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Smilax rotundifolia

Fluorescence UV Lighting
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#1 Mark

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Posted 17 May 2017 - 13:56

In a previous thread (http://www.ultraviol...ation-in-uvivf/) I established that my UV source contributes practically no VIS to my UVIVF images. So, I still have no idea why I am seeing so much of what looks like VIS contamination in my UVIVF images (versus what should look like strictly fluorescence, whatever that case may be).

To give you an example I shot this Greenbrier using my usual go-to setup for UVIVF images. This includes a triple-filter stack on the camera lens (two longpass filters - one with a cut-on at 425nm, and another with a cut-on at 435nm, and a Baader UV/IR cut on the top). The UV source is an MTE-U303, filtered via Hoya U340. I also took a second shot with the addition of an AndreaU-MKII filter (so, Hoya U340 + AndreaU-MKII stack) to help cut out any VIS that might be getting through the U340 alone.
Attached Image: uvivf-VIScolortest11.jpg

The leaves show the tell-tale far-red fluorescence of chlorophyll - but, that green stem. Its green(ish) - in VIS, and UVIVF?! (a little more towards blue in the UVIVF images). That just doesn't seem right. Seems to me that something is illuminating it and overwhelming my UVIVF image.

Also to note, aside from a slight decrease in luminosity/fluorescence intensity, the two filtration options look the same. Which tells me that the choice of filter isn't an issue; and probably that the UV LED source isn't pushing VIS through the filter(s).

To check for background/ambient illumination which could account for this contamination I took a control shot under the same exposure settings. And it is dark. Black. The top half of the image is SOOC, and the bottom is with pushed development settings in PP (pushed to max exposure compensation, and +50% brightness in NX-D). Virtually nothing there; which was good to see.
Attached Image: control1.jpg

I tried again with more of a focus on the stem itself (no pun intended). The results were even more dramatic here, in that the stem looks practically the same color green in all shots - except, here I noticed (after shooting) in the corners of the image that different parts of the stem react differently. Note the main stem with the thorns looks green/VIS contaminated - but, there are two shoots (in either corner) that are fluorescing blue(ish).
Attached Image: uvivf-VIScolortest21.jpg

So, are my UVIVF images VIS contaminated? If so, where is it from? If not, why does that main stem look green(ish) in both the VIS and UVIVF image? Could it actually be fluorescing green? What may be going on here?

Camera
- Nikon D750 [broadband] + 50 mm Nikon series E lens + 20 mm extension tube
Lens filters

425 nm longpass + GG435 longpass + Baader UV/IR [stack]
Illumination/Irradiation
- VIS: Sigma EF-500 DG Super flash

- UVIVF: [MTE-U303 + U340 / AndreaU-MKII]
Exposures
- ISO320 / f11 / 15s

#2 Cadmium

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Posted 18 May 2017 - 07:30

Mark, the two stems look different colors in visual also... that same stem is also more green visually. And I notice that further up that stem is a newer growth that also looks blue like the other stem...
With your eyes, do they look the same in UVIVF as they do in the UVIVF photos? One more green?
I trust my eyes. Perhaps not all our eyes see the same colors visually, but still...

Edited by Cadmium, 18 May 2017 - 07:31.


#3 Mark

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Posted 18 May 2017 - 12:13

@Cadmium: You're right, the two stems do look differently colored in VIS. I think its the shoots with leaves which look blue'ish, while the main stem sections with thorns look green. At this point my guess would be that the thorny stems are strictly structural and unlike the shoots, do not contain chlorophyll (thus do not fluoresce). And I'll mention also that those thorny stems, aside from being protective for the plant, are amazingly resilient... I cut this length with one of my keys (didn't have a clipper on me at the time), and it was very much a hack cut. I then folded the stem in half and stuffed it in my pack until I got home later that day. When I took it out it looked understandably twisted/mangled; though I only needed a small section to shoot so it didn't matter much to me. I stuck the stem in bottle of water as I set up to shoot it. Twenty minutes later it had risen back to its former state, leaves shining, tendrils reaching out. I was impressed. It took a beating and came back swinging like nothing had happened. In fact, I still have it in the water, and it still looks like its ready for anything.

