Histology and its methods of study Flashcards
What is the extracellular matrix and its functions?
ECM is made up of lots of macromolecules
Supports cells:
- contains fluid to transport nutrients to cells and carry away waste and secretory products
What are the main processes for visualising cells on a microscope?
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
What happens during filtration and provide examples of fixatives?
small pieces of tissue are placed in chemical solutions that cross link proteins and inactivate degradative enzymes
LM fixative- formalin
EM fixative - glutaraldehyde
What are some example embedding materials?
Parafin for LM
Plastic resins for LM and EM
What happens in a operating theatre to biopsies ?
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
What do basic dyes termed basophilic stain and provide examples?
Stain cell components with a net negative charge
e.g. nucleic acids
Toluudine blue, alcian blue and methylene blue, haematoxylin
What do acidic dyes termed acidophilic stain and provide examples?
Stain cationic components such as proteins with lots of ionised amino acids
eosin, orange G, acid fuchsin
What is the periodic acid shiff (PAS) reaction?
uses hexose rings of polysaccharides and other carbohydrate rich tissue and stains such macromolecules purple or magenta
How are lipid rich structures visualised?
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
What are the different types of light microscopy (LM)?
bright-field fluorescence phase-contrast confocal polarizing ALL based on interaction of light with tissue components to reveal tissue structure
What is bright-field microscopy?
Method most commonly used by both students and pathologists- uses ordinary light and the colours are imparted by tissue staining
What is fluorescence microscopy?
uses UV light under which only fluorescent molecules are visible, allowing localisation of fluorescent probes which can be much more specific than routine stains
What is phase-contrast microscopy?
uses the differences in refractive index of various natural cell and tissue components to produce an image without staining, allowing observation of living cells
What is confocal microscopy?
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
What is polarising microscopy?
allows you to visualise stained or unseating structures made of highly organised subunits - produces an image only of material having repetitive periodic macromolecular structures
How does electron microscopy work?
It is based upon interactions between tissue components and beams of electrons
What is TEM?
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
What is SEM?
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
What is autoradiography?
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
What can autoradiography do in combination with either LM or TEM?
It permits unique studies of processes such as tissue growth (using radioactive DNA precursors) or cellular pathways of macromolecular synthesis
What is cell/tissue culture and why is it beneficial?
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
Why are cell/tissue cultures important clinically?
Widely used in research to study molecular changes in cancer, analyze infectious viruses, and some protozoa and routine genetic analysis
What is the perl’s prussian blue reaction?
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)
What are the PAS-amylase and alcian blue reactions used to detect?
for polysaccharides (detect glycogenosis and mucopolysaccharidosis) and reactions for lipid and sphingolipids (detect sphinoglipidosis)
What enzyme classes are commonly used in enzyme histochemistry?
phosphatases, dehydrogenases, and peroxidases (these often conjugate to antibodies in immunohistochemistry)
What are the following molecules used to visualise?
- Phalloidin
- Protein A
- lectins
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
What is immunohistochemistry?
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)
What is the difference between direct and indirect immunohistochemistry?
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
What is in situ hybridisation?
Label specific gene sequences or mRNAs of cells microscopically using labelled complementary DNA (cDNA)
