Fleeting fern fluorescence

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Lou Jost
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Fleeting fern fluorescence

Post by Lou Jost »

Red and infrared chlorophyll fluorescence are conspicuous components of UV-excited fluorescence in plants. Because chlorophyll is part of a complex photochemistry system, which evolved to capture light and turn it into plant food, its fluorescence is not passive but active; it depends on what the whole system is doing with the excitation photons. The excitation photons produce an excited state, whose energy can be lost by, among other things, making food or by emitting a fluorescence photon. In addition, plants have feedback mechanisms that control these processes, affecting the fluorescence. The variation of chlorophyll fluorescence with time is a major subject of investigation in biology because it gives clues about the mechanisms of photosynthesis.

But for us photographers, the practical effects of this variation are more important than its scientific meaning. For reasonable photographic levels of UV illumination, chlorophyll fluorescence fades very fast, much faster than the time it would take to do an ordinary stack.

My fastest stacking method is to use the camera's own focus-bracketing function and an MFT camera, in this case the G9. This method requires the use of a native MFT tube lens. The choices are very limited, but I have found the Panasonic 45-175mm zoom to be ideal, because it has internal focusing and internal zooming, and covers a good range. Like most zooms used as tube lenses, it vignettes quickly when the focal length is reduced from its maximum, but at least it gives a little freedom when framing an image.

Here I am using it with the Mitutoyo QV 2.5x 0.14 objective, which has become one of my favorites for this purpose. I used 0.5s shutter speed at ISO 400, with no delay between photos as the camera ran the stack. I turned on the UV light about 1s before I started the stack. I under-exposed to shorten the stacking time. Here are the first seven frames of the stack, and the last frame:
matrix.jpg
And here is graph of luminance (measured in Photoshop) versus time from my experiment. Edit: Note this graph is only rough. The later times might be off, due to buffer filling at some point. But the first part is accurate since the buffer is not filled.
Lum vs time.jpg
The initial red chlorophyll fluorescence is already much dimmer after just 4 seconds, and after 20 or 30 seonds it drops to about 1/5 its initial value (as measured by the L value in the Lab representation of the color in the image). From this point the intensity of the fluorescence reaches an almost stable level, decreasing very slowly and linearly. After 110s, the fluorescence drops to 50% of its value at 20s. That's as far as I needed to go for this stack. Note that the blue non-chlorophyll fluorescence did not decline much over this period.

How can we compensate for this rapid decline? Stacking by focus bracketing (tube lens refocusing) is very fast and works for low m, but not for high m because there is not enough focus throw. Also, it always runs from near to far rather than from far to near, while the chlorophyll fluorescence in typical fern photos is in the background. In my completed stack above, I used Zerene's "Brightness" adjustment function to adjust all the later images in the stack to match the brigthtness of the first image. That seemed to help. Here is the complete stack, with heavy post-processing:
2021-05-23-23.33.59 ZS PMax UDRv2.jpg
Perhaps readers who have used flash for fluorescence can comment on whether a strong short flash burst has less effect on fluorescence decline than contiuous light that contains the same amount of energy. Certainly if the bursts were spaced long enough for the fern to re-adapt to darkness, then we could capture the full brilliance of the chlorophyll fluorescence in a stack, but by the time the stack is done, the leaf will be wilted or, if underwater, will begin to rupture its cells. But maybe a really short burst might not trigger some of the fluorescence-quenching mechanisms of the plant. If so, this would be the best way to capture fugitive fluorescence. An extra advantage is that this technique can be used with ordinary stacking by rail, since there is no light during rail movement. Another advantage is that this method can be arranged to start the stack at the farthest part of the subject (where the chlorophyll usually is) rather than the nearest. A stack controller that turned off the light between shots would also allow rails to be used.