To my eyes (behind UV blocking glasses) the stem looks nearly the same as it does in VIS. I'm actually quite surprised at how much VIS my UV LED source seems to produce (which is case and point my concern in all this). Practically speaking, I know the UV LED source isn't projecting much of any VIS at all, especially through the U340 filter I have installed on it. Which makes me wonder, why, in an otherwise darkened room, why does it look like there is a ton of VIS shining on my subjects under my UV source? I keep coming back to the conclusion that it must be fluorescence from the subject itself. Its just that, at least in this case, I'm surprised to see the same(ish) colors in UVIVF as in VIS. I've even made it a point to remove as much dust/lint as possible from the filter on the UV source, since when UV hits that lint it fluoresces blue-white strongly - which I suppose can illuminate my subjects within a 15 second exposure.

I'm not done with this yet. This UV LED source is still relatively new to me, so I'll have to keep trying it on other subjects too and then see what comes of it.

#4 Andrea B.

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Posted 18 May 2017 - 13:57

Do you have another UV illumination source with which to make comparisons?

I keep coming back to the conclusion that it must be fluorescence from the subject itself.
I think you need to use some subject for which you know the fluorescence in advance. For example, try one red-fluorescing leaf together with a PTFE disk (as suggested previously). If the red chlorophyll fluorescence is producing enough light to illuminate other subjects, you will see that on the PTFE disk. Try several shots with the PTFE disk closer/further from the leaf.

It is very difficult for me to think that the red fluor in your photos would illuminate the green stem and it would still look green. Occam suggests that either the stem fluoresces or you have vis light from somewhere??

I'm sure you will get to the bottom of this little mystery. It's quite interesting!!
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#5 Andrea B.

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Posted 18 May 2017 - 17:33

The viewfinder is closed????


Do those longpass filters fluoresce??

I'll dig mine out and see if I can replicate this.
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#6 Andrea B.

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Posted 18 May 2017 - 18:12

In the course of my experiment I just (re-)discovered how very easy it is to get a bit of torchlight reflecting back into the lens wreaking all sorts of havoc. Lens shade is helpful in preventing this.

Do you think you could accidentally be getting torchlight directly into the lens or reflecting off something back into the lens?

These UV-LED torches are strong. I cannot block my UV-LED with 4 stacked Baader UV/IR-Cut filters plus 2 extra longpass if the torch is shined near the edge of the lens or if reflection is strong back into the lens.
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#7 Mark

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Posted 19 May 2017 - 17:38

Unfortunately, I'm scrambling to get ready for a business trip right now, so I don't have time to go out and get a fresh Smilax r. sample - and the one I have has lost much of its vitality. Nevertheless, this test still doesn't answer my question, and at the same time brings up an interesting point (pun to be explained below).

This test scene incorporated a snippet of the thorny Greenbrier stem imaged previously, a fresh green leaf from some random weed near the edge of my yard, a 0.5" diameter white PFTE target which I've used in the past for WB'ing UVIVF images, and a 1.5" diameter stainless steel impregnated PTFE (SS-PTFE) target which I will strongly consider using from now on for both WB and stray light contamination/control confirmation in my UVIVF images.

In the VIS image you can see that the thorny vine in question is currently turning brown, at least partially. Something to note here on that vine... near the far right, just before the edge/limit of the image, you may notice a very slight/subtle indentation on the vine. This is the spot where I had held it in the teeth of a spring clip while previously photograhing it. Hold that thought.
Attached Image: tmp_5479-05-19-2017_00-20-281819265460.jpg

In the UVIVF image cholorphyll fluorescence is bright and clearly dominates the coloration of the leaf, as expected and hoped for. WB here was set from the central, white PTFE disk (I still have a bit of testing to do with the other, SS-PTFE disk, as the two produced slightly different WB results). The SS-PTFE disk is dark in this image - indicating that not only is it not generating fluorescence, but also that it is not being illuminated by any stray light... not even the light from the chloropyll fluorescence. Only one minor exception there: if you look at the broken thorn just in front of the PTFE disks you will see on it a very brightly fluorescing bit of lint. So bright in fact that its fluorescence does faintly reflect off the SS-PTFE disk. This tells me that induced fluorescence must be very strong to have any secondary illuminating effect in the scene - certainly it is not to blame for the overall question in this thread.
Attached Image: tmp_5479-05-19-2017_00-03-24-111173511.jpg

So, still there remains the question of why this stem looks green - of course, less so in this image given its condition, but still, to the point... it looks practically the same in my UVIVF image as it does in the VIS image. The SS-PTFE disk and the metal clip holding it both look dark/not-illuminated, save for the trace of light from all the lint/dust on them. This shows that nonfluorescent things look, well, they don't look like anything. They're dark/black, as they should be. Which brings me back to the stem, and the why?, and the searching for an answer.

