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Cells and Tissues > Microscopy > Flashcards

Microscopy Flashcards

1
Q

What is a condenser lens?

A

Focuses light onto the specimen

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2
Q

What is an objective lens?

A

Collects the light scattered by the specimen

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3
Q

What is the limit of resolution of the human eye?

A

About 200um

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4
Q

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

A

less than 0.2um

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5
Q

How would you achieve greater detail in your specimen?

A

By slicing them thinner so more light comes through them

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6
Q

What are the three parts of the light microscope?

A
  1. Condensor lens
  2. Objective lens
  3. Eye piece
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7
Q

What is phase contrast microscopy?

A

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

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8
Q

When would you use phase contrast microscopy?

A

Buccal smears or cell cultures

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9
Q

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

A

Firm of hard

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10
Q

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

A
  1. Prepare frozen sections

2. Chemically fix material and embed in hard embedding agent

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11
Q

What is the advantage of freezing sections of embedding?

A

Fastwr

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12
Q

What is the advances of chemically fixing materials over freezing?

A

Easily stored

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13
Q

What kind of fixation do you use for human tissue?

A

Immersion fixation

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14
Q

Give an example of immersion fixative?

A

Formaldehyde or glutaraldehyde

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15
Q

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

A

Small blocks to improve penetration of fixative

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16
Q

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

A

To preseve lipids e.g. using osmic acid

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17
Q

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

A

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

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18
Q

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

A

Alcohols and acetate

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19
Q

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

A

60-100% alcohol

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20
Q

Why would you embed a specimen?

A

To prevent it collapsing

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21
Q

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

A

Microtome

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22
Q

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

A

Resin, paraffin or wax

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23
Q

What preparation would come before getting section 7um thick?

A

Paraffin wax embedding or freezing of the sample

24
Q

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

A

Plastic resin embedded material cut with diamond knives

25
Q

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

A

1um thick

26
Q

What is the most common stain?

A

H and E (Haemotoxylin and Eosin)

27
Q

How does haemotoxylin stain work?

A

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

28
Q

What colour does haemotoxylin stain?

A

Purple

29
Q

What things do haemotoxylin stain?

A

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

30
Q

What colour does haemotoxylin stain the cell nucleus?

A

Purple/black

31
Q

Is haemotoxylin acidic or basic?

A

Basic

32
Q

Is eosin acidic or basic?

A

Acidic

33
Q

What does eosin bind to?

A

+vely charged groups i.e amino acids in proteins

34
Q

What things does eosin stain pink?

A

Collagen fibres and cytoplasm

35
Q

What does Van Gieson stain stain and what colour?

A

Stains collagen red and muscle yellow

36
Q

What is van gieson often used in combination with?

A

A stain for elastic fibres

37
Q

What is an example of something stained with silver stain?

A

Reticular fibres

38
Q

What happens in silver staining?

A

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

39
Q

What does PAS stand for?

A

Periodic acid-Schiff

40
Q

What sorts of molecules does PAS stain?

A

Carbohydrates and proteins

41
Q

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

A

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

42
Q

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

A

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

43
Q

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

A

Alcian blue dye

44
Q

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

A

Secreted by epithelial cells identified by alcian blue dye

45
Q

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

A

The extraceullular matrix of glycosaminoglycan matrix

46
Q

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

A

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

47
Q

How does flourscence/immunoflourescence work?

A

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

48
Q

How do the colours flourescene?

A

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

49
Q

What kind of cells autoflourescence?

A

red blood cells

50
Q

What is confocal light microscopy?

A

Spatial filtering eilminates out of focus light

51
Q

What are the two types of electron microscopy?

A

Scanning and transmission

52
Q

What are the two types of specimen preparation for EM?

A

Freeze fracturing with a knife or sectioning with a diamond knife

53
Q

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

A

An electron beam and it relies on density

54
Q

What is the difference between scanning and transmission EM?

A

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

55
Q

What confocal scanning microscopy?

A

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

Cells and Tissues (10 decks)

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