L1: Microscopy

Question 1

Main Types of Light Microscopy (+ resolution of LM)

Answer 1

Brightfield, Fluorescent, Advanced, Confocal (200nm)

Question 2

Slide Prep: Conventional LM

Answer 2

Fix, embed, section, stain

Question 3

Slide Prep: TEM

Answer 3

Fix (primary), wash, fix (secondary), dehydrate, infiltrate with resin, polymerise resin, section, stain (with electron dense material, heavy metals such as Pb, Ur, Os)

Question 4

How does a TEM function?

Answer 4

Under vacuum, electrons beam passes through sample.

Question 5

Slide Prep: SEM

Answer 5

Fix (glutaraldehyde), dehydrate, gold coat (to protect against beam)

Question 6

How does a SEM function?

Answer 6

Under vacuum, electron beam fired at sample which scatters electrons producing a 3D image of surface. Image is sometimes ‘false coloured’ for impact

Question 7

Types of electron microscope (+ resolution of each)

Answer 7

SEM (1nm), TEM (0.1–0.2nm)

Question 8

Advantages/Disadvantages of LM (Discuss)

Answer 8

-

Question 9

2 examples of chemical stains

Answer 9

Haematoxylin (basic amino acids – purple nuclei) and Eosin (acidic molecules – pink cytoplasm)

Question 10

Application of immunolabelling - what does it show?

Answer 10

“Attaching dyes to antibodies for fluorescent microscopy.
Shows both location and quantity of Ab in tissue.”

Question 11

Immunolabelling (4 steps)

Answer 11

1) Prepare sample and place on microscope slide
2) Incubate with primary Ab; wash away unbound Ab
3) Incubate with fluorochrome–conjugated secondary Ab; wash away unbound Ab
4) Mount specimen and observe in fluorescence microscope

Question 12

“Microscopes which ‘break the resolution limit’:
– SIM
– STED
– PALM”

Answer 12

SIM: Structured illumination microscopy, STED: Stimulated emission depletion microscopy, PALM: Photo–activated localization microscopy

Question 13

Special TEM techniques (and their suitabilities)

Answer 13

– Metal shadowing/negative staining (molecules, viruses, cell components)
– Cryoelectron (unfixed, unstained samples)
– Freeze fracture (membrane interior)

Question 14

TEM wholemounts

Answer 14

When a sample is sufficiently small (e.g. molecules, bacteria, viruses) it can be mounted directly onto microscope. Requires special coated grid of ‘formvar’ on the mesh

Question 15

Million Volt TEM

Answer 15

Same resolution as X–ray crystallography

Question 16

Cryo–electron microscopy

Answer 16

Electron beam fired at frozen protein solution, scattering electrons resulting in an image which is magnified by lens