Electron Microscopy 1

Question 1

1mm =

Answer 1

100um = 1,000,000nm = 10,000,00 angstroms

Question 2

Mammalian cell sizes

Answer 2

7-30um

Question 3

Describe lm

Answer 3

From ancient times, man has wanted to see things far smaller than could be perceived with the naked eye.
Invention of optical microscope:
Use of glass to magnify things dates at least to the Romans with the invention of glass in the first century.
In 1665 Hooke published Micrographia, a book describing observations made with microscopes and telescopes, as well as some original work in biology.
It is widely believed that the Dutch spectacle makers, Zacharias Jansen and his father Hans were responsible for making the first compound microscope in the late 16th century.

Question 4

What can we see with lm

Answer 4

Histology - tissue organization
Phase contrast, differential interference contrast

Question 5

Why can’t we build a lm that can see v small things

Answer 5

Bc resolution

Question 6

What is reolsution

Answer 6

Smallest distance between 2 points that can still be dinstinguished
- less resolution = becomes single dot

Question 7

Resolution formula

Answer 7

R = 0.61lamda / n sin theta
Want smaller value - if high res = small value

Question 8

Describe theta of resolution

Answer 8

Half of angular width of cone of rays collected by objective lens from typical pint in specimen

Question 9

What is n in formula

Answer 9

Refractive index of medium separating specimen from objective and condenser lens
Increase = makes resolution better, but cannot increase n much

Question 10

Describe lambda in formula

Answer 10

Wavelength of light used
High energy= short wavelengths = high spatial resolution

Question 11

Why use electrons as probe

Answer 11

Resolution = high ernergy with short wavelengths = can observe nanoscale features in samples
Electrons interact strongly with matter = gives v good contrast of what we can see
Easy to produce high brightness electron beams - cheap and easy
Electron beam can be manipulated using electron magnetic field - can focus it

Question 12

Em history

Answer 12

• Potential of using electrons for imaging was understood the beginning of the 19th century.
• The first electron microscope was built in 1931 by Ernst Ruska and Max Knoll at the Berlin Technische Hochschule.
• The initial instrument was capable of magnifying objects by only 400 times, demonstrating the principles of an electron microscope.
• Two years later, Ruska constructed an electron microscope that exceeded the resolution possible with an optical microscope
• It was greatly developed through the 1950s and has allowed great advances in the natural sciences and physics.
  In north America - in Canada, rca microscopes

Question 13

Name components of tem

Answer 13

Electron source
Sample illumination - condenser lenses
Imaging lens - objective
Magnification and projections - intermediate projector lens
Detectors - eyes/camer

Question 14

What does tem use

Answer 14

Electrons instead of photons - works in vacuum, magnetic lenses instead of glass

Question 15

Why is tem good

Answer 15

Improves resolution

Question 16

Why is tem bad

Answer 16

Electrons are destructive = cause damage
Radiation damage, sample preservation

Question 17

Why tem so big

Answer 17

Bc when e- hits sample = give off x rays - so need to protect from this
Also bc e- interacts strong with matter- so if low vacuum = e- would collide with everything in air
= need it to be empty inside = vacuum

Question 18

What is sample prep requirements for ‘em

Answer 18

Immobilize sample
Electron resistant
Good contrast
As intact as possible - so represents cell well

Question 19

What are biological samples chemically made out of

Answer 19

Proteins(~chainsofaminoacids).
DNA/RNA/nucleotides(~chainsofnucleotides).
Sugar
Lipids
Water(70%ofthecells)
COHNP

Question 20

Describe biological samples characteristics

Answer 20

Aq-hydrated
Soft
Light elements - cohhsp - will not defract electrons properly
Large
= need to be trasnfer into a solid state - which preserves functions and structure of living state

Question 21

Describe em characteristic

Answer 21

High vacuum
Sensitive to vibration
Electron beam
Limited penetration

Question 22

What are reqs for sample prep

Answer 22

Resistant to high vacuum
Immobilized
Resistant to e- beam
Thin = <300nm for tem
Good contrast

Question 23

Name classical sample prep steps

Answer 23

Fixation
Dehydration
Embedding
Thin sectioning
Staining
Tem

Question 24

Describe fixation

Answer 24

Formaldehyde - bc much smaller so penetrates faster than glutaldehyde (although its better fixer)
Formaldehyde = lm
Glutadldheye = em
Immobilize sample
Solvent dissolve biological matter

Question 25

Describe dehydration

Answer 25

Replace water with solvent bc would evaporate and also for embedding = cannot have water since plastic solubilized in solvent not watere Solvent dissolve biological matter

Question 26

Describe embedding

Answer 26

Solidify sample - embed in block Plastic only Need solid specimen

Question 27

Describe thin sectioning

Answer 27

<100nm

Question 28

Describe staining

Answer 28

Can do heavy metal Contrast enhancement Then tem and imaging

Question 29

Describe ex of sample prep

Answer 29

Glutaraldehyde fixation - perfusion Odium tetroxide= fix and that preserves cell membrane Propylene oxide - 3x10 mins Liquid resin Polymerizaion - oven 60degres - depends on expoxy Polymerized resin block = can cut it

