Histology and its methods of study

Question 1

What is the extracellular matrix and its functions?

Answer 1

ECM is made up of lots of macromolecules
Supports cells:
- contains fluid to transport nutrients to cells and carry away waste and secretory products

Question 2

What are the main processes for visualising cells on a microscope?

Answer 2

Cutting- small tissue pieces to ensure all cells are preserved
Fixation - to preserve tissue structure and prevent degradation by enzymes
Dehydration - tissue is transferred through increasing concentrations of alcohol solutions ending in 100% to ensure all water is removed
Clearing - alcohol is removed in organic solvents in which both alcohol and paraffin are miscible
Infiltration - tissue is placed in melted parafin until it becomes completely infiltrated with this substance
Embedding - parafin-infiltrated tissue is placed in a small moved with melted parafin and allowed to harden
Trimming - resulting paraffin block is trimmed to expose tissue for slicing on a microtome
Mounting- placed the slice onto protective glass coverslip on a plate with clear adhesive

Question 3

What happens during filtration and provide examples of fixatives?

Answer 3

small pieces of tissue are placed in chemical solutions that cross link proteins and inactivate degradative enzymes
LM fixative- formalin
EM fixative - glutaraldehyde

Question 4

What are some example embedding materials?

Answer 4

Parafin for LM

Plastic resins for LM and EM

Question 5

What happens in a operating theatre to biopsies ?

Answer 5

They are fixed in vials of formalin for processing in a laboratory
However, if results are need before the end of the procedure (e.g. determine if tumour is malignant), the biopsy is frozen in liquid nitrogen and a cryostat (microtome) slices the tissue and rapid staining is undertaken

Question 6

What do basic dyes termed basophilic stain and provide examples?

Answer 6

Stain cell components with a net negative charge
e.g. nucleic acids
Toluudine blue, alcian blue and methylene blue, haematoxylin

Question 7

What do acidic dyes termed acidophilic stain and provide examples?

Answer 7

Stain cationic components such as proteins with lots of ionised amino acids
eosin, orange G, acid fuchsin

Question 8

What is the periodic acid shiff (PAS) reaction?

Answer 8

uses hexose rings of polysaccharides and other carbohydrate rich tissue and stains such macromolecules purple or magenta

Question 9

How are lipid rich structures visualised?

Answer 9

By avoiding processing steps which remove lipids- heating and organic solvent
and by using lipid soluble dyes such as sudan black
Useful for diagnosing metabolic diseases

Question 10

What are the different types of light microscopy (LM)?

Answer 10

bright-field
fluorescence
phase-contrast
confocal 
polarizing 
ALL based on interaction of light with tissue components to reveal tissue structure

Question 11

What is bright-field microscopy?

Answer 11

Method most commonly used by both students and pathologists- uses ordinary light and the colours are imparted by tissue staining

Question 12

What is fluorescence microscopy?

Answer 12

uses UV light under which only fluorescent molecules are visible, allowing localisation of fluorescent probes which can be much more specific than routine stains

Question 13

What is phase-contrast microscopy?

Answer 13

uses the differences in refractive index of various natural cell and tissue components to produce an image without staining, allowing observation of living cells

Question 14

What is confocal microscopy?

Answer 14

involves scanning the specimen at successive focal planes with a focused light beam, often from a laser and produces a 3D reconstruction from the images

Question 15

What is polarising microscopy?

Answer 15

allows you to visualise stained or unseating structures made of highly organised subunits - produces an image only of material having repetitive periodic macromolecular structures

Question 16

How does electron microscopy work?

Answer 16

It is based upon interactions between tissue components and beams of electrons

Question 17

What is TEM?

Answer 17

High resolution imaging technique- allowing resolution around 3nm
A beam of electrons are focused using electromagnetic lenses passes through tissue section to produce and B&W image with intermediate grey regions

Question 18

What is SEM?

Answer 18

high resolution view of the surfaces of cells, tissues and organs
produces and focuses a very narrow beam of electrons but it does NOT pass through the tissue - instead specimen is dried and sprayed with a very thin layer of heavy metal which reflects the electrons which are then captured by the detector creating B&W images

Question 19

What is autoradiography?

Answer 19

Method of localising newly synthesized macromolecules
It localises cell components synthesised using radioactive precursors by detecting silver grains produced by weakly emitted radiation in a photographic emulsion coating the tissue

Question 20

What can autoradiography do in combination with either LM or TEM?

Answer 20

It permits unique studies of processes such as tissue growth (using radioactive DNA precursors) or cellular pathways of macromolecular synthesis

Question 21

What is cell/tissue culture and why is it beneficial?

Answer 21

cells can be grown in vitro from newly explanted tissues (primary culture) or as long established cell lines
They are observation of cellular behaviour under phase contrast microscopy

Question 22

Why are cell/tissue cultures important clinically?

Answer 22

Widely used in research to study molecular changes in cancer, analyze infectious viruses, and some protozoa and routine genetic analysis

Question 23

What is the perl’s prussian blue reaction?

Answer 23

An enzyme histochemical procedure for iron to diagnose iron storage diseases, hemochromatosis (body loads too much iron), hemosiderosis(iron overload disorder causing accumulation of hemosiderin)

Question 24

What are the PAS-amylase and alcian blue reactions used to detect?

Answer 24

for polysaccharides (detect glycogenosis and mucopolysaccharidosis) and reactions for lipid and sphingolipids (detect sphinoglipidosis)

Question 25

What enzyme classes are commonly used in enzyme histochemistry?

Answer 25

phosphatases, dehydrogenases, and peroxidases (these often conjugate to antibodies in immunohistochemistry)

Question 26

What are the following molecules used to visualise?

• Phalloidin
• Protein A
• lectins

Answer 26

Phalloidin- interacts with actin protein of microfilaments
Protein A- binds to the Fc region of antibody molecules
Lectins- bind to carbohydrates with high affinity and specificity

Question 27

What is immunohistochemistry?

Answer 27

Based on specific reactions between an antigen and antibodies labelled with visible markers, often fluorescent compounds or peroxidase (for LM) or gold particles (for TEM)

Question 28

What is the difference between direct and indirect immunohistochemistry?

Answer 28

Direct: primary antibody binds directly to the specific antigen
Indirect: uses unlabelled primary antibody which binds to antigen and a secondary labelled antibody binds to the primary antibody for visualisation

Question 29

What is in situ hybridisation?

Answer 29

Label specific gene sequences or mRNAs of cells microscopically using labelled complementary DNA (cDNA)