Microscopy and Epithelia Flashcards
How do light microscopes work
They use visible light which passes through the specimen and is collected by the image forming optics to reveal the structure of living cells and tissue
How do you estimate the size of a structure under a light microscope
Imagine how many can fit into the diameter of a field of view
Or
By comparing it to a structure of known size (eg human blood cell with a diameter of 7μm). This method is best when using EM
What is fixation
A procedure whereby a given cellular structure is preserved at the expense of other structures
Fixatives that coagulate protein preserve the cytoplasm and nucleus
Can fixatives and use chemical changes to the tissue
Yes
Alcohol and other organic solvent extract fat and cause a fat droplets to look empty
Name 2 commonly used fixatives
Formaldehyde
Glutaraldehyde
What are the two methods of tissue fixation
Immersion
Perfusion
Describe immersion fixation
Tissues are immersed in a fixative a solution for the fixing to diffuse into the tissue
For immersion how do you ensure rapid penetration of the fixative into every cell
What can happen
The tissue is rapidly chopped up with a razor blade into small fragments before immersion
Structures in the deeper parts of tissue may be less preserved and some crushing and mechanical damage to cells is unavoidable
Why are capillaries difficult to identify in immersion
Capillary space is squashed
True or false: blood cells are preserved when a tissue is fixed by immersion
True
Some blood vessels may appear full of blood cells
How does perfusion work
The fixative reaches tissues through the blood vessels as it is pumped in via the largest arterial supply
Every cell is fixed virtually instantaneously without cell stress other than induced by the fixative
The capillary blood volume is preserved as it is in vivo but most of the blood cells flushed out
When are distortions introduced
During slide preparation
What does basophilic mean
Eg?
Refers to cellular structures stained by basically dyes
Nucleus, rough ER, RNA, DNA, acid glycoproteins
What do you call structures stained by acidic dyes
Acidophilic
Eg Mitochondria, collagen, cytoplasmic proteins, Secretory granules
Which combination of two days is the most commonly used
Why is it good
H and E
(Haematoxylin and eosin)
H is a basic dye and E is an acidic dye
H dyes acidic structures blue
E dyes basic structures pink
What is good about Masson trichrome
Shows nucleus and cytoplasm very well as well as connective tissue including collagen
This helps to differentiate collagen from smooth muscle
What colours does Masson trichrome stain
Basophilic: blue
Cytoplasm, muscle, RBC, keratin: bright red
Collagen: green/ blue
What colours does Van Gieson stain
Collagen: red
Nuclei: blue
Cytoplasm and RBC: yellow
When is Alcuin blue used
To stain mucin and cartilage
Often combined with H and E or Van Gieson
When used with Van Gieson,, Alcian blue changes times what colour?
Green
What is PAS
Periodic acid-Schiff reaction
It is used to detect polysaccharides, glycoproteins, glycolipids and neutral mucins
What does the Schiff reagent comprise
Basic fuchsin , HCL and sodium metabisulphite
What is Giemsa
Commonly used for staying in blood cells
Nuclei: dark blue
Cytoplasm: pale blue
RBC: pale pink
What is osmium tetroxide
A good fixative and staying for lipids in membranous structures and vesicles
Commonly used to stain myelin with a brown-black colour
How is osmium tetroxide
In electron microscopy both as a fixative and a heavy metal stain
How thick is a plasma membrane
10nm
What is the endoplasmic reticulum
A system of intra cellular membranes which serves both two separate special areas from the cytosol and to provide a service to support organised arrays of enzymes
How does the name endoplasmic reticulum reflect its nature
Endoplasmic reflects its position in the cell and the fact that parts of it joined together to form a loose arrangement and it links to the etymology of reticulum (from the Latin meaning net)
What is the cytosol
The colloid solution of substances in which the organelles are set
What is the rER
The rough endoplasmic reticulum
The region of the ER with ribosomes attached to the cytosol surface
Membranes are arranged as large flattened sacs called cisternae
These regions are specialised for synthesis and segregation of proteins destined to be stored or secreted by the cell
What makes the rER rough
The ribosomes which are attached to the membrane
What is the sER
The regions of the ER with a smooth appearance that are not associated with ribosomes
These regions are tubular and form anastomosing network which may join onto bicycles or sister night of the rER
What does the sER provide
The space for the synthesis of lipids,lipoproteins and steroid substances; The formation of glycogen and for the segregation of some ions within the cytoplasm
Which ions in particular are segregated in the smooth endoplasmic reticulum
Calcium
Describe ribosomes
Rounded structures about 15 mm in diameter particularly abundant in cells including large amount of protein
