Investigative techniques

Question 1

What are the requirements of light microscopy?

Answer 1

• Preservation
• Embedding
• Staining

Question 2

How are tissues preserved for imaging?

Answer 2

Formalin

Question 3

What is the purpose of melted paraffin in tissue processing?

Answer 3

Embeds the tissue allowing it to be sliced into 5 micrometers (um) once the paraffin has set.

Question 4

What is tissue stained with for light microscopy?

Answer 4

Haemotoxylin and eosin

Haemotoxylin - stains nucleus strongly blue

Eosin - stains cytoplasm and extracellular matrix pink

Question 5

What is a frozen section?

Answer 5

Used during surgery for quick histological techniques.

• Specimen is placed on a metal disc and frozen rapidly to -20 to -30C
• Cryostat (microtome in a freezer) slices the specimen into thin slices
• Stained with haemotoxylin and eosin

Question 6

What are the differences between traditional staining and frozen section?

Answer 6

• Frozen sections are quicker than traditional methods (10 minutes vs 16 hours)
• Technical quality of frozen sections is much lower

Question 7

How is gout diagnosed?

Answer 7

Polarised light microscopy visualises monosodium urate crystals commonly in the metatarsophalangeal joint.

Crystals are needle-shaped, strong negative birefringence yellow when parallel to compensator ray.

Question 8

What is gout?

Answer 8

Inflammatory arthritis developing when someone has high levels of uric acid in the blood.

Question 9

How is pseudogout diagnosed?

Answer 9

Calcium pyrophosphate dihydrate (CPPD) crystals.

Rod or rhomboid, weak positive birefringence. Blue when parallel to compensator ray. Most commonly affects the knee joint.

Question 10

What is fluorescence microscopy?

Answer 10

Fluorophores emit light when irradiated by a specific wavelength. Used for cancer cell type differentiation. Molecules are excited by a specific wavelength to emit a certain light.

Question 11

What is confocal microscopy?

Answer 11

Optical imaging technique used to build up a 3D image of a specimen by taking images of multiple planes. Resembles the imaging process in computed axial tomography scanning (CAT scans).

Question 12

What is immunohistochemistry?

Answer 12

Selectively images tissue using antibodies to test for specific antigens. Antibodies are normally linked to enzymes or dye for visualisation. Aids diagnosis of cancer.

Question 13

What is direct immunofluorescence?

Answer 13

Antibody is labelled with fluorescent dye before being added to the tissue.

Question 14

What is indirect immunofluorescence?

Answer 14

Antibody is labelled after it has bound to the antigen through anti-immunoglobulin serum.

Question 15

What is autoradiography?

Answer 15

Use of an x-ray to detect radioactive materials.

• photographic emulsion is used to visualise molecules labelled with a radioactive marker
• marker is injected into the live animal/cell culture
• histological section is coated with the emulsion

Question 16

What is a radionuclide scan?

Answer 16

A way of imaging bones, organs and other components of the body through a small dose of radiation. Example: Iodine¹³¹ scan shows enlarged thyroid gland.

Question 17

What is the resolution and magnification of modern light microscopy?

Answer 17

• Magnification: 1000x
• Resolution: 0.2um

Question 18

What is ultrasound scan?

Answer 18

Also called a sonogram. High frequency sounds waves are emitted by an ultrasound probe which then echoes off structures of the body to be received in an image.

Question 19

When does the frequency of wavelengths affect the resolution of an image?

Answer 19

The image of two particles cannot be seen individually if it is smaller than the wave length.

Question 20

What is transmission electron microscopy?

Answer 20

Microscopy technique using beams of electrons in a vacuum to create an image. Portions of the beam that passes through the tissue appears bright, portions that have absorbed or scattered the tissues appear dark.

Resolution: 400 times light microscopy (0.2um)

Magnification: 250,000 x

Question 21

What is the preparation for a transmission electron microscope?

Answer 21

• Fix: Glutaraldehyde
• Embed: Epoxy resin
• Stain: Osmium tetraoxide
• Microtome: diamond knives

Question 22

What is a freeze fracture electron microscopy?

Answer 22

Tissue is frozen -160C and then fractured by hitting a knife edge. Interior of the cell membrane is then exposed which can be imaged.

Question 23

What is scanning electron microscopy?

Answer 23

The surface is scanned with a focused beam of electrons which is reflected back to a cathode ray tube to create an image.

Question 24

What is x-ray crystallography?

Answer 24

Determines the atomic and molecular structure of a crystal by firing x-ray beams to diffract into different directions which can be measured. Diffraction is dependant on electron density

Question 25

What is nuclear magnetic resonance (NMR) spectroscopy?

Answer 25

Gives details of the electronic structure of a molecule as the intramolecular magnetic field around an atom changes the resonant frequency.

Question 26

What is magnetic resonance imaging?

Answer 26

A type of NMR spectrometry. Strong magnetic fields and radio waves are used to produce detailed images of inside the body.

Used to examine:

• brain and spinal cord
• bones and joints
• breasts
• heart and blood vessels
• internal organs (liver, womb or prostate gland)