My first post

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jin
Posts: 107
Joined: Sun Aug 26, 2012 6:35 am
Location: Singapore

My first post

Post by jin »

Hi, here is my first photomicrogaraphy image posting of a butterfly egg. Please let me know how to improve the sharpness and why is there 'milky whitish cloudy' around the parameter of the egg.

Equipment and set up info:
1. Canon 450D with tube length ~170mm, live view and remote cable,
2. Olympus M20, 0.4 objective on BHM microscope,
3. EX580 II as light source with DIY tube diffuser
4. Helicon focus trail version
5. manual stacking at~2um interval, ~100 images.

Rgds,Image

rjlittlefield
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Location: Richland, Washington State, USA
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Post by rjlittlefield »

jin, welcome to image posting! :D

These eggs of lycaenid butterflies are wonderful things to see up close. I used to get so frustrated looking real time through the microscope and having to fly the focus knob up and down. Now with image stacking I can see the whole thing at once in high resolution. It's wonderful technology!

The image you have here is a good start.

The white fuzzy regions you mention look like what we call "stacking mush". It's a defect often found in Helicon Focus methods A and B, in which the software loses track of low contrast detail and ends up selecting out of focus frames instead of the ones it should have.

Since you're still trialing, I recommend that you also try Zerene Stacker because its PMax method and retouching capabilities were specifically designed to handle deep high magnification stacks like this one. (Standard disclaimer: I wrote Zerene Stacker so I'm biased. On the other hand it's optimized for the sort of work I like to do, and this egg is typical of that sort.)

Other things I notice...

The lighting looks great. You're getting good modeling on the egg, little or no blowout of the whites, and good delineation of those trichomes on the plant, with bright outlines on both sides of most of them.

I see quite a bit of color fringing, especially on the right side of the image. Most likely that's lateral CA (chromatic aberration) in the objective. You can reduce it a lot by by post-processing in Photoshop. The recipe is Filter > Lens Correction... > Custom > "Fix Red/Cyan Fringe", and move the slider all the way left. The amount of fringing is more than Photoshop can handle at one time, so you'll have to repeat the process two or three times to get rid of it.

I hope this helps!

--Rik

jin
Posts: 107
Joined: Sun Aug 26, 2012 6:35 am
Location: Singapore

Post by jin »

Rik, thanks for the comments and tips, much appreciated :-)

I tried processed it with the Z.Stacker (trial) and indeed I can see more details and the fuzzy regions around is almost gone this time but observed the exposure seem a litter brighter than I got with the H.F., see pic below for your comment.Image

rjlittlefield
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Posts: 23626
Joined: Tue Aug 01, 2006 8:34 am
Location: Richland, Washington State, USA
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Post by rjlittlefield »

Ah yes... This looks much better.

There are two common reasons for the shift in brightness.

1. By default Zerene Stacker adjusts the brightness & contrast of each image to match the first one it processes. This is intended to compensate for variation in exposure, typically due to flash illumination. But with deep stacks where the image appearance changes a lot due to focus variation, the adjustment can also introduce systematic changes in brightness and contrast. To prevent this from happening, go to Options > Preferences > Alignment, and remove the checkmark on "Brightness".

2. The PMax stacking method often changes contrast, brightness, and color saturation as a side effect of identifying and retaining detail at all size scales. These changes cannot be avoided. Most of the time they're not a problem. If they are, then one workaround is to select "Retain full dynamic range" when saving, and then apply your own curves/level adjustment in post-processing. With this technique you may want to save the file as 16-bit TIFF since curves/level adjustment in 8-bit mode can introduce posterization in some cases.

--Rik

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