Illumination Techniques (L5) Flashcards
Illumination techniques are AKA?
Contrast-enhancing techniques.
Types of illumination techniques? (7) BP²FLD²
• Bright field.
• Dark field.
• Phase contrast.
• Polarized light.
• DIC.
• Fluorescence microscopy.
• LSCM.
Bright field?
= makes use of intensity (amplitude) differences between light rays passing through different parts of the specimen.
Bright field attributes? (2)
• Works well on high contrast specimens (eg, well-stained specimens).
• Works poorly on low contrast specimens (egs, unstained sections & live specimens).
Mention 3 out of many illumination techniques that have been used to overcome the problems of the Bright field?
• Dark field.
• Phase contrast.
• Polarized light.
Phase contrast?
= makes use of phase differences between light rays passing through the specimen & light rays scattered (diffracted) by the specimen.
Phase contrast attributes? (4)
• 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.
Halo effect?
= the appearance of a bright edge for positive phase contrast or a dark edge for a negative phase contrast around large phase objects.
Polarized light?
= 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”).
Polarized light attributes? (2)
• Crystals found on living organisms are often birefringent.
• Some biological structures are themselves birefringent.
Birefringent?
= optimal property of a material that has a refractive index that depends on the polarization & propagation direction of light.
Fluorescent microscopy?
= where the specimen is illuminated with light of one wavelength (usually UV, blue or green) [the exciting illumination].
Fluorescence microscopy attributes? (5)
• 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.
LSCM stands for?
Laser Scanning Confocal Microscopy.
LSCM is AKA?
Confocal microscopy.
LSCM attributes? (3)
• 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.
Dark field?
= where illumination is arranged so as to miss the objective lens.
Dark field attributes? (3)
• 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.
Pro of Dark field?
Very high contrast can be achieved with suitable specimens.
DIC?
= technique that makes use of phase differences & interference between light rays from adjacent parts of the specimen.
DIC stands for?
Differential Interference Contrast.
DIC is AKA?
Nomarski Interference Contrast.
DIC attributes? (3)
• Needs special equipment.
• Gives shadowed “3-D” effect.
• Does not have the halo problem of phase contrast.
