Microscopy

Question 1

What is a condenser lens?

Answer 1

Focuses light onto the specimen

Question 2

What is an objective lens?

Answer 2

Collects the light scattered by the specimen

Question 3

What is the limit of resolution of the human eye?

Answer 3

About 200um

Question 4

How far apart do two specimens have to be in order to distinguish them in a light microscope?

Answer 4

less than 0.2um

Question 5

How would you achieve greater detail in your specimen?

Answer 5

By slicing them thinner so more light comes through them

Question 6

What are the three parts of the light microscope?

Answer 6

1. Condensor lens
2. Objective lens
3. Eye piece

Question 7

What is phase contrast microscopy?

Answer 7

Where differences between refractile index of cells and water enviroment causes shift in the phase of wavelengths and creates and image

Question 8

When would you use phase contrast microscopy?

Answer 8

Buccal smears or cell cultures

Question 9

What state does the tissue have to be in before preparation can be done?

Answer 9

Firm of hard

Question 10

What two ways are there to make the tissues firm/hard?

Answer 10

1. Prepare frozen sections

2. Chemically fix material and embed in hard embedding agent

Question 11

What is the advantage of freezing sections of embedding?

Answer 11

Fastwr

Question 12

What is the advances of chemically fixing materials over freezing?

Answer 12

Easily stored

Question 13

What kind of fixation do you use for human tissue?

Answer 13

Immersion fixation

Question 14

Give an example of immersion fixative?

Answer 14

Formaldehyde or glutaraldehyde

Question 15

What size should your tissue be before using immersion fixation and why?

Answer 15

Small blocks to improve penetration of fixative

Question 16

Why would you use a second fixative? What is an example of one?

Answer 16

To preseve lipids e.g. using osmic acid

Question 17

Why is it necessary to have a dehydration process in tissue preparation?

Answer 17

Most components dont mix with water so you should remove the water content

Question 18

What would you use to remove the water content of tissues during dehydration?

Answer 18

Alcohols and acetate

Question 19

What kind of dehydration agent would you use during paraffin embedding?

Answer 19

60-100% alcohol

Question 20

Why would you embed a specimen?

Answer 20

To prevent it collapsing

Question 21

How would you cut the specimen once it has been embedded?

Answer 21

Microtome

Question 22

What is the choice of media used in embedding that is molten and has the potential to harden/

Answer 22

Resin, paraffin or wax

Question 23

What preparation would come before getting section 7um thick?

Answer 23

Paraffin wax embedding or freezing of the sample

Question 24

What preparation would come before getting sections that are 1um thick?

Answer 24

Plastic resin embedded material cut with diamond knives

Question 25

How thick should the specimens be to be suitable for transmission electron microscopy?

Answer 25

1um thick

Question 26

What is the most common stain?

Answer 26

H and E (Haemotoxylin and Eosin)

Question 27

How does haemotoxylin stain work?

Answer 27

The dye has +ve charge and will bind to -ve charged species

Question 28

What colour does haemotoxylin stain?

Answer 28

Purple

Question 29

What things do haemotoxylin stain?

Answer 29

Phosphates in DNA, proteins with lots of carboxyls or sulphur groups (all negative)

Question 30

What colour does haemotoxylin stain the cell nucleus?

Answer 30

Purple/black

Question 31

Is haemotoxylin acidic or basic?

Answer 31

Basic

Question 32

Is eosin acidic or basic?

Answer 32

Acidic

Question 33

What does eosin bind to?

Answer 33

+vely charged groups i.e amino acids in proteins

Question 34

What things does eosin stain pink?

Answer 34

Collagen fibres and cytoplasm

Question 35

What does Van Gieson stain stain and what colour?

Answer 35

Stains collagen red and muscle yellow

Question 36

What is van gieson often used in combination with?

Answer 36

A stain for elastic fibres

Question 37

What is an example of something stained with silver stain?

Answer 37

Reticular fibres

Question 38

What happens in silver staining?

Answer 38

The silver nitrate is reduced by biological compotents to form black deposits of sliver

Question 39

What does PAS stand for?

Answer 39

Periodic acid-Schiff

Question 40

What sorts of molecules does PAS stain?

Answer 40

Carbohydrates and proteins

Question 41

What components can PAS be used to stain and what colour are they stained?

Answer 41

Basement membranes and the muscous cells of the stomach which as strongly PAS positive and are stained magentina

Question 42

Why cant H&E stains be used to stain basement membranes?

Answer 42

Because they are only 0.05mm thick so the dye is not absorbed well

Question 43

What can PAS be combined with to distinuish between acid and neurtal eputhelial mucins?

Answer 43

Alcian blue dye

Question 44

What are acid mucins and what stain can be used to identify them?

Answer 44

Secreted by epithelial cells identified by alcian blue dye

Question 45

Apart from acid mucins what else can alcian blue dye stain?

Answer 45

The extraceullular matrix of glycosaminoglycan matrix

Question 46

What happens to the tissue after it has been fixed and stain?

Answer 46

It is covered with a mounting medium and a coverslip is applied and sealed

Question 47

How does flourscence/immunoflourescence work?

Answer 47

Antibodies are tagged with a flourescent dye (flurochrome) which can be then used to tag a protein

Question 48

How do the colours flourescene?

Answer 48

The dyes absorb light at one wavelength and reflects the opposite colour which is will show

Question 49

What kind of cells autoflourescence?

Answer 49

red blood cells

Question 50

What is confocal light microscopy?

Answer 50

Spatial filtering eilminates out of focus light

Question 51

What are the two types of electron microscopy?

Answer 51

Scanning and transmission

Question 52

What are the two types of specimen preparation for EM?

Answer 52

Freeze fracturing with a knife or sectioning with a diamond knife

Question 53

What kind of beam is used to EM and what does it rely on as opposed to colour?

Answer 53

An electron beam and it relies on density

Question 54

What is the difference between scanning and transmission EM?

Answer 54

Scanning reveals a surface by penetrating a metal covered with electrons whereas transmission passes through the the specimen

Question 55

What confocal scanning microscopy?

Answer 55

A narrow laser beam scans specimens of separate planes and merges them together into one image