Lecture 4

Question 1

What can we see in LM?

Answer 1

-Tissue organization
-Only a few organelles

Question 2

Why can’t we build an LM to see very small things?

Answer 2

Resolution: Resolution is the smallest distance between two point that can still be distinguished

Question 3

Low resolution vs High resolution

Answer 3

Low: pixelated
High: looks human

Question 4

What does a larger resoltuion mean?

Answer 4

More pixelated image

Question 5

Limit of the resolution of the LM using an eye objective?

Answer 5

200 nm

Question 6

Resolution equation?

Answer 6

R = 0.61λ/nsinθ

Question 7

How to improve resolution?

Answer 7

Use something with a smaller wavelength

Question 8

T/F: High energy = short wavelengths

Answer 8

Yes

Question 9

Why do we use electrons as probes?

Answer 9

1. High energy electrons have short wavelngth = better resolution
2. ELectrons interact strongly with matter to give good contrast
3. Easy to produce high brightness electron beams
4. Can focus electron beams at specific point using magnetic field

Question 10

First electron microscope

Answer 10

1931

Question 11

How is EM different from LM?

Answer 11

-Electron source(EM)/Light source(LM)
-Condenser for both
-imaging lens(EM)/Objective lense(LM)
-Magnification lenses in both
-Detectors for EM

Question 12

Why are elctrons bad?

Answer 12

Cause radiation damage which makes sample preservation harder

Question 13

Why is the EM large?

Answer 13

Electrons interact very strongly with matter, so if you have a low vacuum in the electron microscope the electron would collide with everything in the air. There needs to be a really high vacuum inside so that the electrons actually hit the sample rather than the air

Question 14

Requirements of a sample for EM?

Answer 14

1. Must be resistant to high vacuum
2. Immoblized
3. Reistant to the elctron beam
4. Thin (<300nm)
5. Good contrast

Question 15

How are samples prior to preparation for EM?

Answer 15

1. Aqueous
2. Soft
   3, Made up of C, O, H, S, P these elements do not refract light properly = low contrast
3. Large

Question 16

Name sample prep steps in order?

Answer 16

1. Fixation
2. Dehydration
3. Embedding
4. Thin sectioning
5. Staining
6. TEM

Question 17

Goal of fixation?

Answer 17

Stop biological processes
Crosslink the sample
Preserve cell morphology

Question 18

How do we fix cells?

Answer 18

Glutaraldehyde/formaldehyde
(usually glutaraldehyde)
or by cryo-fixation

Question 19

Goal of dehydration

Answer 19

Remove water from the specimen
Resin is insoluble in water

Question 20

How do we dehydrate the sample?

Answer 20

Using Ethanol or Acetone to 100%
Can affect ultrastructure

Question 21

Goal of embedding?

Answer 21

Harden the sample for cutting without distoring it

Question 22

How do we embedd the sample?

Answer 22

1. Epoxy resins (Epon Spurr, LR white)
   -High temp
2. Lowicryl
   -Low temp embedding

Question 23

How do we section the sample?

Answer 23

-Ultra-microtome
-Cut in trapezoid to know which sections were cut first

Question 24

Goal of staining?

Answer 24

Introduce contrast

Question 25

How do we stain a sample using heavy metal salts?

Answer 25

-Heavy metal salts increase mass density differences of various components(increases scattering of electrons) -More dense structures get more contrast

Question 26

How do we stain a sample using conventional double staining?

Answer 26

-First uranyl acetate then lead citrate - Or osmium then tannic acid

Question 27

Why do carbohydrates appear white on EM?

Answer 27

Normally the stain used has low specificity for carbohydrates

Question 28

Stain is bound look dark vs no stain is white?

Answer 28

Yes

Question 29

What can we see using TEM?

Answer 29

1. Tissue organization 2. Cell organization 3. Organelle morphology 4. Big protein complexes(nuclear pores, cytoskeleton)

Question 30

Immunogold EM

Answer 30

1. Section is incubated with primary antibody 2. Secondary antibodies are linked with a gold nanoparticle

Question 31

Disadvantage of immunogold EM?

Answer 31

The antigen of interest has to be at the surface of the section

Question 32

Advantage of EM immunogold vs Fluorescent LM

Answer 32

Immunogold: -Easily seen due to high resolution of EM -EM allows us to also see the surrounding environment of the protein whereas in LM we would need to create more antibodies to see the surrounding

Question 33

How can we label multiple proteins using immunogold?

Answer 33

-Use secondary antibodies coupled to different size of gold

Question 34

Immunogold Labelling limitations

Answer 34

-Proteins might not be recognizied by the antibodies due to processing

Question 35

Tokayasu's cryo-sections

Answer 35

Processing is done at cold temperature (nothing denature), sample is kept partially hydrated, embedding is done once the sample has been cut This is better for immunogold

Question 36

TEM as a diagnostic tool

Answer 36

-EM can detect how morphology of cells change in disease -Immunogold can be used to see protein mis-localization -Viruses can be identifies