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Well, I cannot really dispute optics with Shalin!  But I can say that I've looked at resin sections under phase contrast and NOT seen this shade-off artefact.  If flat and uniform they've appeared uniform in shade, and different from the background.  But these would be much larger than 20 x 20 µm.

                                               Guy

Guy Cox, Honorary Associate Professor
School of Medical Sciences

Australian Centre for Microscopy and Microanalysis,
Madsen, F09, University of Sydney, NSW 2006

-----Original Message-----
From: Confocal Microscopy List [mailto:[log in to unmask]] On Behalf Of Shalin Mehta
Sent: Thursday, 20 March 2014 3:10 AM
To: [log in to unmask]
Subject: Re: Phase contrast microscopy

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> I have a few questions about phase contrast microscopy:
>
> 1. Can a phase-contrast microscope see a single Rayleigh-scattering 
> particle, for example, a 40 nm glass bead suspended in water?
> http://en.wikipedia.org/wiki/Rayleigh_scattering

It is possible to see, but it will be small blip (positive or negative depending on the sign of phase delay in the phase ring) on a large background.  You will need high dynamic range camera (e.g., 16-bit
sCMOS) so that the intensity modulation because of the bead spans three bits. The shading in illumination can make things worse. It should help to move the bead out of the view, acquire background, and then subtract the background from the image with the bead in the view.
 It may be easier to start with dark-field to locate the bead and then switch to phase-contrast. I am assuming you are interested in optical assessment rather than practical use.

>
> 2. Suppose you've etched a perfectly smooth square trough (or a raised 
> square ridge) into your coverslip surface, perhaps one micron high and
> 20x20 microns wide. I'm confident the edges of the trough/ridge would 
> show up nicely under phase-contrast microscopy; would the middle of 
> the trough/ridge appear to be a different brightness than the 
> surrounding coverslip?
>

Yes, the middle of the square will approach the background. This is because of shade-off artifact (http://www.microscopyu.com/tutorials/java/phasecontrast/shadeoff/).
It is because some low spatial frequencies diffracted by the specimen get modified by the phase-ring in objective.

>  I'm primarily interested in first-hand experience rather than 
> theoretical predictions, so if you know the answers for 
> similar-in-spirit but not identical samples, please let me know.
>
> I have followup questions, assuming the answers to 1 and 2 are 
> straightforward.

Best
Shalin