Phase contrast on diatom slides - limitations and applications

Here you can discuss different microscopic techniques and illumination methods, such as Brightfield, Darkfield, Phase Contrast, DIC, Oblique illumination, etc.
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MicroBob
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Phase contrast on diatom slides - limitations and applications

#1 Post by MicroBob » Sat Nov 14, 2020 3:47 pm

Hi together,
in the german forum the topic came up whether it makes sense to use phase contrast on diatom slides. I think this might depend on how strong ans massive the diatom frustules are, on their refractive index and on the r.i. of the mountant and the type of phase contrast system. I have used phase contrast only occasionally on diatoms and never made a thorough comparison. It would be interesting to hear how your results were when using phase contrast on cleaned diatoms. It would be especially interesting to hear of someone who can compare different phase contrast systems on the same diatom.

Bob

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75RR
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Re: Phase contrast on diatom slides - limitations and applications

#2 Post by 75RR » Sat Nov 14, 2020 6:15 pm

.
Have you read this article by David Walker

http://www.microscopy-uk.org.uk/mag/ind ... -test.html

The whole article is well worth a read, but the part that refers to your query is about halfway down and it titled: "The loss of resolution in phase contrast"
Zeiss Standard WL (somewhat fashion challenged) & Wild M8
Olympus E-P2 (Micro Four Thirds Camera)

MicroBob
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Re: Phase contrast on diatom slides - limitations and applications

#3 Post by MicroBob » Sat Nov 14, 2020 7:02 pm

Hi Glen,
I knew this article, but I think that there are more factors than just phase vs.brightfield. The Zeiss West phase system uses phase annulli ph1, ph2 and ph3, but ph2 is used for a range of objectives from 16:1 to 40:1. I don't think that resolution will be affected the same for all of them, one example. For very strong/massive diatoms one can use mountants with less extreme r.i. to avois overly strong contrast in the image. So it will probably also be different how well individual diatoms will be visible under phase contrast. When I think of Hobbyst46-Dorons phase contrast diatom images and what I have observed myself I don't really second David Walkers conclusion of severe resolution loss.

Bob

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Re: Phase contrast on diatom slides - limitations and applications

#4 Post by Hobbyst46 » Sat Nov 14, 2020 7:42 pm

A preliminary qualitative viewpoint if I may:

When the cleaned diatom frustules are in water, many of them are almost invisible in brightfield (BF). My default mode is then phase contrast (PC). PC improves visibility a lot.

When they are mounted in Pleurax, visibility varies with the genus, distance from the objective lens, illumination mode and objective NA. For example, in BF, Gyrosigma is barely invisible. Again, PC makes Gyrosigma visible. I can then switch to oblique or DF.

My very few comparisons between oblique and PC (same objective) are (at least qualitatively) in line with theory and with David Walker's article.
I follow literature about removal of halos from PC images, there are publications every now and then in the last two decades, did not find anything applicable to hobby microscopy.

Would love to see comparisons of different PC modes on diatoms! maybe Apochronaut has already posted some ?
Bob, I hope you do not mind me planting a recent PC image of a Japanese diatom (Wakura Beds). It is flat and thin, so appropriate. It was taken with unfiltered white light and slightly post-processed. Diameter ~80um. Unknown (to me) ID.
By contrast, Pleurosira, for example, appears encircled by a bright halo reminiscent of Saturn's rings (sort of), because it is fairly thick.

Edit: guessed ID of diatom in photo: Coscinodiscus radiatus.
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Stack of 3, Planapo 63X1.4 oil, Ph3 phase contrast, tweaked-resized-cropped-sharpened.JPG
Stack of 3, Planapo 63X1.4 oil, Ph3 phase contrast, tweaked-resized-cropped-sharpened.JPG (62.54 KiB) Viewed 247 times
Zeiss Standard GFL+Canon EOS-M10, Olympus VMZ stereo

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