UV reflectance z-stacking

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dlg
Posts: 26
Joined: Wed Nov 04, 2015 11:47 pm

UV reflectance z-stacking

Post by dlg »

I've been playing around with UV reflectance z-stacking. Some may find it interesting to see where the issue are and how to overcome them.

Hardware: Canon 5D2, modified to full spectrum.
MP-E 65: works, but about 10x (3stop) lower transmission and seems to cut off at a longer wavelength.
Nikon EL 80 mm f/5.6. A M39 enlarger lens that has good UV transmission. cost me $40 off Ebay, a bit cheaper than the dedicated UV lenses for >$5K. Be sure to get the older version.
M39/M42 adapter
M42 bellows (Practica, $20 off eBay)
M42 to EOS adapter.
Gets me to about 4:1, which is just about the diffraction limit for f/5.6.
Baader U filter, 2"
From eBay, adapter from Nikon EL80 with 34.5 mm thread to Baader U with 48 mm thread. Aluminum, has tendency to freeze the filter, so applied some WD40 creeping oil.
Cogynysis StackShot: no explanations necessary here, I assume.
365 nm LED flashlight, 5W

Shooting is a straightforward z-stack. The issue is, that the Baader U is visual opaque, so no view finder focus. The LED flashlight is rather dim, but you can focus with live view and get critical focus for start and end point with the magnifying function.

Exposure manually, in the 1-2s range. Autoexposure does not work, of course, so judge by test shot and fire away. Save as RAW.

Illumination: the typical paper reflector does not work, because of UV brighteners in most paper that fluoresce the UV back in as blue light. Plastic does not work, because plastic absorbs UV readily. However aluminum foil works very well.

Processing: Process RAW images (exposure, curve, etc.) in DxO Optics pro and export as tif.

One problem is that during camera conversion, some of the Bayer filter elements most likely got damaged. This is no big deal for single captures, but if you stack 50 or more, one gets a lot of "creative" color streaks. That leads to a lot of post-processing after stacking. I thought of doing a dust delete data in the 5D2 body, but that does not work, because during conversion, the dust shaker hardware is removed, and without it, the dust delete data cannot be obtained.

But because these color dots are in fixed positions, they can be screened out with a Gaussian blur mask. Do the following in Affinity Photo:
- Open a dozen images in AP.
- Add a Gaussian Blur live filter on top of the first image.
- Add a mask to the Gaussian Blur live filter. Blur of about 2.5 pixels worked well for me, but that depends a lot on your camera and the type of Bayer damage you have.
- Fill mask layer only with black.
- Paint with a small white brush (4 pixels worked well for me) over any color spots in the mask layer. Do that at 150-300%. You'll spend some quality time at this. Don't bother fix those color spots right at the edge of the frame. The Gaussian Blur from the edge of the image is going to mess it up.
- Once done with first image, copy-paste Gaussian blur filter with Mask into next source image.
- Check for pesky color spots in second image and refine mask.
- Copy-refine for the the rest of the images.

Open one of the tif source images. Now start macro recording and paste the final version of the Gaussian Blur live filter with mask on top of the image layer. Stop macro recording and save it as something like "UV prep". The cool thing in AP is, that the live filter with mask is saved in the macro step. So the macro does not paste from computer memory, but the stored filter-mask. That means even if you quit the application, the Blur-Mask is still stored. Super cool.

In Open-batch, select the Macro, select your output format (e.g., tif for Zerene), add files, and press OK [Another cool thing in AP, saving/export is not part of macros, but is in the batch dialog. No more infuriating confusions about save functions as in PS]. I am extremely impressed with AP batch processing. All 12 cores on my MacPro desktop go continuously full throttle (100%) for several minutes. I have not seen that with any other application. They really program the batch processing well.

I also tried APs focus merge function, and am quite impressed. PS is worse than useless for serious macro stacking, so most people use either ZereneStacker or HeliconFocus. AP is quite impressive, but Zerene still seems to have the edge. I have not found a way to touch up the final stack layer by layer, as you can in ZS or HF. If I stack with the color artifacts still in place, AP has trouble with proper alignment, while Zerene nails it. Last but not least, tone mapping is much better in Zerene than in AP. This could be fixed before feeding the files into AP.

One problem with APs tif export is, that Zerene only reads them as B&W file (or single channel?). Feeding the same files into AP gives me a color image. Not sure yet what is going on there. The solution is to run the AP cleaned tifs once more through DxO, and feed those secondary DxO tifs into Zerene. Voilà!

Below visible light z-stack of some about 2 mm flowers taken on Stereomicroscope. Oberonia costeriana.
Image

Below UV reflectance z-stack of same plant with method above. No difference, but that is still interesting from a botanical research perspective.
Image

Enjoy. I'm off to the greenhouse taking more images, now that the methods are sorted out.

rjlittlefield
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Joined: Tue Aug 01, 2006 8:34 am
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Re: UV reflectance z-stacking

Post by rjlittlefield »

dlg wrote:One problem with APs tif export is, that Zerene only reads them as B&W file (or single channel?). Feeding the same files into AP gives me a color image. Not sure yet what is going on there. The solution is to run the AP cleaned tifs once more through DxO, and feed those secondary DxO tifs into Zerene. Voilà!
I don't know what's going on with that, but as support@zerenesystems.com I would be interested to explore the problem. In the meantime, try toggling the setting at Options > Preferences > Preprocessing > "Use External TIFF Reader" to see if that affects the behavior.

--Rik

dlg
Posts: 26
Joined: Wed Nov 04, 2015 11:47 pm

Post by dlg »

Hi Rik,

un-checking the external tif reader solved the problem! Thanks!!!

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