Histology & its methods of study (Lecture 1) Flashcards
What are the properties of Epithelial tissue?
- Aggregated polyhedral cells
- Small amount in the extracellular matrix
- Lines surfaces and body cavities
- Facilitates glandular secretion
What are the properties of Connective tissue?
- Several types of fixed and wandering cells
- Abundant amount in the extracellular matrix
- Supports/Protects tissues and organs
What are the properties of Muscle tissue?
- Elongated contractile cells
- Moderate amount in the extracellular matrix
- Strong contraction
- Facilitates body movements
What are the properties of Nervous cells?
- Elongated cells with extremely fine processes
- Very small amount in the extracellular matrix
- Transmits nerve impulses
What are the steps for the preparation of tissues for study?
- Fixation
- Dehydration
- Clearing
- Infiltration
- Embedding
Why is ‘fixation’ essential in the preparation of tissues?
Fixation is used in order to prevent cell denaturing due to enzymes and lysosomes
What should cells NOT have when collected?
Nerves or blood supply
What stain does H&E stand for?
Hematoxilin and Eosin
What stain does PAS stand for?
Periodic Acid-Schiff
What are the two interacting components of tissues?
Cells and extracellular matrix (ECM)
What is the role of the extracellular matrix (ECM)?
The ECM supports the cells and contains the fluid transporting nutrients to the cells and carrying away the waste and secretory products
Describe what happens during ‘Fixation’
Small pieces of tissue are placed in solutions of chemicals that cross-link proteins and inactive degradative enzymes, which preserve cell and tissue structure
Describe what happens during ‘Dehydration’
The tissue is transferred through a series of increasingly concentrated alcohol solutions (ending in 100%) which removes all H2O
Describe what happens during ‘Clearing’
Alcohol is removed in organic solvents in which both alcohol and paraffin are miscible
Describe what happens during ‘Infiltration’
Tissue is placed in melted paraffin until it becomes completely infiltrated
Describe what happens during ‘Embedding’
Paraffin-infiltrated tissue is placed in a small fold with melted paraffin and allowed to harden
Describe what happens during ‘Trimming’
The resulting paraffin block is trimmed to expose the tissue for sectioning
Define “Basophilic”
Cell components with a net negative charge (anionic) have an affinity for basic dyes
Define “acidophilic”
Cell components with a net positive charge (cationic) stain more readily with acidic dyes
What is the mechanism of dyes?
Dyes stain material more or less selectively behaving like acidic or basic compounds and forming electrostatic (salt) linkages with ionisable radicals of macromolecules in tissues
What do basic dyes dye?
Basophilic components such as DNA, RNA and glycosaminoglycans
What do acid dyes dye?
Acidophilic components of tissues such as mitochondria, secretory granules and collagen
What is the mechanism behind the dye Hematoxylin and Eosin (H&E)?
Hematoxylin stains DNA in the cell nucleus, RNA-rich portions of the cytoplasm and the matrix of cartilage producing a dark blue/purple colour. Eosin stains other cytoplasmic structures and collagen pink.
What is the mechanism of the periodic acid-Schiff (PAS) reaction?
It utilises the hexose rings of polysaccharides and other carbohydrate-rich tissue structures and stains it purple
What is the Feulgen reaction?
a modified version of the periodic acid-Schiff (PAS) reaction that can specifically stain the DNA of cell nuclei
Define ‘Resolving Power’
The smallest distance between two structures at which they can be seen as separate objects
What is the mechanism for Bright-Field Microscopes?
Stained tissue is examined with ordinary light passing through the preparation. Quality of images is dependant on magnification and resolving power.
What is the mechanism for Virtual Microscopy?
Regions of a glass-mounted specimen are captured digitally in a grid-like pattern at multiple magnifications using a specialised slide-scanning microscope and saved as thousands of consecutive image files. Software allows access, visualisation and navigation of the original slide.
What is the mechanism for Fluorescence Microscopy?
Tissue sections are irradiated with UV light with emission in the visible portion of the spectrum; the fluorescent substances appear bright on a dark background and are analysed.
What is the mechanism of Phase-Contrast Microscopy?
