Microscopy Principles Flashcards
What determines the brightness and colour of the light?
Brightness: amplitude
Colour: wavelength
What are the 2 ways to make visible a colourless section on a slide?
- Staining: using chemical dyes or heavy metals
- Manipulating the optical pathway: phase contrast, darkfield, differential interference contrast (DIC)
What is the role of the “condenser” on the ms?
Focuses light from the light source onto specimen, and directs light to the objective lens
What are the “edge effects” that produce detail and contrast in ms?
Diffraction, refraction, reflection
Briefly explain the principle of Kohler illumination.
It is a method using transmitted and reflected light that creates an evenly illuminated field of view while illuminating the specimen with a very wide cone of light. It filters out the image of the light source (eg halogen lamp filament), and basically ensures that the paths of light is parallel and start off with the same excitation properties.
What are the types of microscopic methods that fall under the Contrast category?
- Bright field
- Phase contrast
- Differential Interference Contrast
- Dark field
Briefly explain dark field ms
The light rays passing through the specimen from the condenser is directed away from the objective lens, therefore if there is no specimen, there won’t be diffracted/refracted light that direct light into the objective. The background will therefore appear black, and the specimen appear as white
What is meant by EPI-illumination?
When the illumination and detection is on the one side of the sample.
Outline the uses of microscopy.
Provide:
- structural info: cellular and macromolecular
- compositional info: histochemistry, elemental analysis
- Functional info: localise gene expression, tract dynamic changes, visualise physiological processes, localise molecular interactions
Outline the types of outcomes achievable by immunohistochemistry and the system of detection specific to each outcome.
- Structural and functional info: histological staining
- Antigen detection: Fluorescent/enzyme-labelled Ab
- Detecting tissue enzyme activity: Enzyme histochemistry
- Detecting nucleic acid transcripts: fluorescent probes
What are the 3 steps in tissue preparation?
Fixation
Processing of tissue
Sectioning
Outline the types of chemical and physical fixation methods
Chemical: using aldehydes (x-link methylene bridges between amino and amide), oxidising agents, protein denaturation (ETOH).
Physical (freezing): Liq Nitrogen, isopentane, propane
Outline the steps involved in tissue processing.
The tissue in fixative and buffers is mixed with ETOH and Xylol (for dehydration, so that it becomes non-polar), and then embedded in mould of wax/resin.
What are the mechanisms that dyes “stain” the samples
- acid-base interactions
- permeability and displacement
- Deposition/impregnation/precipitation
- Reaction between tissue and colourless dye to form a colour compound
Which molecule form backbone of dyes?
Benzene
Which dye is based on the acid-base principle, and explain the dye.
Haematoxylin and eosin dye.
Haematoxylin is blue and is basic, therefore binds acidic structures like the negatively charged DNA (in nucleus) and RNA. (Neg-charged therefore gave off H+ ions, ie proton donors = acids). (Basic Binds Basophilic Becoming Blue)
Eosin is pink and acidic, therefore binds basic structures such as proteins in the cytoplasm.
(Acidic Attaches Acidophilic Applying Auburn)
What are the 3 disadvantages of using H&E dyes, and how do we get around this?
- Cannot distinguish tissue components of similar charges: Use Trichrome dyes
- Cytoplasm and extracellular fibres show similar staining therefore EC fibres not visible: use metal salts/deposition
- Mucopolysaccharides (and lipids) are unstained: PAS stain (histochemical reaction)
Explain the principles of the Trichrome staining method
All dyes are acidic, ie stains basic components
Based on principle of permeability and displacement:
Diffusible dyes go into compact structures, and colloid dyes go into permeable structures.
Larger molecules displace smaller molecules from permeable structures.
Red = densely packed, pink/yellow = moderately loose packing, blue = loose packing,
Explain the principles of using metal staining
Steps: first pre-treat with oxidising agent to increase affinity of metal binding, then apply metal solution (which is reduced to pure metal at site), and lastly enhance the metal deposit to make it visible.
Explain the principles of the PAS stain
A chemical reaction is induced at specific sites in the tissue (oxidation of -OH group to the aldehyde group) which subsequently interact with colourless solution (Schiff agent) containing benzene-derivative molecules to form a red-purple product.
Stains for: glycogen, mucin, fungus, basement membrane, corporal amylacea
Explain the principle of enzyme histochemistry.
This is used to measure tissue enzyme activity. Active enzymes is tissue react to form a primary reaction product that is invisible. This interacts with a dye salt to form insoluble colour precipitate.
Explain the use of the Orange G stain
Used for background staining: bright orange if acidic or neutral and red if pH > 9
Outline the use of the PAAB dye
Performic acid alcian blue.
water-soluble, stains sulfated and carboxylated mucopolysaccharides and glycoproteins
