Illumination Techniques (L5)

Question 1

Illumination techniques are AKA?

Answer 1

Contrast-enhancing techniques.

Question 2

Types of illumination techniques? (7) BP²FLD²

Answer 2

• Bright field.
• Dark field.
• Phase contrast.
• Polarized light.
• DIC.
• Fluorescence microscopy.
• LSCM.

Question 3

Bright field?

Answer 3

= makes use of intensity (amplitude) differences between light rays passing through different parts of the specimen.

Question 4

Bright field attributes? (2)

Answer 4

• Works well on high contrast specimens (eg, well-stained specimens).

• Works poorly on low contrast specimens (egs, unstained sections & live specimens).

Question 5

Mention 3 out of many illumination techniques that have been used to overcome the problems of the Bright field?

Answer 5

• Dark field.
• Phase contrast.
• Polarized light.

Question 6

Phase contrast?

Answer 6

= makes use of phase differences between light rays passing through the specimen & light rays scattered (diffracted) by the specimen.

Question 7

Phase contrast attributes? (4)

Answer 7

• Provides significant contrast increases for some specimens.

• Useful on living, unstained specimens & some stained specimens.

• Requires special equipment.

• Suffers from halo effects at the edges of specimen structures.

Question 8

Halo effect?

Answer 8

= the appearance of a bright edge for positive phase contrast or a dark edge for a negative phase contrast around large phase objects.

Question 9

Polarized light?

Answer 9

= where polarized light is positioned below the specimen & an analyzer above the objective is arranged so that light does not get through unless the specimen is birefringent and rotates the plane of polarization (polarizer/analyzer is “crossed”).

Question 10

Polarized light attributes? (2)

Answer 10

• Crystals found on living organisms are often birefringent.

• Some biological structures are themselves birefringent.

Question 11

Birefringent?

Answer 11

= optimal property of a material that has a refractive index that depends on the polarization & propagation direction of light.

Question 12

Fluorescent microscopy?

Answer 12

= where the specimen is illuminated with light of one wavelength (usually UV, blue or green) [the exciting illumination].

Question 13

Fluorescence microscopy attributes? (5)

Answer 13

• Specimen fluoresces at a longer wavelength.

• A few specimens are naturally fluorescent, but most have to be stained with fluorescent dye (fluorochrome).

• Dye can be attached to an antibody allowing location of a specific antigen in the specimen.

• Specimen is observed by the fluorescent light.

• Today, epi-fluorescence is the illuminating technique of choice.

Question 14

LSCM stands for?

Answer 14

Laser Scanning Confocal Microscopy.

Question 15

LSCM is AKA?

Answer 15

Confocal microscopy.

Question 16

LSCM attributes? (3)

Answer 16

• Used mostly for fluorescence microscopy of fluorochrome stained specimens.

• Provides especially clear images even of quite thick specimens.

• Allows 3D reconstructions of specimens in the two-dimensional.

Question 17

Dark field?

Answer 17

= where illumination is arranged so as to miss the objective lens.

Question 18

Dark field attributes? (3)

Answer 18

• Specimen is viewed only by scattered light & appears light on a dark background.

• Directs light away from your specimen.

• Very high contrast can be achieved with suitable specimens.

Question 19

Pro of Dark field?

Answer 19

Very high contrast can be achieved with suitable specimens.

Question 20

DIC?

Answer 20

= technique that makes use of phase differences & interference between light rays from adjacent parts of the specimen.

Question 21

DIC stands for?

Answer 21

Differential Interference Contrast.

Question 22

DIC is AKA?

Answer 22

Nomarski Interference Contrast.

Question 23

DIC attributes? (3)

Answer 23

• Needs special equipment.

• Gives shadowed “3-D” effect.

• Does not have the halo problem of phase contrast.