Microscopy Principles

Question 0

What determines the brightness and colour of the light?

Answer 0

Brightness: amplitude
Colour: wavelength

Question 1

What are the 2 ways to make visible a colourless section on a slide?

Answer 1

• Staining: using chemical dyes or heavy metals

- Manipulating the optical pathway: phase contrast, darkfield, differential interference contrast (DIC)

Question 2

What is the role of the “condenser” on the ms?

Answer 2

Focuses light from the light source onto specimen, and directs light to the objective lens

Question 3

What are the “edge effects” that produce detail and contrast in ms?

Answer 3

Diffraction, refraction, reflection

Question 4

Briefly explain the principle of Kohler illumination.

Answer 4

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.

Question 5

What are the types of microscopic methods that fall under the Contrast category?

Answer 5

• Bright field
• Phase contrast
• Differential Interference Contrast
• Dark field

Question 6

Briefly explain dark field ms

Answer 6

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

Question 7

What is meant by EPI-illumination?

Answer 7

When the illumination and detection is on the one side of the sample.

Question 8

Outline the uses of microscopy.

Answer 8

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

Question 9

Outline the types of outcomes achievable by immunohistochemistry and the system of detection specific to each outcome.

Answer 9

• Structural and functional info: histological staining
• Antigen detection: Fluorescent/enzyme-labelled Ab
• Detecting tissue enzyme activity: Enzyme histochemistry
• Detecting nucleic acid transcripts: fluorescent probes

Question 10

What are the 3 steps in tissue preparation?

Answer 10

Fixation
Processing of tissue
Sectioning

Question 11

Outline the types of chemical and physical fixation methods

Answer 11

Chemical: using aldehydes (x-link methylene bridges between amino and amide), oxidising agents, protein denaturation (ETOH).

Physical (freezing): Liq Nitrogen, isopentane, propane

Question 12

Outline the steps involved in tissue processing.

Answer 12

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.

Question 13

What are the mechanisms that dyes “stain” the samples

Answer 13

• acid-base interactions
• permeability and displacement
• Deposition/impregnation/precipitation
• Reaction between tissue and colourless dye to form a colour compound

Question 14

Which molecule form backbone of dyes?

Answer 14

Benzene

Question 15

Which dye is based on the acid-base principle, and explain the dye.

Answer 15

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)

Question 16

What are the 3 disadvantages of using H&E dyes, and how do we get around this?

Answer 16

• 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)

Question 17

Explain the principles of the Trichrome staining method

Answer 17

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,

Question 18

Explain the principles of using metal staining

Answer 18

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.

Question 19

Explain the principles of the PAS stain

Answer 19

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

Question 20

Explain the principle of enzyme histochemistry.

Answer 20

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.

Question 21

Explain the use of the Orange G stain

Answer 21

Used for background staining: bright orange if acidic or neutral and red if pH > 9

Question 22

Outline the use of the PAAB dye

Answer 22

Performic acid alcian blue.

water-soluble, stains sulfated and carboxylated mucopolysaccharides and glycoproteins