BL - Investigative Techniques (done to slide 29)

Question 1

How is cut tissue preserved to stop it from rotting?

Answer 1

Formalin is used

Question 2

What is used to allow tissue to be cut very thinly for microscopy? (5 micrometers)

Answer 2

Melted paraffin which sets hard when cooled

Question 3

What does haematoxylin stain and what colour does it stain it?

Answer 3

It stains the nucleus blue

Question 4

What colour does eosin stain things, and what does it stain?

Answer 4

Cytoplasm and extracellular matrix, pink

Question 5

What is a “frozen section”?

Answer 5

A specimen is placed on a metal disc and then frozen to -20 to -30 degrees C. It is then stained with H+E. It is very fast, but technical quality is low.

Question 6

Two samples are taken from two different patients and viewed using polarised light microscopy. One shows long thin crystals and one shows rhomboid crystals. Which is gout?

Answer 6

Long thin = gout.

Rhomboid = pseudogout

Question 7

True or false - fluorescent microscopy can be used to diagnose cancer?

Answer 7

True.

Question 8

True or false - confocal microscopy is never combined with fluorescent microscopy?

Answer 8

False, they often are.

Question 9

What makes confocal microscopy different to normal microscopy?

Answer 9

A pinhole is placed art the confocal plane of the lens to eliminate out of focus light, which enables sharper 3D images to be created.

Question 10

How does immunohistochemical staining work?

Answer 10

Antigens in the cells of a tissue section can be detected by labelled antibodies binding to them.

Question 11

Describe the process of autoradiography.

Answer 11

A photographic emulsion is used to visualise molecules labelled with radioactive marker. The marker is first injected into the live animal/cell culture. The histological section is then coated with the emulsion.

Question 12

True or false - higher frequency (shorter wavelength) gives a better resolution?

Answer 12

True

Question 13

Why does transmission electron microscopy have a better resolution than light microscopy?

Answer 13

The wavelength is 1 nanometre compared to light microscopy which uses 400 nanometres.

Question 14

Why do some portions of an image created by TEM seem dark?

Answer 14

These portions have absorbed electrons, while the beam has passed straight through the light sections.

Question 15

How is a sample prepared for viewing with a TEM?

Answer 15

Fix with glutaraldehyde, embed in epoxy resin, stain (eg. Osmium tetroxide), use microtome with diamond knives.

Question 16

What is “freeze fracture electron microscopy”?

Answer 16

The tissue is frozen to -160 degrees C and fractured by hitting it with a knife edge. The fracture line passes through the plasma membrane, exposing its interior.

Question 17

How does scanning electron microscopy differ from TEM?

Answer 17

In SEM, the electrons are reflected back rather than through a sample. They create a 3D image of a living sample.