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 JoVE Biology

Phase Contrast and Differential Interference Contrast (DIC) Microscopy

1

1Core Optical Imaging Facility, University of Texas Health Science Center at San Antonio (UTHSCSA)

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    Summary

    This protocol highlights the principles and practical applications of Phase and Differential Interference Contrast (DIC) Microscopy

    Date Published: 8/06/2008, Issue 18; doi: 10.3791/844

    Cite this Article

    Centonze Frohlich, V. Phase Contrast and Differential Interference Contrast (DIC) Microscopy. J. Vis. Exp. (18), e844, doi:10.3791/844 (2008).

    Abstract

    Phase-contrast microscopy is often used to produce contrast for transparent, non light-absorbing, biological specimens. The technique was discovered by Zernike, in 1942, who received the Nobel prize for his achievement. DIC microscopy, introduced in the late 1960s, has been popular in biomedical research because it highlights edges of specimen structural detail, provides high-resolution optical sections of thick specimens including tissue cells, eggs, and embryos and does not suffer from the phase halos typical of phase-contrast images. This protocol highlights the principles and practical applications of these microscopy techniques.

    Protocol

    The complete text protocol for this experimental approach is available in Current Protocols in Cell Biology.

    Disclosures

    The authors have nothing to disclose.

    Acknowledgements

    Several video clips demonstrating phase contrast and DIC microscopy were taken from another video-article, entitled Layers of Symbiosis.  JoVE graciously acknowledges this contribution made by the Leadbetter lab. 

    Comments

    9 Comments

    how much will each cost
    Reply

    Posted by: AnonymousSeptember 15, 2008, 1:23 PM

    The systems vary widely to correlate with your needs.  I would be happy to help you further. 631-547-8501, Ned Jastromb, Product Manager, Nikon Instruments, Inc.
    Reply

    Posted by: AnonymousSeptember 16, 2008, 11:15 AM

    Excellent - I'm definitely going to use this for my students, who are from a mixed background (biologists, physicists, chemists, engineers).
    Reply

    Posted by: AnonymousSeptember 18, 2008, 8:40 AM

    I've seen that there we're colored images obtained using the DIC microscopy that looks like a 3-D image. Is there an approximation using DIC to measure the depth or height difference by just interpreting color differences?
    Reply

    Posted by: AnonymousMay 27, 2009, 7:42 AM

    I've seen that there we're colored images obtained using the DIC microscopy that looks like a 3-D image. Is there an approximation using DIC to measure the depth or height difference by just interpreting color differences?
    Reply

    Posted by: AnonymousMay 27, 2009, 7:59 AM

    In article abstract, the phase-contrast microscopy is claimed to have been invented in 194². This is incorrect. The origins of phase-contrast date back to 1934 (not a microscopy yet) and 1935 (microscopy):

    Zernike F. (1934)
    Diffraction theory of the knife-edge test and its improved form, the phase-contrast method.
    Monthly Notices of the Royal Astronomical Society 94 (5), 377-3­84 (originally published in Dutch).
    (http://esoads.eso.org/journals_service.html)

    Zernike F. (1935)
    Phase-contrast method in microscopical observation (in German).
    Physikalische Zeitschrift 36 (²²/²3): 848-851
    Zeitschrift für Technische Physik 16 (11): 454-457
    (two identical papers)

    Furthermore, the following reports (published when Zernike was only 16 or 17 years old) utilize, in a way, the principle of phase-contrast microscopy, even though only for absorbing (i.e., not phase) objects:

    Rheinberg J. (1904 and 1905)
    On the influence on images of gratings of phase differences amongst their spectra.
    J. Roy. Microsc. Soc.; volumes-not-numbered (Part 4), 388-390; and (Part ²), 15²-155.
    (www.biodiversitylibrary.org/bibliography/7413)

    Conrady A. (1905)
    An experimental proof of phase reversal in diffraction spectra.
    J. Roy. Microsc. Soc.; volume-not-numbered (Part ²), 150-15².
    (www.biodiversitylibrary.org/bibliography/7413)
    Reply

    Posted by: AnonymousMay 4, 2010, 11:14 AM

    Your presentation is excellent and locked in my neurons , the corresponding text in books do not give that effect
    Reply

    Posted by: AnonymousNovember 12, 2011, 10:19 PM

    Nice video.
    I may be mistaken, but one of the images (around 3:47) shown as DIC appears to be phase contrast.
    Also, when mentioning setting up KŒhler illumination during DIC (around 4:30), the video shows the alignment of phase rings. This is not needed (since you do not need to see the back focal plane of the microscope to establish KŒhler) nor, in fact, what would be observed when setting up KŒhler for DIC (there would be no phase annulus in the condenser if it is set up for DIC).
    Reply

    Posted by: AnonymousDecember 7, 2011, 10:36 AM

    The video explained well what i was trying to learn in detail....
    it is really nice
    Thanks
    Reply

    Posted by: AnonymousApril 5, 2012, 2:35 AM

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