Microbiology 2: (Patel) Microscopes

Question 1

Types of light microscopy and uses?

Answer 1

Brightfield
- Dark specimen, light surroundings -> used to analyse stained or naturally pigmented specimens

Phase Contrast
- intensifies contrasts of transparent and colourless objects by influencing the optical path of light (i.e makes invisible -> visible) -> useful to study LIVE cells

Differential Interference Contrast (DIC)
- also enhances contrast of transparent samples

Question 2

What is meant by resolution?

Answer 2

The ability to see 2 very small and closely spaced together objects as separate entities

Question 3

Purpose of putting oil on lens / cover slip?

Answer 3

Increases the amount of light collected by the objective lens -> increases resolution

Question 4

Types of NON-light microscopy? Uses?

Answer 4

Fluorescence microscopy
- Analyse fluorescently tagged specimens

Confocal microscopy

• Used for imaging complex 3D cells and tissues
• Overcomes problem of ‘out-of-focus’ light

Electron microscopy (Scanning EM, Transmission EM)
- Used for studying ultrastructure of cells

Question 5

Types of fluorescence microscopy?

Answer 5

Immunofluorescence Microscopy
- Direct immunofluorescence
- Indirect immunofluorescence
(Depend on availability of antibody specific to molecule of interest)

GFP-tagging of proteins in live cells

Question 6

Monoclonal and Polyclonal antibody differences?

Answer 6

Polyclonal (Recognise multiple sequences of aa’s)

• inexpensive
• skills required for production low,
• quick to produce,
• non-specific binding often occurs
• made in host such as rabbit by injecting antigen

Monoclonal (Recognise one specific aa sequence)

• More expensive to produce,
• Training required to use tech
• hybridoma tech (B lymphocyte fusion with tumour cell, myeloma) -> produces many identical Ab’s
• Once hybridoma is made, can be re-used

Question 7

Describe method of Direct Immunofluorescence microscopy - what is it best used for?

Answer 7

Cells are fixed (preserved) using glutaraldehyde or formaldehyde.
- these react with free -NH2 groups of proteins, fixing adjacent proteins

Cells are permeabilised through addition of detergent

Cells incubated with 1% w/v Bovine Serum Albumin
- albumin binds to other proteins to block non-specific binding sites

Add FITC-conjugated antibody -> Ab only binds to target antigen

Cell mounted on coverslip in mounting medium -> analysed with fluorescent microscope

USED FOR HIGHLY ABUNDANT PROTEINS

Question 8

Describe method of Indirect Immunofluorescence microscopy - what is it best used for?

Answer 8

Same as direct to begin with ->

• Preservation with Formaldehyde
• Permeabilisation with Detergent
• Blocking with BSA

Add unlabelled primary antibody which binds only to target protein

Add FITC-conjugated secondary antibody. Multiple 2nd Abs bind to a single primary, resulting in signal amplification

Analyse with fluorescence microscope

Question 9

How is the secondary antibody made?

Answer 9

Primary antibody injected into animal model (DIFFERENT FROM PRIMARY ANTIBODY)
-> response antibodies then collected, labelled and used as secondary antibodies

Question 10

How is GFP used to determine the location of a protein within a cell?

Answer 10

cDNA of GFP and target protein (e.g. alpha-tubulin) are ligated together IN FRAME using compatible restriction enzymes

• Recombinant DNA is then re-introduced back into mammalian cell via transfection
• DNA is the transcribed and translated to form GFP-alpha-tubulin fusion protein