Investigative Techniques

Question 1

Define Limit of Resolution

Answer 1

Smallest distance by which 2 objects can be distinguished as separate objects

Question 2

Identify 3 requirements for tissue imaging

Answer 2

• Formalin to prevent tissue putrefaction
• Paraffin to embed tissue in a substance that allows thin slicing
• H&E staining to clearly see structures

Question 3

When is fluorescent microscopy most commonly used and what are the advantages?

Answer 3

• Differentiation of different cancer cell types
• Makes it easy to see tagged cells/ objects
• Often used with confocal microscopy

Question 4

Give advantages of the following types of microscopy

• Phase contrast
• Dark field
• Confocal

Answer 4

• Phase: To study unstained samples
• Dark: Live unstained samples, Almost artefact-free, Easy to see bright objects
• Confocal: Sharp, 3D, Live Images

Question 5

How is a Frozen Section prepared?

Answer 5

• Rapidly frozen
• Cut with cryostat
• Stained with H&E

Question 6

Outline process of Transmission Electron Microscopy

Answer 6

• Electron beam is passed through tissue

Section appears BRIGHT if electrons pass through

Section appears DARK if electrons are absorbed or scattered

Question 7

How is a Freeze Fracture produced for Electron Microscopy?

Answer 7

• Frozen
• Fractured with knife edge
• Where fracture line goes through membrane, the membrane interior is exposed

Question 8

Outline process of Scanning Electron Microscopy

Answer 8

• Electron beams are reflected from surface of specimen

- Received by a cathode ray tube (CRT)

Question 9

What methods can be used to collect tissues?

Answer 9

1. Scraping (Curettage, Endometrium)
2. Sharp needles (Pipelle- Endometrium, lining of womb)
3. Dissecting a sample from surgery (Biopsy)
4. Venupuncture
5. Collecting bodily fluids

Question 10

Outline the process of Fixation

How can an artefact be made like this?

Answer 10

• Removes H20 from sample, stiffening it
• Done with Formalin (Formaldehyde + Isotonic 0.9% Saline)
• Cross links proteins
• Fixating for more than 24-48 hours may cause tissue to shrink and produce “Fixation Artefacts”

Question 11

Outline the process of Embedding

Answer 11

1. Specimen dehydrated in varying alcohol concentrations
2. Immersed in hot, dissolved paraffin wax overnight
3. Placed in a mould, more hot wax added
4. Cool to room temp.
5. Cut with microtome into very thin sections

Question 12

What is the effect of staining with H&E?

Answer 12

Hematoxylin stains NUCLEUS BLUE
Eosin stains CYTOPLASM + ECM PINK

When combined, nuclei are clearer and more detailed cytoplasm

Stains RBC with Red edges and White inner circle

Question 13

Compare Paraffin-embedded tissue section VS Frozen section

Answer 13

Paraffin:

• Frozen tissues
• Takes longer to prepare
• Higher quality image
• Can be stored permanently

Frozen:

• Fresh tissues (Surgeries)
• Faster to prepare
• Lower quality image
• Can only be stored for a few months

Question 14

How does immunohistochemistry work?

Answer 14

• Primary antibody binds to proteins
• Labelled secondary antibody binds to Primary
• Substrate adde, converted to coloured product by enzyme on secondary antibody

Question 15

How does Immunofluorescence work?

Answer 15

• Primary Antibody labelled with fluorescent tag binds to proteins
• Light shone onto sample causes signal to be released