• Skip to primary navigation
  • Skip to main content
  • Skip to primary sidebar
  • Skip to footer

iBiology

Bringing the World's Best Biology to You

  • Start Here
    • Explore iBiology
    • All Playlists
    • All Speakers
    • All Talks
    • What’s new at iBiology?
  • Research Talks
    • Talks by Topic
      • Biochemistry
      • Bioengineering
      • Biophysics
      • Cell Biology
      • Development and Stem Cells
      • Ecology
      • Evolution
      • Genetics and Gene Regulation
      • Human Disease
      • Immunology
      • Microbiology
      • Neuroscience
      • Plant Biology
      • Techniques
      • Archive
    • Talks by Series
      • Bench to Bedside
      • Famous Discoveries
      • Great Unanswered Questions
      • Young Scientist Series
  • Stories
    • Background to Breakthrough
    • Interviews and Profiles
    • Science and Society
  • Courses
  • Careers
    • Professional Development Talks
    • Professional Development Courses
    • Career Exploration
    • NRMN Resources
    • Biomedical Workforce
  • Educators
  • About
    • Mission
    • iBiology Team
    • Board of Directors
    • Make a Donation
    • iBiology Funders and Partnerships
    • Contact Us
Home » Courses » Microscopy Series » Contrast Generation for Transmitted Light

Darkfield and Phase Contrast Microscopy

  • Duration: 34:14
  • Downloads
    • Hi-Res
    • Low-Res
This Talk
Speaker: Ted Salmon
Audience:
  • Researcher
Recorded: July 2012
More Talks in Microscopy Series
  • Shinya Inoue Polarized Light and its Interaction with Material
    Polarized Light and its Interaction with Material
  • Polarization (Edward Salmon)
    Polarization Microscopy
  • Differential Interference Contrast (DIC) Microscopy Edward Salmon
    Differential Interference Contrast (DIC) Microscopy
All Talks in Microscopy Series
Share

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

  1. 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
  2. True or false: In transmitted light microscopy, an image is formed by undiffracted light interfering with diffracted light from the specimen.
  3. 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
  4. 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
  5. 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
  6. True or false. One phase condenser annulus will suffice for 20x, 40x, and 60x objective lenses on most microscopes.
  7. What produces halos in a phase image?
  8. In dark field microscopy, why must the N.A. of the objective be less than the N.A. of the condenser?
  9. 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.)?
  10. Which of the following statement is false about phase contrast microscopy?
  1. The phase ring retards or advances the phase of the illuminating light relative to the scattered light by half of a wavelength.
  2. The phase ring reduces the intensity of the illuminating light
  3. Phase rings are built into the objective lens
  4. Phase microscopy requires a matching annulus in the condenser turret.

Answers

View Answers
  1. B
  2. True
  3. 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.
  4. 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.
  5. 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.
  6. 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.
  7. Diffracted light from the specimen hitting the annulus of the phase ring.
  8. 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.
  9. 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.
  10. A

Speaker Bio

Ted Salmon

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

Playlist: Microscopy Series

  • Correcting for Spherical Aberration with a Correction Collar Stephen Ross
    Correcting for Spherical Aberration with a Correction Collar
  • Shinya Inoue Polarized Light and its Interaction with Material
    Polarized Light and its Interaction with Material
  • Polarization (Edward Salmon)
    Polarization Microscopy
  • Differential Interference Contrast (DIC) Microscopy Edward Salmon
    Differential Interference Contrast (DIC) Microscopy

Reader Interactions

Leave a Reply Cancel reply

Your email address will not be published. Required fields are marked *

Primary Sidebar

iBiology is Hiring:

XBio Director Position

Like our Website?

You'll love our newsletter...

  • Sign up for:
    • Exclusive iBiology content
    • 10 Must See Biology Videos
    • Talks by 20+ Nobel Winners
    • Our young Scientist Survival Toolkit
    • Talks by trailblazing Women in Biology
    • Updates on exciting iBiology projects in development
    Tyler Allen
  • This field is for validation purposes and should be left unchanged.

Privacy Policy

Help us keep bringing the world’s best biology to you!

Footer

Primary Funding

Partners

Start Here

  • Talks for Everyone
  • Talks for Students
  • Talks for Research
  • Talks for Educators

Explore

  • Explore
  • All Playlists
  • All Speakers
  • All Talks

Talks By Topic

  • Biochemistry
  • Bioengineering
  • Biophysics
  • Cell Biology
  • Development and Stem Cells
  • Ecology
  • Genetics and Gene Regulation
  • Human Disease
  • Immunology
  • Microbiology
  • Neuroscience
  • Plant Biology
  • Techniques

Talks by Series

  • Bench to Bedside
  • Famous Discoveries
  • Great Questions
  • Young Scientist Series

Career

  • Professional Development
  • Career Exploration
  • NRMN Resources
  • Biomedical Workforce

Courses

  • Microscopy Series
  • Short Microscopy Series
  • Open edX Courses
  • Cell Biology Flipped Course
  • Engineering Life Flipped Course
  • Evolution Flipped Course

Educators

  • Educator Registration
  • Educator Resources
  • Log In

About Us

  • About Us
  • iBiology Team
  • Wonder Collaborative
  • Contact Us
  • Make a Donation
  • Mission
  • Privacy Policy

This material is based upon work supported by the National Science Foundation and the National Institute of General Medical Sciences under Grant No. MCB-1052331.

Any opinion, finding, conclusion, or recommendation expressed in these videos are solely those of the speaker and do not necessarily represent the views of iBiology, the National Science Foundation, the National Institutes of Health, or other iBiology funders.

© 2019 - 2006 iBiology · All content under CC BY-NC-ND 3.0 license · Privacy Policy · Terms of Use · Usage Policy

 

Power by iBiology

 

 

This website uses cookies to remember you and improve your experience. To find out more see our Privacy Policy.Accept