A ribosome is an enzyme complex composed of RNA molecules and protein
Where can free ribosomes be found
In the cytosol
They are often linked by mRNA to form a polyribosome
Describe the Golgi apparatus
How does it appear
A large organelle highly developed in secretory cells
As a series of flattened, smooth membrane sacs stopped upon one another, surrounded by a number of membrane icicles and support proteins to and from the apparatus
Name some functions of the Golgi
Condensation of proteins to form secretion droplets or granules,
the recycling and alteration of cytoplasmic membrane is,
the addition of the carbohydrate to glycoproteins, glycoproteins and mucins
What are lysosomes
Spherical organelles which contain a large amount of hydrolytic enzymes involved in the intracellular digestion of macro molecules, membranes and organelles
What are lysosomes known as initially
Primary lysosomes
These are spherical organelles which appear homogenous in content and do not contain particles or membranes
What do primary lysosomes do
Fuse with material taken into the cell by phagocytosis or pinocytosis
They also give rise to secondary lysosomes (which have a diverse morphology and contain particles undergoing enzymatic digestion)
What are peroxisomes
AKA micro bodies
Small spherical organelles which contain enzymes involved oxidative reactions, possibly forming hydrogen peroxide
How is the concentration of hydrogen peroxide from peroxisomes regulated
By the enzyme catalase (present in peroxisomes in high concentrations)
Which 2 organelles Were thought to be the same for many years
Why
Lysosomes and peroxisomes
Due to the morphological similarity
Name 2 fuel reserves in cells
Glycogen granules
Lipid droplets
What does glycogen appear as
Rosettes which are larger and darker than ribosomes in an EM
Why do lipid droplets aggregate
Lipid cannot mix well with the aqueous materials in the cytoplasm
What is resolution
The minimum distance that must exist between two small structures before they can be distinguished a separate entities
What is embedding
What does it facilitate
The procedure to infiltrate the tissue with a stiff flexible material that can be cut
Thin section of the tissue with a microtome
What is often used for embedding
Paraffin
What was to happen before a tissue is embedded
Because you must be dehydrated by passing it through a series of graded alcohol is ending in 98% alcohol
Tissue is then immersed into melted paraffin wax in the oven at about 6°C. This causes the spaces within the tissues to become impregnated with paraffin
When the tissue is taken out of the oven the impregnated paraffin solidifies
What is sectioning normally done with
How thin should it be
A microtome
Thin enough to transmit light (~7μm)
Which dyes are generally used to dye each biochemical component
Alcian blue- mucopolysacchrides
Eosin - acidophilic components
Haematoxylin - basophilic components
Ponceau S - elastin
Osmium tetroxide- lipid
Give five important differences between transmission electron microscopy and LM
A beam of electrons rather than a beam of light is used
Prevent the destruction of electrons, the electron beam operates in a vacuum
As a vacuum is employed, specimen must always be dead. Specimens must be fixed in plastic. The vacuum and the electron beam can be damaging to the specimen
A high resolution and higher magnification can be achieved
What resolution and magnification can be achieved with transmission light microscopy
Resolution: 0.2nm
Magnification: x500,000
What does SEM provide
Scanning electron microscopy provides 3D high resolution images of cells and tissue
How is the diameter of SEM determined
By the diameter of the electron beam (2.5-7.5nm)
How are specimens for SEM prepared
Fixed and dehydrated but not embedded
The surface has to be coated with heavy metal before examination
How thin are specimens for EM
How is sectioning done
How is staining done
(50-80nm)
Using ultramicrotomes
By exposing the sections to have a metal salts which render the various cellular constituents electron opaque
Different constituents are revealed with Darius degrees of contrast according to that degree of impregnation with these salts
What is freeze fracture etch
Specimens are quickly frozen prior to a embedding (below -150° C) then fractured
At this temperature breakage tends to occur along the natural lines of weakness, between the phospholipid bilayer of the cell membrane
Action is done by exposing the material in a vacuum
Water sublimes and the relief of the surface is enhanced
A heavy metal replica is made to preserve the surface which is then examined by TEM
What is the depth of field of a typical electron microscope
5μm
When do membranes form sharp images in an electron micrograph
When they are running vertically through the section i.e. parallel to the electron beam
What is immunocytochemistry
A technique used to detect the presence of specific proteins or antigens in cells sampled by use of antibodies but bind to get a target with high specificity and affinity
