Electron Microscopy

•! SEM = Scanning Electron Microscopy •! TEM = Transmission Electron Microscopy

Sara Henriksson, UCEM 2016-02-18

E. coli, William E. Bentley, Maryland, USA

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Stereocilia on a hair cell in the inner ear Light microscop

SEM

TEM

Figure 9-50 Molecular Biology of the Cell (© Garland Science 2008)

For more information, kindly refer to UCEM website: http://www.kbc.umu.se/platforms/electron-microscopy.html

TEM – Electron Tomography SEM

Figure 9-51 Molecular Biology of the Cell (© Garland Science 2008)

TEM

Figure 12-9c Molecular Biology of the Cell (© Garland Science 2008)

Figure 9-58 Molecular Biology of the Cell (© Garland Science 2008)

Figure 12-9c Molecular Biology of the Cell (© Garland Science 2008)

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Celler et al, 2013, J Bac

Full cell volume of Fission yeast Johanna Höög Celler et al, 2013, J Bac

100 nm

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+GTP Celler et al, 2013, J Bac

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Celler et al, 2013, J Bac

In vitro polymerized microtubules, Linda Sandblad

Tubulin: Nogales et al. 1998

The organells in a eucaryotic cell

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Celler et al, 2013, J Bac

Tubulin: Nogales et al. 1998 Tubulin: Nogales et al. 1998 Figure 9-45 Molecular Biology of the Cell (© Garland Science 2008)

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Golgi apparatus

Mitochondria

Datum

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Datum

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Light Microscope

Light Microscopy is limited by the diffraction limit

TEM

Cathode

Resolve Details 0,2 µm apart Limited by the wavelength of light

CCD Camera Figure 9-3b Molecular Biology of the Cell (© Garland Science 2008)

Figure 9-42 (part 1 of 2) Molecular Biology of the Cell (© Garland Science 2008)

Resolution

Electrons Electrons are used instead of light waves for illumination

Resolution is determined by the wavelength of the illumination (electron source)

Electrons are easily absorbed & scattered by different forms of matter, e.g. heavy metals or proteins, this interaction forms the image Electrons have a low penetration depth->sectioning is necessary

High vacuum is necessary because electrons have an extremely low mass and easily give up their energy in collisions with gas atoms and molecules

Higher kV of electron source -> shorter wavelength ->smaller details can be resolved The amount of electrons (current density) and the size of the electron beam (spot size) determines resolution The source of electrons determines the coherence of the electron beam Resolution is normally limited by imperfections in the optics, noise and sample preparation artifacts!!

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Electron microscopy for higher resolution

JEOL TEM 1230

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Datum

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2D projection of a 3D object

Datum

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Bacteria

Electrons that reach the fluorescent screen forms a bright spot and electrons that do not reach the screen form dark spot The varying degree of intensity of electrons form the image with a varying degree of grey Since biological materials generally have a low atomic number, the dispersion is poor Very poor dispersion means very poor contrast in the image formation

How do biologists increase the image contrast??? 24

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Bacteria

Proteins in solution

3 ways to work with biological/soft/hydrated material •! Drying and staining with heavy metal salts – Negative Staning •! Dehydration and embedding in plastic – Ultra microtome sectioning •! Cryo – Vitrification - HPF + AFS, HPF + cryo ultra microtome sectioning, plunge freezing

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Microtubule – Negative staining electron microscopy

Negative staining Protein in solution

Linda Sandblad

100 nm

K5 + K14

What you see in the TEM

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Recombinant human keratin

K1 + K10

Heavy metal salt

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Adenovirus

100 nm

Carin Årdahl & Linda Sandblad

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From a living cell to a EM specimen Chemical fixation:

Preserve the cell morphology Protect tissue against disruption Keep antigens at their original localization Glutaraldehyde and paraformaldehyde

Osmification:

Fixative and provides contrast to membranes

Dehydration:

Replace water by organic solvent

Resin infiltration:

A plastic material The support needed for sectioning Plasticity - specimen becomes less brittle

Polymerization:

Heat or UV-light crosslink polymers

Sectioning:

Ultra microtome and diamond knife

Post staining:

Contrasting of proteins, membranes and sugar 32

3 ways to work with biological/soft/hydrated material •! Drying and staining with heavy metal salts – Negative Staning •! Dehydration and embedding in plastic – Ultra microtome sectioning •! Cryo – Vitrification - HPF + AFS, HPF + cryo ultra microtome sectioning, plunge freezing

