Microscopy

Question 1

What are the steps involved in tissue processing?

Answer 1

1. fixation
2. dehydration
3. clearing
4. embedding
5. sectioning
6. section mounting
7. section staining

Question 2

What is the process of fixation?

Answer 2

avoid autolysis/degradation by bacteria and to preserve the structure and molecular composition

Question 3

What are the two chemical methods of fixation?

Answer 3

1. LM-formaldehyde: reacts with amine groups
2. TEM-glutaraldehyde: cross-links proteins; addition of osmium tetroxide

Question 4

What is the process of dehydration?

Answer 4

water is extracted from fixed tissue fragments by bathing them successfully in series of ethanol (70% -> 80% -> 90% ->abs. ethanol)

Question 5

What is the process of clearing?

Answer 5

ethanol tissue replaced with xylene which makes tissue transparent

Question 6

What is included in the process of embedding?

Answer 6

1. paraffin: tissue placed in melted paraffin; heat causes xylene to evaporate and spaces in tissue get filled with paraffin that hardens
2. epoxy resins: tissues cleared using plastic solvents

Question 7

What is the process of sectioning?

Answer 7

cool/hard paraffin tissue is sectioned on slicing machine (mirotome for LM- 6 thick ; ultramicrotome for TEM- .06 thick)

Question 8

What is the process of section mounting?

Answer 8

tissue sections are mounted on glass slides (light microscope) or copper wired grid (TEM)

Question 9

What does the process of section staining include?

Answer 9

1. paraffin must be dissolved with xylene and sections must be rehydrated with alcohols stained with hematoxylin in water
2. eosin staining after dehydrating sections because counterstain is more soluble in alcohol

Question 10

What does hematoxylin stain? What color?

Answer 10

cell nucleus, nucleic acid, RNA rich organelles; blue/purple

Question 11

What does eosin stain? What color?

Answer 11

mitochondria, secretory granules, extracellular material; pink

Question 12

Which chemical stain is acidic dye, carries “-“ charge, stains “+”, and reacts with cationic groups?

Answer 12

eosin

Question 13

What are the characteristics of hematoxylin?

Answer 13

basic dye, carries “+” charge, stains “-“, and reacts with anionic groups

Question 14

What are some neurotransmitters stained with?

Answer 14

heavy metal

Question 15

What is the ONLY organelle visible at light level?

Answer 15

nucleus; the rest leave an impression

Question 16

What are the artifacts of preparation?

Answer 16

shrinkage and artificial spaces

Question 17

What is the problem with interpretations of tissue sections?

Answer 17

distortion (shrinkage)

Question 18

During interpretation of tissue sections, artificial spaces are caused by ….

Answer 18

shrinkage and loss of molecules

Question 19

What can cause shrinkage?

Answer 19

fixation, dehydration in ethanol, heat of melted paraffin

Question 20

What are the artifacts of sectioning?

Answer 20

1. a sectioned ball-shaped structure will appear as a circle
2. a sectioned tube looks like a ring
3. sections taken through a solid ball-like structure/ taken through a solid cylinder are similar

Question 21

What is resolution (resolving power)?

Answer 21

smallest distance between 2 particles

Question 22

What does resolving power depend on?

Answer 22

the quality of the objective lens

Question 23

The quality of an image depends on ….

Answer 23

resolving power

Question 24

What is a light microscope composed of ?

Answer 24

condenser, ocular, and objective lens

Question 25

What is the resolving power and magnification of a TEM?

Answer 25

max resolving power: 3nm
max magnification: 400,000x
magnification: 120,000x

Question 26

What is the resolving power and magnification of a light microscope?

Answer 26

resolving power: .2 microns
magnification: 1000 - 1500x

Question 27

What are confocal microscopes used for?

Answer 27

immunoflurescence

Question 28

What are the types of microscopes used?

Answer 28

1. polarizing microscopy
2. confocal
3. light
4. electron
5. ultra high voltage TEM

Question 29

What is the exception for an electron microscope?

Answer 29

if .1nm or less, then NOT able to resolve