Methods in Light Microscopy

Question 0

State the meaning of the term ‘tissue’

Answer 0

A collection of cells specialised to perform a particular function
(Collection of tissues = organ)

Question 1

State the relationship between milli-, micro- and nanometers

Answer 1

Cm --> mm --> um --> nm
Millimetre 10-3m
Micrometre 10-6m
Nanometre 10-9m
Angstrom 10-10m
(Most cells - 10-20um)

Question 2

Explain the value of histology in diagnosis

Answer 2

Gold standard - histology is proof

Question 3

Describe common biopsy techniques (e.g. curettage, needle, transvascular etc.) giving examples of tissues which can be sampled by each method

Answer 3

Biopsy - the removal of a small piece of tissue from an organ or part of the body for microscopic examination
Curettage - endometrial lining of uterus
Needle - brain, breast, liver, kidney, muscle
Transvascular - heart, liver
Smear - cervix, buccal cavity
Direct incision - skin, mouth, larynx
Endoscopic - lung, intestine, bladder

Question 4

Explain why tissues need to be fixed and state which fixatives are commonly used

Answer 4

So they don’t mould otherwise microbes and bacteria will break it down
Glutaraldehyde, formaldehyde
Biopsy –> fixation –> dehydration –> clearing –> embedding –> sectioning –> rehydration –> staining –> dehydration –> mounting

Question 5

Describe how tissue processing can lead to the formation of shrinkage artefacts

Answer 5

Shrinkage artefacts can occur as a result of tissue processing of any tissue
Recognise such shrinkage, don’t confuse shrinkage artefact spaces with spaces that occur naturally in living tissues

Question 6

Discuss the value of histological staining and state the components of tissue stained by the Periodic acid Schiff reaction (PAS) and by Haematoxylin and Eosin (H&E) staining

Answer 6

PAS - stains carbohydrates and glycoproteins magenta

Haemotoxylin stains acidic components of cells blue/purple, eosin stains basic components pink

Question 7

Outline the advantages conferred by phase contrast, dark field, fluorescence and confocal light microscopy

Answer 7

Phase contrast - see detail in unstained living cells - heightened contrast
Dark field - image is from scattered light (detection of syphillis and malaria)
Fluorescence - can see where individual substances/structures lie within cells
Confocal light - image optical sections via a laser to show 3D structure - illumination and eliminates out ‘focus flare’