Introduction to microscopy and staining

Question 1

the relationship between millimetres, micrometres and nanometres

Answer 1

Millimetres 10-3 (mm)

Micrometres 10-6 (um) - most human cells are 10-20um
-> RBCs (7.2um) , Neutrophils (11um), Keratinocyte (25um), Oocytes (100um)

Nanometres 10-9 (nm)
-> small molecules, lipids, proteins, and viruses

Question 2

Define the term ‘limit of resolution’

Answer 2

-> the minimum distance two objects can be distinguished / resolved and seen as distinct entities.

Question 3

Explain why electron microscopes are capable of finer resolution than light
Microscopes

Answer 3

Electron microscopes differ from light microscopes in that they produce an image of a specimen by using a beam of electrons rather than a beam of light.

-> Electrons have much a shorter wavelength than visible light, and this allows electron microscopes to produce higher-resolution images than standard light microscopes.

Question 4

limit of resolution for light and electron microscopes

Answer 4

Electron microscope: 1 nanometre is the limit of resolution

Light microspore: 200 nanometres is the limit of resolution

Question 5

What is a Biopsy?

Answer 5

The removal of a piece of tissue for microscopic examination.

Question 6

types of Smear biopsies?

Answer 6

cervix or buccal cavity

Question 7

types of Curettage Biopsy

Answer 7

endometrial lining of the uterus

Question 8

types of Direct incision biopsy

Answer 8

Direct incision – e.g. skin or mouth or larynx

Question 9

Examples of Biopsy Procedures

Answer 9

Needle – e.g. brain, breast
Endoscopic – e.g. intestine or bladder
TransVascular – e.g. heart or liver

Question 10

Explain why tissue needs to be fixed

Answer 10

Tissues need to be fixed (chemically preserved) using chemicals such as formaldehyde, glutaraldehyde, alcohol a

-> (macromolecules are cross-linked, cellular structure is preserved, no autolysis or putrefaction).

Question 11

Biopsy to Microscopy steps

Answer 11

1.Collection – biopsy (e.g. needle biopsy fresh, wet and bloody)

2.Fixation- chemical preserving e.g. formaldehyde to preserve structure

3.Embedding and Processing - dehydration with ethanol, Xylene removes ethanol making it easier to embed the tissue in paraffin wax which removes xylene and solidifies the tissue into a paraffin block

4.Sectioning – cutting the solidified tissue using a single turn of the microtome drive wheel usually around 4μm in thickness

5.Staining – colouring the tissue section:

Question 12

Haematoxylin

Answer 12

BASIC

-> It stains acidic components of cells

a purple/blue colour

e.g. nucleolus (rRNA) chromatin (DNA) are basophilic.

Question 13

eosin

Answer 13

ACIDIC

-> It stains basic components of cells

shades of pink colour

e.g. most cytoplasmic proteins and extracellular proteins (such as collagen) are strongly eosinophilic.

Question 14

PAS – Periodic acid-Schiff

Answer 14

Stains complex carbohydrates

Dark Red or magenta

Identifies glycogen in cells

e.g. hepatocytes and muscle cells

Question 15

Masson Trichome

Answer 15

Stains supporting tissue particularly collagen

-

Question 16

What colour does Masson Trichome stain collective tissue?

Answer 16

Stains connective tissue blue

Question 17

What colour does Masson Trichome stain nucelli

Answer 17

Stains nucelli dark red or purple

Question 18

What colour does Masson Trichome stain cytoplasm

Answer 18

Stains cytoplasm, keratin, erythrocytes, muscles red/pink

Question 19

Acain Blue stains…

Answer 19

Stains acidic polysaccharides blue

Question 20

Van Gieson

Answer 20

Often used to stain blood vessels and the skin

Useful to visualise collagen and differentiate muscle from collagen

• Collagen appears pink
• Elastic fibres appear black
• Nuclei appear blue
• Erythrocytes , muscle and cytoplasm and fibrin appear yellow

Question 21

Giemsa stain

Answer 21

Standard stain for blood and bone marrow smears

• Erythrocytes appear pale pink
• Cytoplasm pale blue
• Nuclei dark blue or violet

Question 22

Sudan Black and osmium

Answer 22

Stains lipid containing structure brown/black

e.g. myelin and nerves, and adipocytes

*useful stain in diagnosing metabolic diseases that result in increased intracellular cholesterol, phospholipids, or glycolipids

Question 23

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

Answer 23

-> artefacts are distortions in the cell tissue architecture and they can be induced through any of the tissue preparations

Question 24

What is histology

Answer 24

the study of tissues and organs using microscopy

Question 25

State the components of tissue stained by the: periodic acid-Schiff reaction (PAS)

Answer 25

This is a special stain which stains carbohydrates and glycoproteins (magneta – colour deep red)

Question 26

Nissl bodies

Answer 26

Using appropriate stains, RER and free ribosomes appear as basophilic, granular areas, called ‘Nissl bodies’.

These are sites of protein synthesis. Nissl bodies are particularly abundant in large nerve cells such as motor neurones.

Chromatolysis (chroma: colour; lysis: splitting) refers to the disintegration of Nissl bodies following motor neurone injury.

Question 27

Frozen sections

Answer 27

used for faster results – but only used urgently as the quality of the slides are not as good.

e.g. during surgery the tissues samples can be analysed quickly before completely the surgery.

The margin of the resected mass is sent to the pathology lab.
->the mass is embedded in optimal cutting temperature compound (OCT)
->frozen in liquid nitrogen
->cryostat is maintained at subfreezing temperature.

->most commonly stained with h and e, methylene blue and/or PAS
->formalin fixed and paraffin embedded.

*turnaround time as fast as 10 minutes.