Lecture 4 - Electron Microscope 1

Question 1

Why use EM?

Answer 1

understand what is a clell and provide clues on what it does

Question 2

size of biological objects

Answer 2

Mammalian cell ~ 20’000 nm
Nucleus ~5’000 to 10’000 nm
Bacteria/Mitochondria ~ 1’000 nm
Virus ~ 10-200nm
Ribosomes ~ 20-30nm
Proteins ~ 1nm(5kDa), 3nm(100kDa), 5nm(500kDa)
Membranes ~ 5-10nm thick
Water molecule ~ 0.2nm
Atom ~ 0.1nm

Question 3

Light Microscopy

Answer 3

see things far smaller than could be perceived with the naked eye.
invention of optical microscope -> glass to magnify things -> first century
Hooke (1665) -> published Micrographia
Zacharias Jansen and Hans -> making the 1st compound microscope in late 16th century

you can study microscopic structure of tissues (histology), we can see cell cultures

but you cannot see anything smaller than 200nm

Question 4

what is resolution?

Answer 4

the resolution (d) is the smalled distance between two points that can still be distinguished.

Question 5

what is the resolution limit?

Answer 5

Resolution = 0.61x(λ)/(nsinθ)
θ : half the angular width of the cone of the rays collected by the objective lens from a typical point in the specimen. max width is 180°, max of sin θ is 1
n : the refraction index of the medium seperating the speciment from the objective and condenser lenses (air(1.0) or oil(1.51))
λ : the wavelength of light used
High energy = short wavelengths = high spatial resolution

Question 6

How to improve Resolution?

Answer 6

Increase θ and n
decrease λ
thats why we use electrons because it has shorter wavelengths. high energy electrons have short wavelengths that allow us to observe nanoscale features in samples

Question 7

why use electron as a probe?

Answer 7

electrons interact strongly with matter
easy to produce high brightness electron beams (e.g CRT TV, electron beams)
electron beam can be manipulated using electron magnetic field, much the same way that optical lenses focus and direct light.

Question 8

History

Answer 8

potential of e microscopy was found in the 19th century
first built in 1931 by Ruska and Knoll in Berlin
first EM could magnify by only 400 times.
two years after -> Ruska exceeded the resolution limit of LM
greatly developed through the 1950’s and gas allowed great advances

Question 9

RCA Microscopes

Answer 9

EM were commercialized with high resolution in the whole of north America.

Question 10

First EM of a biological sample

Answer 10

you start seeing things you couldnt see before.
1935: Phages infecting bacteria.
Now you can see molecules

Question 11

Components of Transmission Electron Microscope
(TEM)

Answer 11

Electron Source
Sample illumination (condenser lenses)
imaging lens (objective)
magnification and projections (intermediate and projector lens)
detectors
similar structure than LM

uses electrons instead of photons to form the image
works in vacuum. magnetic lenses instead of glass
Good: better resolution
Bad: Electrons are destructive (Radiation dammage)

Question 12

sample preparation requirement for EM

Answer 12

• Immobilization (formaldehyde)
• electron resistant
• good contrast
• as intact as possible

Question 13

Chemical constituents of the biological samples

Answer 13

we have proteins, DNA/RNA/nucleotides, Sugar, Lipids, Water
so there is a lot of Carbon, Oxygens, Hydrogen, Nitrogen, and Phosphate (99% of the total atoms in the body)

Question 14

Dilema of sample preparation

Answer 14

Biological Samples : Aqueous/hydrated, Soft, Light element, Large
the samples need to be transfered into a solid state, which preserves the structures as a function of the living states. It needs to be Resistant to high vacuum, immobilized and resistant to electron beem, thin and good contrast.

Question 15

Classical Sample Preparation

Answer 15

1. Fixation (immobilize the sample, with glutaraldehydeI and osmium tetroxide)
2. Dehydration (replacing water with solvent, higher concetrations of ethanol)
3. Embedding (solidify inside resin and let it polymerize and oven)
4. This sectioning (create a thin section < 100nm)
5. staining (contrast enhancement)
6. TEM (imaging)

Question 16

Fixation

Answer 16

Goals: stop the biological processes in the cell as quick as possible, immobilize the sample and preseve cell morphology
Methods : chemically with formaldehyde or glutaraldehyde. you can also rapid freeze (cryo-fixation)

Question 17

dehydration

Answer 17

goal: remove water completely because water is difficult to cut. resin is solube in solvent not water. resins are polymers whoch can be hardened but this reaction is inhibited by water
method: the specimens can be dehydrated with ethanol or acetone to 100% to remove moister. this process may have consequences for ultrastructure preservation and immunocytochemistry.

