More Diatom Illumination Experimentation

Here you can discuss different microscopic techniques and illumination methods, such as Brightfield, Darkfield, Phase Contrast, DIC, Oblique illumination, etc.
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rnabholz
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More Diatom Illumination Experimentation

#1 Post by rnabholz » Fri Aug 05, 2016 1:32 am

In a continuation of the experimentation started here viewtopic.php?f=28&t=3560 , I tired a few new things tonight.

Using just one stand tonight, I shot this Encyonema in Brightfield, Phase Contrast, Phase Contrast with Green Filtration, Oblique using the 10x Phase Annulus with the 100x Objective.

If I were presenting these as something other that experimental I would likely have cleaned things up a bit more, spots, halos, deritus etc, but I was primarily interested in just trying some different approaches. So here they are for whatever you may take away from them.

AO 20, 100x Oil Phase Objective, Phase Condenser, Objective and Condenser both oiled, Canon 70D, Single Frames.

Encyonema p. leibleinii 54µm
Brightfield
Brightfield
DIT Encyonema Brightfield 793.JPG (106.72 KiB) Viewed 6303 times
Brightfield Converted to Gray Scale
Brightfield Converted to Gray Scale
DIT Encyonema Brightfield Gray Scale 793.JPG (76.57 KiB) Viewed 6303 times
Oblique using 10x Annulus
Oblique using 10x Annulus
DIT Encyonema Oblique 786.JPG (79.63 KiB) Viewed 6303 times
Phase Contrast
Phase Contrast
DIT Encyonema Phase Contrast 781.JPG (80.24 KiB) Viewed 6303 times
Phase Contrast w/ Green Filter
Phase Contrast w/ Green Filter
DIT Encyonema PC Green 781.JPG (88.4 KiB) Viewed 6303 times

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KurtM
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Re: More Diatom Illumination Experimentation

#2 Post by KurtM » Fri Aug 05, 2016 1:56 am

Do you focus for each shot, or set it once then leave it?

It's a horse race between the last two, for my money. And it's neck and neck, since the phase image yields more areaolae contrast in the central area whereas the phase-plus-green filter gives a bit more at the apices. The main thing I was searching for is what's happening with the raphae at the apices, which is always the toughest to discern, seems like. I still can't quite tell, but at least I'm certain the focus couldn't be better.

Another very, very cool set, and superbly done! Lovely subject too. 8-)
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Kurt Maurer
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email: ngc704(at)gmail(dot)com
https://www.flickr.com/photos/67904872@ ... 912223623/

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rnabholz
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Re: More Diatom Illumination Experimentation

#3 Post by rnabholz » Fri Aug 05, 2016 3:10 am

Thanks Kurt.

I typically find that I need to tweak focus between methods. Brightfield the condenser is closed, Phase is wide open, Oblique is open but diffraction causes some difference in focus, they all seem to be just a tick or two off.

For all images I used the central raphe as the focus point. Obviously that selection could vary per your agenda or interest, but of course you gotta pick one for photos.

My experience using green filters would always make it the goto choice if my mission was pure science, to my eye it just always performs best with my Achro objectives. Aesthetically, it is less desirable.

Thanks for the interest.

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KurtM
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Re: More Diatom Illumination Experimentation

#4 Post by KurtM » Fri Aug 05, 2016 4:13 am

I used the central raphe as the focus point.
For single frames that's exactly what I do. You pretty much have to, otherwise it'd be like a bird photo with the eye(s) out of focus, and I know you know what that accomplishes.

I expected you were tweaking focus for each shot - heck, I'd bet you bracketed each one. Thinking back, it was rather a silly question. :)
Cheers,
Kurt Maurer
League City, Texas
email: ngc704(at)gmail(dot)com
https://www.flickr.com/photos/67904872@ ... 912223623/

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gekko
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Re: More Diatom Illumination Experimentation

#5 Post by gekko » Fri Aug 05, 2016 10:23 pm

I can't decide: they are all different and they are all very nice. When you say "oblique using 10x annulus" do you mean phase annulus centered [COL] or phase annulus off-center? Probably dumb question: why did you use the 10x rather than, say, the 40x phase annulus with the 100x objective?

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rnabholz
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Re: More Diatom Illumination Experimentation

#6 Post by rnabholz » Sat Aug 06, 2016 2:42 am

gekko wrote:I can't decide: they are all different and they are all very nice. When you say "oblique using 10x annulus" do you mean phase annulus centered [COL] or phase annulus off-center? Probably dumb question: why did you use the 10x rather than, say, the 40x phase annulus with the 100x objective?
Thank you Gekko.

The Phase Annulus was centered in this case.

The decision to use the 10x annulus was simply that I thought it provided the best effect, I did try all of them. I don't know if that will always be the case, but it is easy enough to switch them all into place and give them a try.

Thanks for the interest.

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zzffnn
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Re: More Diatom Illumination Experimentation

#7 Post by zzffnn » Sat Aug 06, 2016 3:55 am

rnabholz wrote: Thank you Gekko.

The Phase Annulus was centered in this case.

The decision to use the 10x annulus was simply that I thought it provided the best effect, I did try all of them. I don't know if that will always be the case, but it is easy enough to switch them all into place and give them a try.

Thanks for the interest.
Rod,

PH1 (10x) condenser annulus will significantly limit effective imaging NA, when used with 100x objective COL. Your resolution would be better by using darkfield annulus decentered. Likely better still, use DIY opague stops under top lens of your swing up condenser. Or put a sufficiently large stop in your brightfield port.

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Re: More Diatom Illumination Experimentation

#8 Post by rnabholz » Sat Aug 06, 2016 12:06 pm

zzffnn wrote:
rnabholz wrote: Thank you Gekko.

The Phase Annulus was centered in this case.

The decision to use the 10x annulus was simply that I thought it provided the best effect, I did try all of them. I don't know if that will always be the case, but it is easy enough to switch them all into place and give them a try.

Thanks for the interest.
Rod,

PH1 (10x) condenser annulus will significantly limit effective imaging NA, when used with 100x objective COL. Your resolution would be better by using darkfield annulus decentered. Likely better still, use DIY opague stops under top lens of your swing up condenser. Or put a sufficiently large stop in your brightfield port.
Thanks zz. I was just experimenting to see what the new scope could do as configured. I have played around with most of the techniques you mentioned in the past .

Culicoides
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Re: Halo ring lights for phase, dark field etc.

#9 Post by Culicoides » Sat Aug 20, 2016 9:06 am

I've been reading an article by David Walker (micscape magazine, August 2016) on the use of halo ring led lights for phase, dark field and rheinberg on "upright" microscopes. Does anyone on the forum any experience of this?

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Re: More Diatom Illumination Experimentation

#10 Post by c-krebs » Sat Aug 20, 2016 6:15 pm

Culicoides wrote:ve been reading an article by David Walker (micscape magazine, August 2016) on the use of halo ring led lights for phase, dark field and rheinberg on "upright" microscopes. Does anyone on the forum any experience of this?
I've tried them (primarily for darkfield experimentation... no phase). They do work, but I found the "quality" of the darkfield light was nowhere near as "nice" as with dedicated darkfield condensers (or, with low powers, a simple black darkfield stop). It would vary greatly with the subject. I realize "quality" and "nice" are not exactly empirical terms :D . It seemed to be a result of the point light sources and the spacing between the LEDs.

I do think that this technique offers some very real creative possibilities, and deserves attention. At the time I was primarily trying to come up with a good simple simple darkfield method for a 2X and 4X objective. When I did not like the "look" for that usage I put them on a shelf.

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