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03 - Body Logistics > 2 - Examining Cells and Tissues > Flashcards

2 - Examining Cells and Tissues Flashcards

1
Q

`What is the limit of resolution?

A

The smallest distance by which two objects can be separated and still be distinguished as two separate objects

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2
Q

Compare light and electron microscopes.

A
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3
Q

How would you produce a specimen to view with electron microscopy?

A
  1. Fix with glutaraldehyte
  2. Embed in epoxy resin
  3. Stain with osmium tetroxide or heavy metal
  4. Cut with microtome with diamond knife for TEM
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4
Q

What is the difference between SEM and TEM?

A

SEM:

  • Can see 3D
  • Lower resolution

TEM:

  • Thin section
  • 2D
  • Can see internal structures
  • Higher resolving power
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5
Q

How do electron microscopes produce images?

A
  • Stain with heavy metal, denser areas will take up more metal
  • Metal reflects electrons and these are collected
  • Denser areas will reflect more
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6
Q

What is freeze fracture EM?

A
  • Tissue frozen to -160 degrees in glycerol to prevent water crystals forming
  • Hit with knife to fracture it
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7
Q

Why do you need to preserve and fix samples, and how do you most commonly do this?

A

Prevent putrefaction, allow storage and allow for thin cutting

- Formalin (37% formaldehyde, 0.9% NaCl)

- Paraffin wax that sets hard

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8
Q

What are common biopsy techniques and what do you do after you have taken the biopsy?

A
  • Preserve in formalin
  • Embed in paraffin
  • Cut with microtome
  • Stain with H and E
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9
Q

How does formaldehyde preserve tissue?

A

Isotonic so penetrates the cell. Reacts with amino acids forming methylated bridges, dehydrates and stiffens it

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10
Q

How can tissue processing produce artefacts?

A
  • Dehydration
  • Cutting
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11
Q

What do H and E stain?

A

H - Basic dye so binds to acids like DNA.

E - Acidic dye so binds to base like proteins and cytoplasm

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12
Q

Why are fats not visible with H and E staining?

A

Xylene and toluene based solvents strip lipophilic molecules

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13
Q

How do you embed tissue?

A
  • Wash specimen and dehydrated in series of alcohol solutions, 0 to 100%.
  • Wash with xyulol or tolulol
  • Immerse in hot paraffin wax and leave to harden over night
  • Repeat hot wax
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14
Q

Why do red blood cells appear white in the middle?

A

They are biconcave and no nucleus, all haemoglobin on the edges

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15
Q

What is a frozen section and what are the advantages and disadvantages?

A
  • Frozen rapidly to -20 to -30 degrees
  • Cryostat (microtome in freezer)
  • Stained with H and E

+ Super fast so can be done in surgery

  • Poor clarity
  • Can’t be stoed indefinitely
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16
Q

Compare a paraffin-embedded section an a frozen section.

A
17
Q

How can you use immunology for staining?

A
  • Labelled antibodies with fluorescence or enzyme for coloured precipitate
  • Very sensitive
  • Only stains the cells the antigen antibody complex is on
18
Q

What are the advantages of the following microscopes:

  • Phase Contrast
  • Dark Field
  • Fluorescence
  • Confocal Light
A
19
Q

Why does electron microscopy have a higher resolution?

A

Lower limit of resolution because of using electrons instead of light

20
Q

How can you make fats visible under the microscope?

A
  • Use periodic-schiff stain
  • Frozen section and dyes soluble in fats
  • Use heavy metals to preserve structure of membrane
21
Q

What is the smallest organelle visible under the light microscope?

A
  • Nucleolus
22
Q

What are the positives and negatives of viewing cells in culture?

A

+ Manipulate environment

+ All cells are the same

+ Don’t need to test on animals

  • 3d Architecture lost
  • Influence of other cells not maintained
  • Can only grow small amounts
  • Dedifferentiation and instability
23
Q

What do you do with a section after you have cut it with a microtome to around 1-5 micrometres?

A
  • Pick up section with paintbrush and float on surface of warm water bath
  • Surface tension and heat cause cross section to stretch to minimise cutting artefacts
  • Glass slide placed under floating section
  • Leave tissue to dry and stain

03 - Body Logistics (20 decks)

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