Sectioning A microtome makes physical sections of large tissue

Glass or diamond

Section thickness 50-80nm: •! Determined by inteference colours •! Best contrast and maximum resolution

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Sections of cells on a EM grid

Figure 9-44 Molecular Biology of the Cell (© Garland Science 2008)

Sections of cells on a EM grid

Figure 9-44 Molecular Biology of the Cell (© Garland Science 2008)

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Sidfot

Linda Sandblad

Sidfot

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Sidfot

Lenore Johansson Sara Henriksson 39

How to interpret your image

Svitlana Vdovikova: Macrophage with Listeria

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Tokuyasu Characteristic non-membrane staining with good possibilities for immuno-gold-labeling

Lenore Johansson and Roland Rosqvist, YopE-immunogold on Yersinia 42

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High Pressure Freezing + Freeze Substitution

3 ways to work with biological/soft/hydrated material •  Drying and staining with heavy metal salts – Negative Staning •  Dehydration and embedding in plastic – Ultra microtome sectioning •  Cryo – Vitrification - HPF + AFS, HPF + cryo ultra microtome sectioning, plunge freezing

Instant fixation (6ms) under high pressure (2000 bar) gives perfect fixation of your sample Sample is dehydrated and embedded in plastic at low temperatures to avoid ice crystal formation

HPF and AFS

Haemophilus influenzae

Gives better and more native structure of your specimen

Chemical fixation RT

More time consuming and expensive. Each type of sample might require optimization to get a nice result

HPF and AFS

Linda Sandblad

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TEM methods @ UCEM Facility service:

•  Chemical fixation, plastic embedding and ultra microtome sectioning •  Tokuyasu frozen “frozen/cryo” ultramicrotome sectioning •  Negative staining •  Immunolabeling for EM

Method development:

Future facility:

•  High pressure freezing (HPF) •  Automated freeze substitution (AFS) •  Correlative light and electron microscopy (CLEM) (Interaction with Umeå Biochemical Imaging facilities)

•  Cryo EM for structure biology •  Electron tomography •  FIB-SEM

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Service and method development: Negative staining with improved resolution for single proteins visualization

How are electrons generated? •  Thermionic emission –  Tungsten (W) filament –  Lanthanum hexaboride (LaB6) filament

•  Field emission gun

Linda Sandblad and Tomas Edgren: Yersinia membrane vesicles and flagella

W hairpin

FEG LaB6 crystal

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Field emissions

The Electron Gun Filament (20-100 KV) Bias (Wehnelt) Cylinder

V1

V2

Anode An extremely high field is produced at the sharp tip of the cathode. This reduces the potential barrier and permits electrons to tunnel out.

stream of electrons originating from outer shell of filament atoms

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Condenser lens system

Electron interaction with the specimen •  Image formation occurs by electron scattering •  Electron strike the atomic nuclei and get dispersed •  This disperse electrons form the image 1.  2. 

3.  The condenser aperture must be centered

Transmi+ed  electrons  (A)  of  the  beam  passes  straight  through  the   specimen  on  to  the  screen   Some  electron  (B)  of  the  beam  lose  a  bit  of  their  energy  while  passing   through  the  specimen  &  get  deflected  a  li+le  from  their  original  axis  of   the  beam  !  inelasCcally  sca+ered  electrons   Some  electron  (C)  interact  with  atoms  of  specimen  &  get  elasCcally   sca+ered  without  losing  energy.  Electron  deviate  widely  

C1 controls the spot size C2 changes the convergence of the beam 54

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6. Some atom emit x-ray & light energy

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Chose a suitable grid

Chose a suitable grid

•! Which metal? •! What surface, open, formvar, carbon, holy carbon •! Mesh size, how large is your sample, how stable is your surface film? Do you need to tilt? •! Finder grids – for correlative microscopy Make your one prefect surface - in the negative staining practical •! •! •! •!

Pull a formvar film on a glass slides Floating a thin film on the water surface Carbon coating Glow discharge 58

EM sample preparation for proteins or microorganisms in vitro

TEM FACILITIES @ UCEM

JEOL 1230 Transmission Electron Microscope Cryo fixation methods Ultra microtome sectioning Carbon coater and glow discharge equipment

Linda Sandblad

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EM sample preparation for microorganisms, cells and tissue

EM sample preparation for microorganisms, cells and tissue

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