Microscopy & Electronic Visualization

Question 1

What are the four basic tissues?

Answer 1

Epithelial
Connective
Muscle
Nerve

Question 2

What is the primary choice for imaging when it comes to cells? What is the secondary choice?

Answer 2

Primary: living or fixed tissues
Secondary: type of probe to interrogate the specimen

Question 3

There are 9 orders of magnitude between gross anatomy and atomic scale. What are they?

Answer 3

20mm, 2mm, 0.2mm, 20 um, 2 um, 0.2 um, 20 nm, 2nm, 0.2nm

Question 4

What are the steps for preparation of Microscopy?

Answer 4

Fixation: stabilize structure
Dehydration: remove water
Solidification: embed in wax (LM)or plastic (EM)
Section: 0.5-10um thick (LM); 0.1-0.5 um thick (EM)
Stain: visible color (LM); density grey scale contrast (EM)

Question 5

LM slide preparation

Answer 5

1) movement of microtome arm
2) specimen embedded in wax or resin
3) fixed steel blade
4) ribbon of sections
5) ribbon sections on glass slide, stained and mounted under a glass coverslip

Question 6

EM slide preparation

Answer 6

1) copper grid covered with Carbon and/or plastic film

2) specimen in ribbon of thin sections

Question 7

Intrinsic Scales of LM and EM images

Answer 7

Human red cell: approx 7 um diameter
Mitochondrion: approx 0.5 um thickness
Ribosome: approx 25 nm diameter
Microtubules: approx 22nm thickness
Plasma membrane: approx 10nm thickness

Question 8

Primary vs secondary antibody

Answer 8

Primary: rabbit antibody directed against antigen A
Secondary: marker coupled antibodies directed against rabbit antibodies

Question 9

What is the set up for scanning electron microscopy (SEM)?

Answer 9

1) heavy metal evaporated form filament “shadows” the specimen
2) a strengthening film of Carbon evaporated from above
3) the replica is floated onto they fraction a powerful solvent to dissolve away the specimen
4) The replica is washed and picked up on a copper grid for examination