Investigative techniques

Question 1

Identify 3 requirements for light microscopy.

Answer 1

Stain (H&E)
Paraffin
Formalin solution

Question 2

What role does formalin solution have in light microscopy?

Answer 2

This preserves the specimen ensuring that no putrefaction occurs.

Question 3

What role does paraffin have in light microscopy?

Answer 3

Embed the tissue in paraffin so that it is easier to cut thin slices to make it easier to see under a microscope as more light can pass through.

Question 4

What does H&E stand for and what is its role in light microscopy/

Answer 4

Haematoxylin and Eosin stain

Stains the specimens so it is easier to differentiate between the different tissues and components.

Question 5

Describe the process of producing a frozen section.

Answer 5

1. Specimen is placed on metal disc and frozen -20 to -30ºC
2. Frozen specimen cut with microtome in cryostat.
3. Stain with H&E

Question 6

Describe what you would see if you used these stains in a light microscopy:

• H&E
• Masson’s trichrome
• Periodic Acid-Schiff stain

Answer 6

H&E: stains acidic components (DNA) blue/purple and basic components (cytosol) red/pink

Masson's trichrome: 
red=keratin & muscle fibres
blue/green= collagen & bone
light red/pink= cytoplasm
brown/black= nuclei

Periodic Acid-Schiff stain: stains anything with a sugar magenta

Question 7

Pros and cons of frozen sections.

Answer 7

+ quicker to do
+preserves fats

• lower quality specimens
• poorer stain retention
• shorter lifespan
• can have ice damage

Question 8

Outline the process of fluorescent microscopy.

Answer 8

1. Specimen has fluorescent molecules bound to it.
2. Fluorescent molecules emit light when exposed to specific radiation wavelength.
3. You would see different colours for different components and cell types.

Question 9

When is fluorescent microscopy most commonly used?

Answer 9

Used for differentiating heterogeneity of a cancer

Question 10

Outline the process of confocal microscopy.

Answer 10

1. Use a fluorescent dye
2. Lasers are projected to specimen and excite the fluorescent molecules.
3. A light is emitted through a pinhole screen to a detector.
4. Only focused part of light reaches detector
5. Creates very sharp image.

Question 11

Pros of confocal microscopy.

Answer 11

• high quality image produced
• 3D images
• can be used for live specimens (time lapse video)

Question 12

Outline the process of autoradiography.

Answer 12

• Live specimen injected with a radioactive marker

- Histological section is coated in an emulsion to see the molecules marker

Question 13

What is ultrasound?

Answer 13

Use of soundwaves to produce an image based on how long it takes for echoes to return.
More reflection (hyperechoic) = white
Less reflection (hypoechoic) = black

Question 14

Describe the process of performing an ultrasound.

Answer 14

1. Transducer emits ultrasound waves towards the tissues
2. Waves echo off the tissue.
3. Based on the density and distance of structures, the reflection times will differ.
4. A black and white image will be produced due to the reflected echoes being recorded.

Question 15

Describe the use and aim of an ultrasound.

Answer 15

Most commonly used to view fetuses or any internal body structures to find/exclude a source of disease.
The use of ultrasound is quick and doesn’t involve any radiation so it is safer to use.

Question 16

Outline the process involved in TEM.

Answer 16

1. Fix, embed, stain, cut
2. e- beam passes through specimen
3. Transmitted beam = lighter image
4. Absorbed/scattered beams = dark image

Question 17

Make a relative comparison of electron & light microscopy in terms of:

• resolution
• magnification

Answer 17

Electron microscopy allows for a much higher resolution and magnification than light microscopy.
E = 400x light resolution
E = 250x light mag.

Question 18

Outline the process involved in freeze fracture electron microscopy.

Answer 18

Used to look at the cell membrane components.

1. Tissue frozen to -160ºC and fractured by hitting the specimen with a knife edge.
2. Fracture line will go through the plasma membrane so the interior structure is exposed.

Question 19

Outline the process involved with SEM.

Answer 19

1. e- projected towards the specimen
2. all e- reflected back to cathode ray tube
3. depending on where e- are reflected will give you a 3D image of the surface of the specimen.

Question 20

What is an MRI?

Answer 20

Non-invasive, non-destructive tool that relies on a magnetic field that produces transverse images of soft tissues.

Question 21

How does an MRI work?

Answer 21

1. H atoms spin in random directions in their own magnetic field
2. MRI produces a magnetic field and the atoms line up north/south.
3. Applied radio frequency pulse matches all the atoms’ direction of spin.
4. When radio freq. turned off, the originally unmatched atoms will spin (relax) to their original direction and emits energy by doing that.
5. Energy sends a signal to the computer which creates an image.

Question 22

When you look at an MRI image, you can see different shades of black, grey and white. How are these different contrasts produced?

Answer 22

The contrasts are created due to the different relaxation times of the H atoms within the different tissues.