02. Electron Microscopy

Buffl

Basic Methods in Cell Biology

by Anna M.

In what range does electron microscopy visualize structures?

What are light microscopy methods limited in resolution?

light diffraction and Abbe´s diffrection limit
—> point spread function

What is the particle - wave dualism?

Each very small particle (proton, electron) with a mass m and velocity v can be attributed a quantum mechanic wave length λ (De-Broglie-wave-length).
This wave length determines the diffraction interference of particles

What is the maximal resolution of electron microscopes?

with electron microscopy it is able to reach pm resolution
for SEM: 2 nm
for TEM: 0,2 nm

What is TEM?

transmission electron microscopy
can achieve atomic resolution
—> by excillerating an electron you get very small wavelenghts that can be used for the high resolution in EM

Beim Auftreffen der Elektronen auf die Probenatome kann es zu folgenden Wechselwirkungen kommen:
—> Strahlelektronen durchdringen die Probe ungehindert
—> Strahlelektron werden durch einen positiv geladenen Kern abgelenkt, verlieren dabei aber wenig oder keine Energie (elastische Streuung)
—> Strahlelektronen treffen auf Elektronen in einer Kernhülle; die getroffenen Elektronen sind durch einen Energieverlust und einer lediglich geringen Abweichung aus ihrer Bahn charakterisiert (unelastische Streuung)

Used for: Internal structure at atomic/nanoscale (crystal structure, defects, nanoparticles, viruses).

Basics: A high-energy electron beam passes through an ultrathin sample. Transmitted electrons are focused by electromagnetic lenses to form a high-resolution image or diffraction pattern.

What sample requirements does TEM have?

very thin sample slices of about 40 nm
—> because the beam has to go through
the vacuum need to be increased because TEM uses even shorter wavelengths wich scatter more easily
labeling for example with osmium because it scatters the electrins so the labled parts appear darker in the detector

How is an electron microscope structured?

kathode as electron source (wolfram wire)
—> so called electron guns

How can you visualize atomic protein structures?

using cryo EM
biggest advantage: no labeling needed like in light microscopy
=> in light microscopy you can only image things you know to exist in the cells and in EM you can see everything

What is SEM?

scanning electron microscopy
—> detectors positioned to detect bachscattered and secondary electrons

Used for: Surface morphology and topography (3D-like images), particle size/shape, surface composition.

Basics: A focused electron beam scans the sample surface. Emitted secondary/backscattered electrons are detected to form an image. Works on bulk samples; often needs conductive coating.

What does an scannling elecron microscope look like?

What kinds of electrons are observed in SEM?

Backscattered electrons (BSE):
—> elasticlly scattered depending on the atomic number Z
—> usefum for viewing different phases and precipates in microstructures
—> does not require the sample to be etched
Secondary electrons (SE):
—> emitted from inelastic collisions between the primary electron beam and the elektrpn sin the k-orbital of the specimens atom
—> usefum for the inspection of the topography of the samples surface
—> lower energy then BSEs but highest resolution
Auger and x-ray effects
—> caused by the ejection of inner shell electrons and the dropping down of a electron of an higher orbital filling the gap

What does the Z Number of an atom matter?

the higher the Z number of an atom the brighter the contrast in SEM
inorganic material gives good contrast in SEM, biological samples have to be coated with gold or platinum

What is High pressure freeze 3D EM and correlative spectroscopy?

quick way of freezing samples
—> prevents the formation of ice cristals
to see microtubuli the fixation process needs to be faster than the depolarisation of the microtubuli (usecase for HPF)
EM data can be quantified
you can still use correlative immunolabeling for immaging