Question 30

What are goals of fixation

Answer 30

Stop biological processes in cell as quick as possible Immobilize - cross link sample Preserve cell morphology

Question 31

What are methods of fixation

Answer 31

With chemicals = glutaraldehyde/formaldehyde By rapid freezing - cryo fixation

Question 32

What are goals of dehydration

Answer 32

Remove water entirely bc hard to cut Resin soluble in solvent - not water Resins = polymers which can be hardened with heat or uv but this reaction inhibited by water

Question 33

What are methods of dehydration

Answer 33

Ethanol or acetone to 100% to remove moisture Process may have consequences for ultarstructure preservation and immunocytochemistry

Question 34

What are goals of resin embedding

Answer 34

Harden sample for cutting without distorting it

Question 35

Describe expoxy method of resin embedding

Answer 35

Resins - Epon, spurr, lr white Water immiscible Polymerization by heat-12-24hrs at 60dgrees - bad preservation of epitopes tho

Question 36

Describe lowicryls resin embedding method

Answer 36

Polar - hydrophilic k4m Or a non polar - hydrophobic hm20 Photopolymerized by uv - 360nm - better for preserving antigens for igg labelling K4m = specimens may be kept in partially hydrated state - polymerization up to 5% water in block Freeze substitution - low temp embedding - below 50degrees

Question 37

Describe sectioning or ultramicrotomy

Answer 37

Thin Tweezers pick up grid diamond knife edge - resin embedded specimen block will be sliced - can adjust thickness then = Sections in water Delicate Trapezoid shape = bc want to know which one early slice vs later slice - this way can tell if have long vertex

Question 38

Goal of staining

Answer 38

Introduce contrast for sample

Question 39

Methods of staining

Answer 39

Thin sections usually stained with solutions of heavy metal salts = enhance scattering of Contrast of specimens by increasing mass density differences of various components of tissues and cells - this increasing scattering of electrons Metal ions of staining solutions form complexes with certain components of cells= increase their density Often - staining has little chemical specificity but contrast of components such as ribosomes and membranes is increased relative to their surroundings= Not very specific - certain stains better for diff things Conventional double staining -first in uranyl acetate followed by lead citrate Also odium and tannic acid

Question 40

Describe staining flow chart

Answer 40

Floating em grid contains the section upside down on top a drop of water and stain solution Em grid = with thin sections —> wash h2o —blot/dry—> 2.5% uranyl acetate —blot/dry—> wash h2o —blot/dry—> Storage/imaging (can store for years)

Question 41

New era in cell biology - what can we see using tem

Answer 41

TIssue organization at high res Cell organization Cell morphology Big protein complexes = npcs, cilia - csk

Question 42

What is weakness of ‘em

Answer 42

How do we know what we are looking at

Question 43

What are we looking at

Answer 43

See dots mainly = important to label proteins and stain them so we know what we seeing

Question 44

Describe lm immunofluorescence

Answer 44

Antigen - primary antibody first then secondary binds primary Use detergent to permeabilzie cell = can be specific to Pam and not nuclear membrane or whatever

Question 45

Describe immunogold em - gen

Answer 45

Very hard Label section Localize molecules in cells, tissues at high res Secondary antibody has gold nano particle - linked to primary antibody

Question 46

Describe immunogold em - staining process

Answer 46

Sections incubated with primary antibody- generated to recognize specific protein Secondary antibodies - conjugated to 5-20nm golf Particles = high scattering = easily seen in tem, amplification purposes, cost/ease of exp

Question 47

What is advantage of em immunogold vs fluorescent lm

Answer 47

See in high res exactly where protein fo interest is Also an see surrounding environment = see whole cell contents Resolution Great

Question 48

How to label multiple proteins sue immunogold

Answer 48

Use secondary antibodies coupled with diff sizes gold beads = see size Also weakness bc hard to see diff sizes- can only label 3-4 diff sizes

Question 49

Describe immunogold limitations

Answer 49

Ability of proteins to be recognized by antibodies can be affected by processes Classical processing not optimal for preserving immunogeneicity = to keep antigens = not easy since heat in oven

Question 50

Describe immunogold limitations - alternative methods

Answer 50

Cryo sectioning = most processing done at cold temp Sample kept partially hydrated Embedding done once sample has been cut Cryo sectioning and embedding - antigen not destroyed

Question 51

Em as tool in cell bio - morphology

Answer 51

See morphology and change in morphology causes by Dsieases Ex = nemaline myopathy 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= can see looks wrong compared to wild type muscle cell

Question 52

Em as tool in cell bio - immunogold

Answer 52

Can see if proteins mislocalized - not v well tho Ex = accumulation of amyloid in alzeihmers = can see protein not distributed properly

Question 53

Em as tool in cell bio - em observation

Answer 53

Fast diagnostic tool = vrisues have their characteristic morphology that allows to identify them easily = see sars, hiv, Ebola , Covid on cilia of lung

Question 54

Describe case study - sars cov2

Answer 54

Saw it on cilia = thought it maybe enters through cilia What effect of covid on cilia Conscious = sars cov2 = triggers pathway that leads to cilia loss and impairs mucus clearance = see morphology purely

Question 55

Why immunogold bad

Answer 55

Antigen = has to be on surface - antibody cannot go in