How does immunocytochemistry work
What can it be used to do
Antibodies which point we specific protein can be generated by me reactions. Such antibodies when isolated and provide contact with radioactive isotopes etc.
To quantify the amount of protein present But must be done in cells that have had the extracellular matrix removed
What is immunohistochemistry
The same as immunocytochemistry but the original architecture of the tissue surrounding the cell is maintained
What in situ hybridisation
When shot nuclear segment of a particular sequence or synthesise chemically and tagged
They bind to coiled complementary DNA in the nucleus or mRNA in the cytosol
This allows gene expression to be determined
What is usually used to localise the probe in in situ hybridisation
Auto radiography
What is autoradiography
When the tissue is fixed, embedded and section and the sections covered in the dark with a photographic emulsion
Molecules in the section with Incorporated radioactivity will activate the silver greens in the emulsion lying in above them
The emotion can then be developed photographically to demonstrate the activated silver grains and the section stained to identify the exact position of the label
What do all epithelia rest on
A basement membrane known as the basal lamina
Describe the basal lamina
I specialise extracellular matrix that marks the boundary between the epithelial cells and the underlying supporting tissue
It anchors down the epithelium to its connective tissue underneath
How is the basal lamina connected to the underlying tissue
Proteins in the cell membrane is attached to proteins in the basement membrane which in turn is linked to the extracellular matrix of connective tissue
Is there blood supply between epithelial cells
No because blood vessels do not cross the basement membrane
Instead epithelial cells rely on diffusion of oxygen and metabolite from capillaries in the underlying supporting tissue
Name one important component of the basement membrane
Type IV collagen
What does it mean to say epithelial cells show polarity
They have two different surfaces: apical and basolateral, each with specific functions and membrane constituents
What is a main common function in of epithelia
To serve as a selective permeable membrane separating compartments that have different chemical compositions and regulating the substances that move between them
Why do most epithelium have stem cells
To replenish the tissue on a continuous basis
Where is simple squamous epithelium found
Lining surfaces at which diffusion occurs
Describe cuboidal epithelium
Round with a centrally located nucleus, often more polygonal than cuboidal
Where is simple cuboidal epithelium found
Lining tubules and ducts that may be involved in secretary, excretory and absorptive processes
Describe simple columnar epithelium
Where is it found
Tall columnar cells with a size that varies depending on location and elongated nuclei that often lies at the base of the cell (may also be at centre or apex)
Lining the large duct of exocrine glands and maybe ciliated at the luminal surface of certain locations e.g. in the Fallopian tube
Describe pseudostratified epithelium
In some simple epithelia, cells can be much taller than others and their nuclei are disposed at different levels, hence there appear to be two or more distinct layers
True or false
Most of the cells of pseudostratified epithelium are ciliated
True
They line the larger airways of the respiratory system
What are cilia
Motile cell projections which develop from basal bodies in the apical portion of the cytoplasm
What is the structure of cilia
A central core of microtubules called the axoneme Which shows a characteristic arrangement of a central pair of microtubules surrounded by nine concentrically arranged microtubular pairs
What do micro villi contain
A core of cross-linked microfilaments
Compare the sizes of microvilli and cilia
Microvilli are 10x smaller and cannot be resolved in LM
The cells of the basal layer of stratified epithelium are generally which shape
How are stratified epithelium classified
Cuboidal
On the basis of the shape of the cells in the surface layers
Describe the stratified squamous epithelium
Have a flat surface layer
The Bazel cells are undifferentiated and divide continuously. The new cells push upwards to replace the dead cells at the top layer which ultimately shed
The cells covering the basal layer of differentiating cells which become highly differentiated as they reach the top layer
As the cells migrate there is a progressive change in cell shape (from cuboidal to squamous)
Why have we adapted stratified squamous epithelium
How
Where is it found
To withstand abrasion so it’s main function is protection
The loss of the surface cells does not compromise the underlying tissue
Found lining the skin, oesophagus, vagina etc
What is the epithelium of the skin covered by
A layer of fibrous protein known as keratin
It is therefore known as stratified squamous keratinised epithelium
What is urothelium?
