Introduction to microscopy and staining Flashcards
the relationship between millimetres, micrometres and nanometres
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
Define the term ‘limit of resolution’
-> the minimum distance two objects can be distinguished / resolved and seen as distinct entities.
Explain why electron microscopes are capable of finer resolution than light
Microscopes
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.
limit of resolution for light and electron microscopes
Electron microscope: 1 nanometre is the limit of resolution
Light microspore: 200 nanometres is the limit of resolution
What is a Biopsy?
The removal of a piece of tissue for microscopic examination.
types of Smear biopsies?
cervix or buccal cavity
types of Curettage Biopsy
endometrial lining of the uterus
types of Direct incision biopsy
Direct incision – e.g. skin or mouth or larynx
Examples of Biopsy Procedures
Needle – e.g. brain, breast
Endoscopic – e.g. intestine or bladder
TransVascular – e.g. heart or liver
Explain why tissue needs to be fixed
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).
Biopsy to Microscopy steps
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:
Haematoxylin
BASIC
-> It stains acidic components of cells
a purple/blue colour
e.g. nucleolus (rRNA) chromatin (DNA) are basophilic.
eosin
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.
PAS – Periodic acid-Schiff
Stains complex carbohydrates
Dark Red or magenta
Identifies glycogen in cells
e.g. hepatocytes and muscle cells
Masson Trichome
Stains supporting tissue particularly collagen
-
What colour does Masson Trichome stain collective tissue?
Stains connective tissue blue
What colour does Masson Trichome stain nucelli
Stains nucelli dark red or purple
What colour does Masson Trichome stain cytoplasm
Stains cytoplasm, keratin, erythrocytes, muscles red/pink
Acain Blue stains…
Stains acidic polysaccharides blue
Van Gieson
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
Giemsa stain
Standard stain for blood and bone marrow smears
- Erythrocytes appear pale pink
- Cytoplasm pale blue
- Nuclei dark blue or violet
Sudan Black and osmium
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
Describe how tissue processing can lead to the formation of artefacts:
-> artefacts are distortions in the cell tissue architecture and they can be induced through any of the tissue preparations
What is histology
the study of tissues and organs using microscopy
State the components of tissue stained by the: periodic acid-Schiff reaction (PAS)
This is a special stain which stains carbohydrates and glycoproteins (magneta – colour deep red)
Nissl bodies
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.
Frozen sections
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.