If the stem is fluorescing, okay, great. I'll take that explanation - but I'd be surprised, because of the color. If it is not fluorescing I'll also be surprised, because I can't imagine where in the world its illumination is coming from - given that this test shows that non-fluorescent things show as non-fluorescent in my UVIVF imaging, and that induced fluorescence in the scene is practically not capable of secondarily illuminating other objects in the scene.

If anyone else out there has these thorny Greenbriers near home I would be very interested to see what kind of results others get from UVIVF imaging this.

Oh, about that 'point' I mentioned... One of the things I found interesting in this test is the point of the one thorn which is still intact, seen in lower center of the frame. In the VIS image the tip is a dark, reddish brown color - but in the UVIVF image it looks like a bright yellow.

Also, the area on the stem where I had previously held it in the teeth of a spring clip... that spot, where the cuts/"bite marks" are left from the clip - they appear to be fluorescing, that same tell-tale red of chlorophyll. So, the inside contains chlorophyll which fluoresces red when exposed, but the outside fluoresces green(ish)? Or there is a mystery light illuminating just the stem, every time I shoot it, in any configuration?

P.S. I covered the camera and viewfinder during this test, thus no stray light got into the camera. And the room was fully darkened (no computer/other lights on anywhere in the room). And, only one of the two longpass filters in my stack fluoresces under UV - but in my stack it is sandwiched in the middle (I've verified already in past tests it does not fluoresce in this configuration).

Edited by Mark, 19 May 2017 - 17:46.


#8 Andrea B.

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Posted 20 May 2017 - 16:23

Excellent test. Interesting results. Good conclusions.
(Covered camera & viewfinder - good Dealt with the fluorescing filter - good.)

Yes, I think that this stem likely does fluoresce -- maybe from chlorophyll, maybe from some other pigment, we don't know -- and that the outer layer of the stem is changing the intensity & coloration of the fluorescence.

The outer layer of a stem may be bark-like or there may be a waxy coating or some other construction. This, of course, can vary. (Consult basic botany book.)

Clearly the previously clamped, damaged area of the stem shows underlying chloro fluor. So it would seem that the next step would be to take a very sharp knife and scrape the stem very lightly to remove some or all of the outer layer on one area of the stem. Then re-photograph it to see what you've got. And perhaps also photograph a cross-section of the stem.
Andrea G. Blum
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#9 Andrea B.

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Posted 20 May 2017 - 16:45

U r busy. I look up 4 u in Wicked Pete.

The epidermis is a layer of cells that covers the leaves, flowers, roots and stems of plants.
Woody stems may have a secondary covering called the periderm. Epidermis and periderm are protective layers.

Epidermal cells are tightly linked to each other and provide mechanical strength and protection to the plant. The walls of the epidermal cells of the above ground parts of plants contain cutin, and are covered with a cuticle. The cuticle reduces water loss to the atmosphere, it is sometimes covered with wax in smooth sheets, granules, plates, tubes or filaments. The wax layers give some plants a whitish or bluish surface color.

The cortex is the outermost layer of the stem or root of a plant, bounded on the outside by the epidermis and on the inside by the endodermis. --->>> Some of the outer cortical cells may contain chloroplasts. <<<---

Bark is consists of the outermost layers of stems and roots of woody plants. Plants with bark include trees, woody vines, and shrubs. Bark is a non-technical term.

******

Your stem doesn't seem woody in the sense of having bark? So I think that it is probably the epidermis and cuticle and any wax which must be scraped off to expose the underlying chlorophyll.

I don't have Greenbrier nearby. But also I don't want to interfere with your Greenbri3r experiment. I might try this on some other vine though.

We look forward to your return from the business trip to continue this interesting experiment!!
Andrea G. Blum
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#10 JCDowdy

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Posted 22 May 2017 - 23:09

Still not getting why your PTFE is showing up white. Seems to me that unless it is emitting UVIVF it should be dark.