Module 3: Specialized Microscopes

Question 1

Fluorescence Microscopy

Answer 1

Use: ID of specific organisms, cells or tissue components (Eg. Bacteria, Lymphocyte subsets, tumor markers)

Fluorescent dye labels that fluoresce when excited by a short wavelength (UV) and emit light of a longer wavelength (Visible)
Typically monoclonal antibody conjugated with a fluorescent label

Exciter (woods) filter removes visible light; only short UV light reaches specimen
Barrier filter prevents UV light from reaching eyepiece; only longer emitted wavelengths to pass

Objects appear brightly coloured on a dark background

Question 2

2 types of fluorescence microscopy

Answer 2

Transmission: light comes from below the specimen

Epi (incident): light comes from side; no condenser; NO KOHLER SET; dichromatic filter

Question 3

Darkfield

Answer 3

Use: examination of unstained, living cells (no fixing)
Special darkfield condenser produces hollow cone of light that is VERY WIDE
Specimen causes light rays to refract enough that they will enter the objective lens
Bright objects on a dark field; typically only see outline

Question 4

Phase contrast

Answer 4

Use: Examination of unstained living cells
Can produce greater internal detail than darkfield (Differences in thickness of internal structures alter light rays as they pass through the sample)
Results in different light waves being out of phase
Phase difference is further enhanced by passing the light waves through a phase plate in the objective (allows for better contrast and detail)

Condenser with ANNULUS (creates hollow cone)
Phase plate in objects (pos and neg)

Question 5

Phase contrast - Phase plates in objectives

Answer 5

Resulting difference between unaltered and diffracted rays is about 1/2 wavelength
Pos: glass disk with thinner grove (most common)
dark object on light grey background

Neg: Glass disk with raised ring
bright object on dark background

Question 6

Polarized light microscopy

Answer 6

Use: ID of crystals and other birefringent substances (able to polarize light themselves)

As polarized light passes through birefringent substance, it re-orientates (polarizes) the light in another plane
Second polarizing filter is 90Deg and only allow “repolarized” light to pass

Components:
Polarizes (first filter)
Analyzer (second filter)

Birefringent substances will display characteristic color patterns depending on the orientation of the 2 polarizing filters

Question 7

Inverted Microscope

Answer 7

Use: examine tissue cell cultures, agglutination reactions
Light source illumination specimen from above
Objectives located BELOW the stage

Specimens are located on the bottom on the container and are viewed from below