Phase techniques

A forum to ask questions, post setups, and generally discuss anything having to do with photomacrography and photomicroscopy.

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gpmatthews
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Phase techniques

Post by gpmatthews »

Image

This is a comparison of different methods of phase contrast.

1. Conventional, Zernicke, phase contrast, Olympus FH microscope with Gillette & Sibert x40 PH objective
2. Wilska phase contrast, using a smoked central spot, Watson x30 parachromat, Olympus FH microscope
3. Wilska phase contrast using a smoked annulus, Watson x30 parachromat, Olympus FH microscope
4. Conventional brightfield using the Gillette & Sibert x40 PH objective, Olympus FH microscope

I have updated my webpages on home-made phase conrast. You can find them at http://www.gpmatthews.nildram.co.uk/mic ... phase.html

A Very Merry Christmas to you all
Graham

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Ken Ramos
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Post by Ken Ramos »

:D Same to you Graham, A Very Merry Christmas and a Prosporous New Year! 8)
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Reposts of my images within the galleries are welcome, as are constructive critical critiques.

Steve West
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Post by Steve West »

Hi Graham,

Merry Christmas to you and all on this list!

The kids are off playing with their stuff, so I took a look at your photos here. This is some great stuff. The first thing that struck me is how your experimental phase photos had many of the attributes of the brightfield photo. For example, the little pores in the interior of the diatom are visible in all except for the Zernike phase image.

If I understand your experiments, they are not true phase since the objective ring must impart a 1/4-wave phase shift to the zero-order light. Your experimental objectives rings and condenser rings seem to amount to some sort of brightfield staining technique it seems to me, and not phase-type interference.

Even so, it looks like the technique used in image 3 is quite astounding in it's effect and looks to be extremely useful as most of the brightfield detail is retained with excellent contrast enhancement.

Maybe I didn't completely understand your experiments. I seem to recall that Ted Clarke was doing something similar to this a while back on the yahoo list, and accidentally rediscovered some sort of staining technique using objective masks. I recall that these masks were almost darkfield masks but cutoff just inside the objective's NA so that very striking colors resulted.

Cheers,

Steve

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gpmatthews
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Post by gpmatthews »

There has indeed been some debate, both at the time Wilska first published his method in Nature, and also more recently, about whether this is a phase or amplitude contrast technique. Dr Barer (Nature (171, 697; 1953)) suggested that this was essentially an amplitude contrast method, but Prof. Wilska was of the opinion that there was actually a phase shift caused by the smoked pattern. He states in his reply to Barer:


"The soot layer appears to cause a considerable phase retardation in addition to the absorption."


This appears to me to be possible if the soot particles are of colloidal size - i.e. significantly less in size than light wavelength - rather in the manner an aerial element of near half wave produces a phase delay in, say, a parasitic array. Any phase shift would, however, be rather unpredictable because of lack of control over the soot particle size and the thickness of the deposited film. This is essentiallly speculation on my part, but the technique certainly seems to produce some useful images, which is, after all, the most important thing from the point of view of the microscopist.

Transcripts of the original Nature correspondence are available on my website at the URL in my original posting.

I'm pleased if this gives some interesting discussion, and maybe tempts some into the odd experiment or two...
Graham

Charles Krebs
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Post by Charles Krebs »

Nice camparison Graham. Thanks for putting is up here!

All three look like they could be very useful and attractive.

So many choices in life :shock:

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