Phase Contrast Microscopy

Posted by Knecht, David on
URL: http://confocal-microscopy-list.275.s1.nabble.com/Phase-Contrast-Microscopy-tp7590859.html

*****
To join, leave or search the confocal microscopy listserv, go to:
http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy
Post images on http://www.imgur.com and include the link in your posting.
*****

I posted this message on the Microscopy listserv and got such confusing answers that I thought I would try this list to see if anyone can clarify.

I thought I understood how phase contrast microscopy works, but then I was reading MicroscopyU (https://www.microscopyu.com/techniques/phase-contrast/introduction-to-phase-contrast-microscopy<https://nam10.safelinks.protection.outlook.com/?url=https%3A%2F%2Fwww.microscopyu.com%2Ftechniques%2Fphase-contrast%2Fintroduction-to-phase-contrast-microscopy&data=02%7C01%7Cdavid.knecht%40uconn.edu%7C80f17dd0f2dd4d83409c08d7e6c53275%7C17f1a87e2a254eaab9df9d439034b080%7C0%7C0%7C637231606945304315&sdata=LkYN7e343%2B7073PGh%2FePzDsc3tWc4gO3E5G0s1Q%2Fo8Q%3D&reserved=0>) and other sites and now I am confused.  My understanding is that it works because of refractive index differences in different parts of the light path (cells vs. surround), leading to retardation of light refracted by cells (slowed about ¼ wavelength) and eventual phase differences relative to un-refracted (surround) light that passes through the thinned area of the phase plate relative to the thick area (another ¼ wavelength) in the objective.  However, in the web site indicated and others, they use refracted light and diffracted light almost interchangeably in explaining phase contrast.  To my “biologist” level understanding, diffraction and refraction are very different phenomenon and I did not think that diffraction changed the optical path length like refractive index differences so would not lead to interference in the same way.  Refraction makes total sense to me in the context of phase contrast, but I don’t see how diffraction is relevant. Can someone explain what I am missing?

Thanks, Dave

Dr. David Knecht
Professor, Department of Molecular and Cell Biology
University of Connecticut
91 N. Eagleville Rd.
U-3125
Storrs, CT 06269-3125