Diatom Illumination Experiment

[rnabholz]

In a recent discussion found here: viewtopic.php?f=6&t=3552 there was interest expressed in seeing the effects of various illumination techniques on diatoms.

Tonight I set out to see if I could provide some images for comparison.

As per the discussion, the test would include Brightfield, Darkfield, Phase Contrast, Oblique and I added one more, a van Egmond mask.

The target was a Gyrosigma, approximately 140µm in length, mounted in Pleurax.

In order to accomplish all of the techniques I had to move across three stands. In all cases the images were shot using 100x Oil Plan Achro objectives, oiled to the slide, with the slide oiled to the condenser. All were taken with a Canon 70D, and are single frames. Images were cropped and resized to 900 pixel width, levels were tweaked and unsharp mask was applied with the same parameters for all images.

These are the set ups for each illumination type.

Brightfield and Phase Contrast: AO 20, 100x Oil Phase Contrast Plan Achro.

Darkfield : AO 10, Ortho-Illuminator, 100 Oil Iris Plan Achro, 214 Darkfield Condenser.

Oblique and van Egmond Mask: AO One Ten, 100x Oil Iris Plan Achro.

Brightfield

DIT Brightfield 746.JPG (153.33 KiB) Viewed 8306 times

Darkfield

DIT Darkfield 755.JPG (100.86 KiB) Viewed 8306 times

Phase Contrast

DIT Phase Contrast 750.JPG (109.58 KiB) Viewed 8306 times

Oblique

DIT Oblique 769.JPG (144.17 KiB) Viewed 8306 times

Blue van Egmond Mask

DIT Blue van Egmond Mask 766.JPG (118.55 KiB) Viewed 8306 times

Please let me know what you think.

Rod

[McConkey]

Really nice effort! I was surprised to see how big of difference each illumination technique actually made. I think the brightfield and the phase contrast show the most detail, really sharp and crisp.
All images are very well done considering they are not stacks! Great single image capture for all of these.

Thank you for sharing and putting in the effort to show us the difference!

[zzffnn]

Great job, Rod!

For 100x oil objective though, dots on Gyrosigma are not small enough (Frustulia or Nitschia would show more difference between illuminations). As a result, most illuminations worked well and difference was small. I expect phase and DIY van Egmond may not be able to resolve Frustulia to dots.

[rnabholz]

Really nice effort! I was surprised to see how big of difference each illumination technique actually made. I think the brightfield and the phase contrast show the most detail, really sharp and crisp.
All images are very well done considering they are not stacks! Great single image capture for all of these.

Thank you for sharing and putting in the effort to show us the difference!

Thanks McC,

Glad you found it interesting. The Gyrosigma is nice and flat and makes a nice target.

Thanks for the interest.

Rod

[rnabholz]

Great job, Rod!

For 100x oil objective though, dots on Gyrosigma are not small enough (Frustulia or Nitschia would show more difference between illuminations). As a result, most illuminations worked well and difference was small. I expect phase and DIY van Egmond may not be able to resolve Frustulia to dots.

Well I will have to see if I can round up another target.

Rod

[75RR]

Thanks for this rnabholz - very interesting comparison.

If you are planing another test I would not go for Frustulia , punctae are silly small.
In any case test was not for resolution but to see what each illumination technique revealed.

Thanks again

[KurtM]

... test was not for resolution but to see what each illumination technique revealed.

Wait ... what...?

VERY cool Rod, you sure know how to pack a world of value into one affordable and easy to digest package! You get an Uber Geek for this one.

[billbillt]

This test seems to have revealed some useful data.. Thanks for sharing!...

BillT

[rnabholz]

Thanks for this rnabholz - very interesting comparison.

If you are planing another test I would not go for Frustulia , punctae are silly small.
In any case test was not for resolution but to see what each illumination technique revealed.

Thanks again

Glad you found it interesting, I did too. The same rays of light, manipulated and producing different results. Amazing to think about, and taken for granted by most.

Thanks

Rod

[rnabholz]

... test was not for resolution but to see what each illumination technique revealed.

Wait ... what...?

VERY cool Rod, you sure know how to pack a world of value into one affordable and easy to digest package! You get an Uber Geek for this one.

Thanks Kurt. I accept the Uber Geek on behalf of all geeks everywhere....

[rnabholz]

This test seems to have revealed some useful data.. Thanks for sharing!...

BillT

Thanks Bill, glad you found it useful.

[gekko]

Interesting comparison and very nice images.

[apatientspider]

Rod,

The brightfield image seemed to my uneducated eyes to be the sharpest and clearest. You write that you oiled the condensers to the slide: Other than the darkfield condenser what other condenser(s) were you using?

And thanks for going to the bother of posting all these. Such comparisons are always interesting.

Jim

[rnabholz]

Thanks Gekko.

Rod,

The brightfield image seemed to my uneducated eyes to be the sharpest and clearest. You write that you oiled the condensers to the slide: Other than the darkfield condenser what other condenser(s) were you using?

And thanks for going to the bother of posting all these. Such comparisons are always interesting.

Jim

Thanks Jim.

The brightfield and phase images were done with the 1201 phase condenser.

The oblique and van Egmond images were through the 1094 Flip Top condenser.

Rod