A similar principle might be useable even with continuous light, if we had a very bright light and we could do an entire stack in less than four seconds. This is actually possible if the stack depth is less than the number of images that the camera buffer can store. My Panasonic G9 can store about 50-60 images in the buffer, so if the shutter speed were very fast, we could do a stack of 50 images in one second. But this might backfire, because the exitation light would have to be very strong to give the same exposure, and this might bleach the chlorophyll just as much as in the slower method. Later I will do the experiment and report the result, unless some reader already knows the answer.
Last edited by Lou Jost on Mon May 24, 2021 1:27 pm, edited 1 time in total.

viktor j nilsson
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Re: Fleeting fern fluorescence

Post by viktor j nilsson »

Haven't thought this through at all but I'll just throw it out there: would it be possible to compensate for the photobleaching effect by starting out at a lower excitation intensity, and ramping it up over the course of the stack? Just like people here build flash trigger circuits, I could see someone building a UV fluorescence trigger unit.

Edit: forgot about the fact that the blue excitation didn't change as much, that would be a problem.

Lou Jost
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Re: Fleeting fern fluorescence

Post by Lou Jost »

"would it be possible to compensate for the photobleaching effect by starting out at a lower excitation intensity, and ramping it up over the course of the stack?"
Not a a bad idea, but as you say, other things don't bleach at the same rate. In fact, the change in chlorophyll fluorescence is not due mostly to bleaching in the normal sense, but due to active processes in the chloroplast.

Lou Jost
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Re: Fleeting fern fluorescence

Post by Lou Jost »

I made two UV flashes this afternoon...when night falls I will be able to see whether flashing helps these stacks or not. Stay tuned. Theoretically I think it should work better than continuous light.

Lou Jost
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Re: Fleeting fern fluorescence

Post by Lou Jost »

I tried my home-made flashes, but they need more work before they can match my flashlights in power. I need some way to collect their very unfocused light and concentrate it on my subject. I do not see any decline in frames during the first 16 shots, but the cumulative energy delivered to the subject may not have matched the cumulative energy delivered between the first and fourth shot of my continuous-light test. Still, somewhat encouraging...

micro_pix
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Re: Fleeting fern fluorescence

Post by micro_pix »

Excellent Lou!

Dave

Lou Jost
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Re: Fleeting fern fluorescence

Post by Lou Jost »

Thanks Dave. After a bit more testing, I see that my home-made UV flashes are not only weak, they are also leaking lots of red (and IR?) light. My UV-pass filters, which I have mounted on the flashes, are the kind that also pass IR. This never mattered when I was using a good UV LED, but it does matter when one tries to pick the UV light out of a source which emits primarily in the visible spectrum. :cry:

Pau
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Re: Fleeting fern fluorescence

Post by Pau »

Lou, this is a very interesting thread, and an excellent stack,BTW
After a bit more testing, I see that my home-made UV flashes are not only weak, they are also leaking lots of red (and IR?) light. My UV-pass filters, which I have mounted on the flashes, are the kind that also pass IR. This never mattered when I was using a good UV LED, but it does matter when one tries to pick the UV light out of a source which emits primarily in the visible spectrum.

This is normal when you use a light source other than LED. Al my old Zeiss illuminators and illluminator filter sets designed for UV Schott black absorption filters like UG1 or UG5 have IR filters* both for protecting the other filters from heat and also I assume because UG filters allow to pass far red and IR.

Complementing your filtering with IR cut could solve it, I guess.

* Schott KG01 and BG38
I've collected some info on filters that could be useful, PM me if interested
Pau

Lou Jost
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Re: Fleeting fern fluorescence

Post by Lou Jost »

Thanks Pau. I'm looking for a good IR-cut filter right now. I think it might be most useful on the camera rather than the light source, since it may not have 100% transmittance in the UV. This would be most economical since I would only need one, rather than one for each flash. The best curves I have found so far for a camera IR-cut filter is the Astronomik L-3 filter, which also has the advantage of blocking UV (of course, this would not work on the flash!). I ordered one and it will arrive in a month or so. I would also be very interested in your recommendations. Will PM you.

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