Class 1 : microscopy & histological techniques Flashcards
Define human histology & state what is a tissue.
Histology is the scientific study of microscopic structures of tissues & organs in the human body.
A tissue is a group of cells that have similar structure and same developmental origin that together carry out a specific function.
Organs are then formed by the functional grouping of multiple tissues.
Name two factors which can influence individual cells.
- Disease process
- Age (many tissues change considerably with age - so that something that is considered normal in an adult might not be normal for a child and vice versa.
Cells have distinctive shapes and functions depending on how they have been differentiated, the proteins they express and their interactions with other cell types. Approx. how many cell types make up the body ?
200
List the type of microscopy available to study the microanatomy of cells & tissues.
- Light microscopy (limited resolution of 0.2 um)
- Electron microscopy
Explain the difference in resolving power of LM and EM and identify which organelles can be visualized w/ each.
Power of resolution is the capacity to resolve between 2 close points. Resolution depends on the wave length of the source of illumination. Electrons have a very short wave length (higher resolution).
Naked eye can see 0.2 mm
LM : 0.2 um
EM : SEM 2.5 nm and TEM 0.5 nm
This means that w/ LM, you can only see cells and their basic structures such as nuclei as well as bacteria but you cant see much more. With EM, you can see ultrastructures & viruses.
List different types of light microscopes.
- Conventional Light microscope
- Phase-Contrast microscope
- Fluorescent microscope (uses UV light to capture fluorescence to generate an image)
- Confocal microscope (uses laser light) - more in dept than fluorescent
- Polarizing microscope (uses polarized light to analyze refraction patterns of specimens.
What is the application of the conventional light microscope
Examines chemically fixed and stained tissue specimens
Discrimination level 0.2 um (means that objects smaller than this or apart from each other by less than 0.2 um cannot be discriminated.
Subcellular details cannot be seen (ribosome, membranes, filaments…)
What is the application of phase contrast microscope ?
Allows examination of living cells.
List the different types of electron microscopes.
Transmission electron microscope (TEM)
Scanning electron microscope (SEM)
How is an electron microscope different from a light microscope? Name and describe two widely used types.
EM use high velocity electrons (short wave) as a source of ‘’illumination’’.
- Transmission Electron Microscope (TEM) : you are scanning the surface of the section by firing right at it. This is useful to see organelles. Highest resolution.
- Scanning Electron Microscope (SEM) : backscattered electrons and secondary electrons emitted form the surface of the specimen are collected to construct a surface image - allowing you to see the surface in 3D. Great to see overall structure.
Outline types of histological sections for the light microscope.
There are 3 techniques for preparation:
- The paraffin technique (most commonly used)
- Frozen sections
- Semithin sections (for really hard tissues such as bone and teeth)
Once the sections are prepared, they are stained. This is needed because otherwise, you cant see nothing much, the optical density of the different tissue components being too similar.
What is the paraffin technique ?
Tissues are fixed and embedded in wax.
This makes the tissue hard and much easier to cut section from.
The section are then stained.
State the basic steps (6) of tissue preparation for light microscopy using the paraffin technique.
- Fixation (Submerge the tissue in a 4% formaldehyde solution for 6-12h. This cross-links proteins and stabilizes the structures.)
- Dehydration (Pass through an ascending series of alcohol (up to 100%) in order to remove water from the fixed tissue sample. During this process lipids are also extracted).
- Clearing ( Remove alcohol w/ xylol/xylene - because it wont set well in paraffin otherwise)
- Embedding/infiltration (place the prepared tissue in paraffin wax - lasting forever at RT)
- Sectioning_(_cut with microtome obtaining 4-10 u paraffin sections
- Staining
What does fixation allows you to do ?
Preservation of cells and other elements in a life-like state.
State the difference between acidophilic and basophilic staining.
H & E is the most common staining system.
Hematoxylin is a basic dye – basic dye stains basophilic structures (which are acidic)
It stains the nucleus (DNA) and acidic components of the cytoplasm (RNA) purple or blue.
Eosin is an acid dye - acid dye stains acidophilic structures (which are basic). This is also called eosinophilic. It stains proteins red or pink, such as the cytoplasm, which contains a lot of them.
What is a trichrome stain?
This histological technique uses 3 dyes which differentiates tissues by tinting them in contrasting colour.
It increases the contrast of microscopic features in cells and tissues, which makes them easier to see when viewed through a microscope.
Collagen is coloured blue / Nucleus is coloured red
Often use to see if there is an increase in collagen tissue in the liver, which would indicate liver cirrhosis.
Know how to recognize this.
Reticular fibres are black and are not wavy as opposed to elastic fibres.
Verhoeff van Gieson stain :
It colours the elastic tissue black and the collagen red.
What is the PAS stain?
Periodic Acid Schiff : It is specific for carbohydrate - so it stains cells containing carbohydrates as well as basement membrane. Structures with a high content of carbohydrates stain with an intense red coloration.
Which kind of fibers are shown here and what is the stain used ?
Elastic fibers.
Orcein stain.
Which stain help identify thick blood smears and chromosomal aberrations ?
Giemsa
MYELIN is another staining technique for special tissues. It stains myelin sheat in myelinated nerve fibres. See below.
It looks like soap bubbles. What you are looking at is nerve fibre bundles.
What staining method is used here ?
H&E.
Typical H&E stain.
What is this stain ?
PAS