A lens system is used to produce visible images from transparent objects and living, cultured cells using the phenomenon that light changes its speed when passing through cellular structures with different refractive indices. This causes structures to appear lighter or darker.
What is the name of the modified version of Phase-Contrast Microscopy that produces 3D images?
Differential Interference Contrast Microscopy
What is the mechanism for Confocal Microscopy?
It achieves high resolution and sharp focus by using:
1. a small point of high intensity light
2. a plate with a pinhole aperture in front of the image detector
and produces a 3D reconstruction from the images.
What is the mechanism for polarising microscopy?
Macromolecules with highly patterned structures that are placed between two polarising filters that are perpendicular to each other rotate the axis of light emerging from the polariser using their repetitive structure. The bright structures appear against a dark background.
Define ‘Birefringence’
The ability to rotate the direction of vibration of polarised light
[a feature of crystalline substances or substances containing highly oriented molecules]
How are contrast and resolution improved in TEMs?
Compounds with heavy metal ions are added to the fixative or dehydrating solutions used for tissue preparation
What is the difference between Transmission Electron Microscope (TEM) and Scanning Electron Microscope (SEM)?
The focused electron beam passes through the specimen in TEMs but is moved sequentially (scanned) across the specimen surface in SEMs
Define ‘Microscopic Autoradiography’
A method of localising newly synthesised macromolecules in cells or tissue sections using radioactive precursors by detecting silver grains produced by weakly emitted radiation in a photographic emulsion coating the tissue section or cells
How does Autoradiography work?
- Slides with radiolabeled cells or tissue sections are coated in a darkroom with photographic emulsion in which AgBr crystals act as microdetectors of the radiation in the same way that they respond to light in photographic film
- AgBr crystals reduced by radiation produce small black grains of metallic silver (under light microscope/TEMs this indicates the locations of radiolabeled macromolecules)
- Permits studies of tissue growth and cellular pathways of macromolecular synthesis
What is Enzyme Histochemistry (or Cytochemistry)?
A method for localising cellular structures using a specific enzymatic activity present in those structures
What is the mechanism behind Enzyme Histochemistry?
- Tissue sections are immersed in a solution containing the substrate of the enzyme to be localised
- The enzyme is allowed to act on its substrate
- The section is put in contact with a marker compound that reacts with a product of the enzymatic action on the substrate
- The final product from the marker, which must be insoluble and visible by light or electron microscopy, precipitates over the site of enzymes identifying their location
What is ‘Hybridisation’?
The specific binding between two single strands of nucleic acid, which occurs under appropriate conditions if the strands are complementary
What is ‘In Situ Hybridisation’?
When nucleic acid sequences in solution are applied directly to prepared cells and tissue sections allowing specific identification of sequences in genes or RNA
What is hybridisation ideal for?
- Determining if a cell has a specific sequence of DNA (e.g a gene or part of a gene)
- Identifying the cells containing specific mRNAs
- Determining the localisation of a gene in a specific chromosome (DNA/RNA must be denatured to become single-stranded and nucleotide sequences are detected with probes consisting of single-stranded complementary DNA)
Why are chemical fixatives important?
They are used to preserve tissue structure by cross-linking and denaturing proteins, inactivating enzymes, and preventing cell autolysis or self-digestion
In a light microscope used for histology, resolution and magnification of cells are largely dependent on which component?
Objective Lens
Cellular storage deposits of glycogen, a free polysaccharide, could best be detected histologically using what procedure?
PAS Reaction
Adding heavy metal compounds to the fixative and ultra thin sectioning of the embedded tissue with a glass knife are techniques used for which histologic procedure?
TEM
To identify and localise a specific protein within cells or the ECM, what would be the best approach?
Immunohistochemistry
What macromolecule is usually lost in the paraffin procedure?
Lipids
Name a widely used fixative for light microscopy
Formalin (a buffered isotonic solution of 37% formaldehyde)
What happens in metal impregnation?
Metal salts bind onto structures which change the colour
What is the mechanism behind SEMs?
Gold is added to the specimen and the electrons are deflected ‘scanned’ across the specimen forming a 3D picture
