Week 1- Methods in Light Microscopy

Question 1

Explain the value of histology in diagnosis.

Answer 1

Histology studies tissue and cell structure. For many diseases, doctors will not give treatment until the histopathologists have given a diagnosis.

Final proof is histology and diagnosis. Histology is the gold standard of diagnosis. Histology can also help type the disease and inform therapy.

Question 2

Describe common biopsy techniques giving examples of tissues which can be sampled by each method.

Answer 2

1. Curettage - Endometrial lining of uterus
2. Endoscopic - lungs, intestine, bladder
3. Needle - brain, breast, liver, kidney, muscle
4. Transvascular - liver, heart
5. Smear- cervix or buccal cavity
6. Direct incision- skin, mouth, larynx

Question 3

Explain why tissues need to be fixed.

Answer 3

Fresh biopsy (e.g. needle biopsy - wet and bloody): need to make it rigid and firm
Fixed biopsy: macromolecules cross-linked, cellular structure preserved, no autolysis or putrefaction

Question 4

Biopsy

Answer 4

The removal of a small piece of tissue from an organ or part of the body for microscopic examination

Question 5

State which fixatives are commonly used for tissue fixing.

Answer 5

1. Glutaraldehyde

2. Formaldehyde

Question 6

Tissue

Answer 6

A collection of cells specialised to perform a particular function

Question 7

Discuss the value of histological staining.

Answer 7

• Differentially stain different components of the tissue

- Most stains are water-soluble

Question 8

State the components of tissue stained by the Haematoxylin and Eosin (H&E) staining.

Answer 8

1. Haematoxylin: stains acidic components of cells purple/blue
   - Nucleolus (RNA), Chromatin (DNA)
2. Eosin: stains basic components of cells pink;
   - Most cytoplasmic proteins, extracellular fibres

Question 9

State the components of tissue stained by the Periodic Acid Schiff reaction.

Answer 9

PAS method stains carbohydrates and glycoproteins magenta.

Question 10

Outline the advantages conferred by phase-contrast microscopy.

Answer 10

It exploits the interference effects produced when 2 sets of waves combine.
It enables non-conspicuous, otherwise invisible organelles of the cell to be seen.

Question 11

Outline the advantages conferred by dark field microscopy.

Answer 11

Light from the side is scattered.

Question 12

Outline the advantages conferred by fluorescence microscopy.

Answer 12

-Antibodies used to fluoresce different parts of the same cell

Question 13

Outline the advantages conferred by confocal light microscopy.

Answer 13

• Used to image tissues which have been labelled with one or more fluorescent probes
• Using a conventional light microscope, the fluorescence in the image away from the region of interest interferes with resolution of structures in focus.
• Able to eliminate ‘out of focus’ flare from thick fluorescently labelled specimens
• Illumination in a confocal microscope can be achieved by scanning one or more focused beams of light, usually from a laser, across the specimen
• Images produced by scanning the specimen in this way: optical sections
• Tissue is effectively sectioned in a non-invasive way by light rather than physical means
• Facilitated imaging of living specimens
• Enabled the electronically automated construction of three-dimensional images from a series of 2D images taken at successive depths.