Question 18

resin embedding

Answer 18

goal: harden the sample for cutting without distorting the sample.
method:
1. epoxy resin - water immiscible, polymerization by heat (but bad preservation of epitopes)
2. lowicryls - polar(K4M) or a non-polar(HM20). photopolymerized by UV, K4M -> sample can be partially hydrated. freeze substitution.

Question 19

EM image

Answer 19

The different shades of gray come from how much the heavy metal stain binds to different structures.
if a region has a lot of heavy metal, its electron dense, scatters many electrons, and fewer electrons reach the detectors, it appears dark (e.g. nucleolus, nucleus)
if a region has little heavy metal, its electron light, allows elctrons to reach the detectors, it appears light. (lipid droplets, vesicles, empty spaces)

Question 19

Sectioning or Ultra-microtomy

Answer 19

cutting with a diamond knife extremely thin slices of a specimen from the resin
usually 50-100nm thick to let electrons pass through
picking up the sections with a grid.

Question 19

Staining

Answer 19

goal: to introduce contrast for the sample
Method :
- thin sections are usually stained with solutions of heavy metal salts to enhance the scattering contract of t specimens by increasing the mass density differences thus increasing the scattering electrons
- the metal ions of the staining solutions form complexes with certain components of the cells, thus increasing their density
- Often, such staining has little chemical specificity, but the contrast of components such as ribosomes and
membranes is increased relative to their surrounding
- conventional double staining : first in uranyl acetate followed by lead citrate
- also, osmium and tannic acid
- Float grid upside down on droplets, Wash H₂O → Uranyl acetate → Wash H₂O, then Blot/Dry
- store and image

Question 20

what can we see using TEM?

Answer 20

tissue organization at high resolution
- nerve tissue (no intracellular space, synapses containing synaptic vesicles, myelinated axon, dendrite containing neurofilaments)
- skeletal muscle (M-band, Z-line, sarcomere)

cell organization
- pancreas cell (golgi complex, condensing vacuoles, secretory granules)
- plant cell (chloroplast)

Organelle Morphology
- Nuclear envelope
- rough ER
- vesicular tubular clustera
- cis Golgi network

Big Protein complexes : nuclear pores or cytoskeleton and cilia for e.g.

Question 21

how to label multiple proteins

Answer 21

using 2nd antibodies coupled with different sizes of gold beads.
for e.g. insulin (20-nm gold)
glucagon (40-nm gold)

Question 21

Immzunogold electron microscopy

Answer 21

to localize molecules in the cells, tissues at high resolution
sections are incubated with primary antibody, to recognize a specific protein
the secondary antibodies are conjugated to 5-20nm gold particles:
- gold particles (high scattering) is easily seen in the TEM
- amplification purposes
- cost/ease of experiment

Question 21

advantages of EM immunogold vs fluorescent LM

Answer 21

immuno-EM provides much better resolution compared to immunofluorescence Gold appears as black dots in TEM Can localize proteins to: * Specific membranes * Specific sides of membranes * Individual organelles * Even specific structural domains

Question 22

immunogold limitations

Answer 22

The ability of the proteins to be recognized by the antibodies can be affected by processing. Classical processing is not optimal for preserving immunogenicity

Question 23

Nemaline Myopathy

Answer 23

morphology and change in morphology can be seen, and could be caused by various diseases The dark rod-shaped subsarcolemmal inclusions seen here in this electron micrograph are known as nemaline rods. The "rods" are composed of aggregates of Z bands

Question 24

Accumulation of intraneuronal β-Amyloid 42 peptides (alzheimer's disease)

Answer 24

Immunogold labeling continues to a tool frequently and routinely used to localize proteins at high resolution

Question 25

EM observation is a fast diagnostic tool

Answer 25

viruses have their characteristic morphology that allows to identify them easily e.g. identify infectious agents like SARS coronavirus, HIV and ebolavirus