New to focus stacking, feedback appreciated

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zachg
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Joined: Fri Nov 06, 2020 1:48 pm
Location: San Diego

New to focus stacking, feedback appreciated

Post by zachg »

Hi there. I'm a biology grad student. In my lab this summer, we made some cool butterfly mutants that we now need to photograph with focus stacking. I just set up our rails last week and tried it for the first time. I don't really know what I'm doing yet but below are a few of my results so far. I used Zerene for the focus stacking followed by minor adjustments in Lightroom. I know that these images aren't perfect and I would like to improve. Any feedback or suggestions anyone here can give me would be much appreciated!

https://drive.google.com/file/d/1h8sHM2 ... sp=sharing
https://drive.google.com/file/d/1h8S4CW ... sp=sharing

rjlittlefield
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Re: New to focus stacking, feedback appreciated

Post by rjlittlefield »

Zach, welcome aboard! I'm the fellow who wrote and supports Zerene Stacker, so I'll be happy to go first here.

Sharpness looks fine, as expected with the low magnification needed for butterflies of this size. You've clearly captured the irregular scaling patterns found in several places especially on the specimen with darker background.

I expect that color is important for this work, so I recommend using a standardized background, illumination, and camera settings. Something like a standard gray card (20% gray) as background would provide a lot of confidence that what a viewer sees is what you intend them to see. Still there are other things that can go wrong with colors, so it's a good idea to photograph some typical parent butterflies as well as the mutants, to facilitate side-by-side comparison. It's also a good idea to photograph some industry color standards such as a ColorChecker target (https://xritephoto.com/colorchecker-targets), because those can expose some issues that simple color balancing cannot.

When color is important, you should be doing the bulk of your stacking with DMap, not PMax. If your illumination and other exposure controls are stable enough, then you should also turn off Brightness correction at Options > Alignment. The reason for these settings is explained in the FAQs on the zerenesystems.com website, for example at "My colors changed a little. Why is that?" .

When using DMap, you can avoid halos in background around the specimen by adjusting the contrast threshold slider so that all the background goes "black in preview", except for a narrow band right next to the specimen. Because your specimens have black also, you may prefer to change the DMap indicator color to be something other than black. See the discussion at http://www.photomacrography.net/forum/viewtopic.php?p=215149#215149 for both of these points. Also read through the "How To Use DMap" tutorial, linked on the Tutorials index page.

No doubt there are other things to consider, so I'm hoping that other people will chime in also.

--Rik

zachg
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Joined: Fri Nov 06, 2020 1:48 pm
Location: San Diego

Re: New to focus stacking, feedback appreciated

Post by zachg »

Thanks, Rik! This is all very helpful.

Lou Jost
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Re: New to focus stacking, feedback appreciated

Post by Lou Jost »

They look pretty good, but I would suggest you use a uniform background (easiest is white or black, but gray also works) so that you can easily make seamless composite illustrations of several individuals for publication. Also don't forget a scale bar!

If you choose to have a white background, it may be nice to light the white background from behind.

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MarkSturtevant
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Re: New to focus stacking, feedback appreciated

Post by MarkSturtevant »

Very late to the thread - only just now saw it. Interesting mutant butterfly. Are the altered color patterns made by RNAi, or by something similar?
I think its fascinating what is being done with experimenting on butterfly color patterns to understand a bit more about biological pattern formation.
Mark Sturtevant
Dept. of Still Waters

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