Electron Micrscopy

Question 1

Resolution (human, LM,EM)

Answer 1

Human: 200um
LM: 200nm
Em: 200pm (0,2nm)

Question 2

LM (pros and cons, resolution - wavelegth -image)

Answer 2

Photons - colour image
Resolution = y/2 (highest resolution 200nm)
High y, a lot of waves mixed because photons from another area are hitting it (blurry)
Cell populations and tissue morphology
Quick preparation
Live cell imaging
Requires correct expression of protein or affective Ab
Only see what you label, no context

Question 3

EM

Answer 3

Electrons
-y — + accelaration voltage — + resolution (120KeV = 112pm =0,1nm)
More context
Beam onto thin slice
1- electron gun - illumination
2- vaccum system - e-
3- electromagnetic lenses - focus and magnify
4 - cooling system - image stability
5- detector
6 - Cryo capabillities

Question 4

TEM

Answer 4

Beam interacts with various apertures and lenses and thin specimen
Electron dense - loss of some electrons (abs or scattering) - + difference in e- loss - + contrast
Cells and their interactions

Question 5

TEM (preparation of samples)

Answer 5

Fixation (unstable)- chemical (glutaraldehyde, paraformaldehyde or osmium) or cryo (HPF or plunge freezing)

Low contrast - electron dense stains (heavy metals - uranium, osmium, lead) or immuno methods (silver, gold, DAB)

Gradual dehydratation (wet)- replace water with resin (ethanol first)

Sectioning (bulky)- polymerise at 60-70°C to harden and ultramicrotomy (<1mm3)

Question 6

Alternative staining - small particles (examples)

Answer 6

Negative staining with uranyl acetate
Dont require slicing and dehydratation
Stains background

Viruses, bacteria, cellular components

Question 7

SEM

Answer 7

Scans beam across surface (larger specimens)
Detecting electrons reflected from sample surface (BSE) or 2nd electrons by interaction with primary beam

-BSE: elemental contrast - higher atomic mass are brighter, higher energies less resolution
-SE: topography and morphology - highest spatial resolution, escape specimen
- X rays: compositional analysis - energy dispersive spectroscopy (colour)

Topographical, crystalline, chemical composition and electrical behaviour
Smaller area - more magnification

Question 8

SEM (preparation of samples)

Answer 8

Fixation (Unstable)
Dehydratation (Wet): can be more because of variable pressure
Drying (CPD or HMDS)
Acquiring conducting (Insulating): build up of e- creates artefact so needs to be mounted (grounds) with conductor materials and sputter coating (prevents build up of a charge)

Question 9

Advanced EM methods

Answer 9

CLEM (correlative light and electron microscopy): LM gives fluorescence imaging with expressed proteins and EM get context - overlay two to see where is expressing and how is behaving within context

EDS (energy dispersive spectroscopy): mapping and elemental spectrum of what is going on (elemental composition)

3D-EM (Volume EM): segmentation and reconstrution of 3D structures - 2D resin sections collected and restocked (different sections and joins them)