Methods Of Light Microscopy

Question 0

Define milli-, micro-, and nanometres in metres.

Answer 0

Millimetres: 1 x 10^-3 metres
Micrometres (u): 1 x 10^-6 metres
Nanometres: 1 x 10^-9 metres

Question 1

What is the value of histology in diagnosis?

Answer 1

• Confirmation/final proof of disease (gold standard of diagnosis)
• Typing of disease informs treatment e.g. cancer chemo v.s. surgery; diagnosis of Crohn’s disease

Question 2

Describe a smear biopsy and give an example of the sample tissue.

Answer 2

Wiping of the surface of a tissue e.g. cervix, buccal cavity (inside of cheek)

Question 3

Describe a curettage biopsy and give an example of the sample tissue.

Answer 3

Curved spoon (curette) scrapes endometrial lining of sample e.g. lesions on the uterus endometrial lining.

Question 4

Describe a needle biopsy and give an example of the sample tissue.

Answer 4

Wide bore needle or fine needle aspiration takes a core of tissue from site e.g. masses in brain, breast, liver, kidney, muscle.

Question 5

Describe a direct incision (excisional) biopsy and give an example of the sample tissue.

Answer 5

Cut out sample with scalpel and forceps e.g. skin, mouth, larynx.

Question 6

Describe an endoscope biopsy and give an example of the sample tissue.

Answer 6

Pass endoscope (directable tube with a camera) and use “jaws” to remove tissue e.g. lungs, intestine, bladder.

Question 7

Describe a transvascular biopsy and give an example of the sample tissue.

Answer 7

Enter major veins of closed systems using an x-ray guided probe e.g. liver, kidney, heart.

Question 8

Why does tissue have to be fixed?

Answer 8

Prevent degradation of proteins, including enzymes (due to autolysis) of the sample, which leads to putrefaction.

Question 9

Name and describe some common stains used in histology.

Answer 9

Haematoxylin & Eoisin - H stains acidic components purple/blue e.g. DNA and RNA; E stains basic components pink e.g. most cytoplasmic proteins.
Periodic Acid-Schiff - stains carbohydrates and glycoproteins magenta e.g. goblet cells, basement membrane.

Question 10

Name the common fixatives used in histology.

Answer 10

Glutaraldehyde & Formaldehyde form cross-links between macromolecules, therefore no putrefaction of sample.

Question 11

What are artefacts, and how may they occur?

Answer 11

Artefacts = unwanted effects of fixation
e.g. removal of adipose tissue (dissolved in xylene & toluene),
shrinking/swelling of cells (fixed/dehydrated too fast) leading to distortion/compression of extraceullar spaces.

Question 12

How does phase contrast microscopy work, and what are the advantages of using it?

Answer 12

Phase contrast microscopy = converts invisible phase shifts in light (due to light passing through a transparent specimen) to variations in brightness of the sample when viewed through a microscope.

Advantage:

• boundaries easier to differentiate than simple brightfield microscope
• view unstained cells

Question 13

How does darkfield microscopy work, and what are the advantages of using it?

Answer 13

Darkfield microscopy = light refracted on dark background.

Advantage: easier to spot live microorganisms e.g. malaria, syphilis

Question 14

How does fluorescence microscopy work, and what are the advantages of using it?

Answer 14

Fluorescence microscopy = use fluorescent stains/markers to label certain components.

Advantage: can differentiate between different molecules and observe and track reaction progress e.g. mitosis.

Question 15

How does confocal microscopy work, and what are the advantages of using it?

Answer 15

Confocal microscopy = laser beam passed through sample, building a 3D image from the optical sections.

Advantage:

• allows living specimens to be observed, as it is non-invasive and no staining is required.
• eliminates “out of focus flare”