Posted by
Kai Schleicher on
URL: http://confocal-microscopy-list.275.s1.nabble.com/Phase-Contrast-Microscopy-tp7590859p7590862.html
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Hi David,
I think that you understanding of phase contrast is correct and that you have explained it yourself properly and in a concise way.
Diffraction and refraction are different indeed. It is in fact the diffraction of light that plays an important role in phase contrast microscopy.
Have a look at the Leica tutorial:
https://www.leica-microsystems.com/science-lab/the-principles-of-phase-contrastand figure 4 in the link you shared:
1. The illumination light passes through the annual ring in the condenser, resulting in a hollow cone of illumination.
2.1 A higher refractive index in the sample causes retardation, on average generating a phase shift of -1/4 λ in the light that interacted with the specimen compared to freely passing light.
2.2 Light interacting with the specimen (cell, granule, nucleus...) is diffracted to the outside of the illuminating light cone.The smaller the object, the larger the angle of diffraction [1].
2.3 Light that does not interact with the specimen is not diffracted and hence stays on the inside of the illumination cone.
3. In the phase plate, only the light on the inside of the light-cone is then advanced +1/4 λ .
4. This result in a phase difference between illuminating light and sample light of 1/2 λ, generating destructive interference and hence maximum contrast in the imaging plane wherever there was a structure in the sample
Here is also a very nice iBology talk on the subject:
https://www.ibiology.org/talks/phase-contrast-microscopyHope this helps!
[1] Abbe Diffraction:
https://www.youtube.com/watch?v=d8Tqoo0S6gc