Transitional epithelium
Specialised epithelium to allow large degrees of stretch and to withstand the toxicity of urine
Why is it known are transitional epithelium
Its appearance changes dramatically between stretched and unstretched states
Unstretched it appears to be four or five cell layers thick but stretched it appears as only two or three cell layers thick and the intermediate and surface layers are flattened
What shape are the base and intermediate cells of transitional epithelium
What about the surface cells?
Cuboidal or polygonal but this shape and size changes with degrees of distension
They are umbrella cells which are large and domed shaped, often overlapping two or more of the underlying cells. They may contain multiple nuclei
What do the surface cells of transitional epithelium contain
Plaques of urine resistant proteins
These are added to the surface to allow the increase in area when the cells change from an unstretched to a flattened stretched shape
How are epithelial cells attached to each other
Laterally by specialised intercellular junctions.
What are the main types of junction present in mammalian epithelia
What is the combination of these known as
Tight junction
Intermediate junction
Spot desmosome
A junctional complex
How are tight junctions and intermediate junctions usually present
What about spot desmosomes?
one under the other as belt structures surrounding the cell
Spot desmosomes occupy discrete limited areas
What are the two other names of a tight junction
Occluding junction
Zonula occludens
What are the two other names of an intermediate junction
Zonula adherens
Belt desmosome
What are the two other names of a spot desmosome
Desmosome
Macula adherens
What do intercellular junctions allow
Intercellular mechanical attachment,
prevention of passage of macromolecules through the intercellular space
allow metabolic cell coupling
What is a tight junction
A belt like zone that runs around the intercellular region immediately below cell’s luminar surface
Holds cells together,
functions as a permeability barrier between the two compartments separated by the epithelium,
and prevents the diffusion of membrane proteins along the plasma membrane
What is an intermediate junction
Located below the T-junction at forms an anchoring belt around the cells
Describe a desmosome
A circular spot of strong mechanical attachment
It is located deeper, below the intermediate junction
There can be a variable number of desmosomes binding 2 adjacent cells in epithelium
What is a hemidesmosome?
A variant of desmosome that provides point attachment to the basement membrane
Which stain allows junctional complexes to be seen
A protein stain eg iron Haematoxylin
They appear as short dark lines between cells
What are gap junctions
What is their primary role
Minute intercellular channels that allow the passage of small molecules and ions directly between cells
To coordinate the activities of adjacent cells
What is the terminal web
What is the function
A specialisation of the cytoskeleton found in certain epithelial cells with microvilli such as intestinal epithelium cells
To allow limited movement of the cell apex and microvilli
Describe a terminal web
At the Junctional complex, fibrous elements of the cytoskeleton are attached at the same membrane. These fibres, mostly actin microfilaments, run across the apex of the epithelial cells, up into any microvilli and attach to the intermediate junction
