Darkfield and Phase Contrast Microscopy

Talk Overview

This lecture describe the principles of dark field and phase contrast microscopy, two ways of generating contrast in a specimen which may be hard to see by bright field. The lecture describes how the phase rings work to generate interference between the diffracted and undiffracted light.

Questions

When Zernicke blocked the illuminating light at the back aperture of the objective, he saw
1. Both the field and the particles (specimen) were bright
2. The field was dark and the particles were bright.
3. The field was bright and the particles were dark.
4. Both the field and the particles (specimen) were dark
True or false: In transmitted light microscopy, an image is formed by undiffracted light interfering with diffracted light from the specimen.
Which of the following might be a good specimen to examine by dark field microscopy?
1. A mammalian tissue culture cell
2. A thin histological section of liver
3. 25 nm microtubules polymerized in vitro
4. Pollen grains
Which of the following statements is false about dark field microscopy?
1. Poor depth of field
2. Is generally a higher resolution technique than phase microscopy
3. Requires a very bright light source
4. Can use both dry and oil condensers and objectives
In phase microscopy, the function of the phase ring in the objective lens is to:
1. Attenuate and produce ¼ l phase retardation of the illuminating light
2. Increase the relative brightness and produce a ¼ l phase retardation of the illuminating light
3. Attenuate and produce a ¼ l phase advance of the illuminating light
4. Attenuate and produce a l phase advance of the illuminating light
True or false. One phase condenser annulus will suffice for 20x, 40x, and 60x objective lenses on most microscopes.
What produces halos in a phase image?
In dark field microscopy, why must the N.A. of the objective be less than the N.A. of the condenser?
If you are just performing fluorescence microscopy, why is a phase objective somewhat less desirable than the comparable non-phase objective (same mag, NA, etc.)?
Which of the following statement is false about phase contrast microscopy?

The phase ring retards or advances the phase of the illuminating light relative to the scattered light by half of a wavelength.
The phase ring reduces the intensity of the illuminating light
Phase rings are built into the objective lens
Phase microscopy requires a matching annulus in the condenser turret.

Answers

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B
True
C: Dark field, which collects diffracted light and focuses it onto a dark background, produces very nice high contrast images of microtubules in vitro, which are small but simple objects. The other objects are more complex and thicker and generate a tremendous amount of scattered light which produces a bright but information poor image of cells or tissues.
B: Dark field has limited resolution since the objective NA must be lower than the NA of the condenser. In practice, this means using objectives with NA below 1. A phase objective does not have such restrictions and one can use higher NA lens.
C: In phase microscopy, you want the light traveling through the transparent sample to interfere with the illuminating light to generate contrast. A thin sample typically produces a ¼ l phase retardation of the illuminating light. If illuminating light is phase advanced by ¼ l, then the specimen and illuminating light will be ½ l out-of-phase, resulting in destructive interference and a darker image than the background. The illuminating light also is attenuated to make it closer in intensity to the weaker diffracted light from the specimen and thus generate better contrast.
False: You need a specific annulus on the condenser that will match the phase ring on each objective. Then, the illuminating light will be confined to and be modified by the phase ring. Furthermore, the condenser annulus must be adjusted (with set screws) so that it is aligned with the objective phase ring in the light path.
Diffracted light from the specimen hitting the annulus of the phase ring.
So that the cone angle of the illuminating light (that angle being determined by the N.A. of condenser) is not captured by the objective. Then the field will appear dark because the illuminating light is not being imaged. Some of the light diffracted by the specimen, however, will be collected by the objective.
Some of the fluorescence light from the specimen is attenuated by the objective phase ring, resulting in a loss of valuable photons if the image is dim. The phase ring also slightly spreads out the Airy disk.
A

Speaker Bio

Ted Salmon

Ted Salmon is a Distinguished Professor in the Biology Department at the University of North Carolina. His lab has pioneered techniques in video and digital imaging to study the assembly of spindle microtubules and the segregation of chromosomes during mitosis. Continue Reading

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Darkfield and Phase Contrast Microscopy

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Ted Salmon

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