Greg, that’s very exciting progress. Your new summary of all the measurements that can be taken with a MultispeQ is excellent. It’s the best description yet of the MultispeQ’s capabilities.

One of the statements there relates to a discussion Robert started at the Public Lab Barnraising in Louisiana. You say:

“…here’s a cool infra-red filtered image showing fluorescing trees and grass – notice that the brightest objects (most emitted infra-red) are the leaves!”

Robert was also saying that “most” of the glow of foliage in the near infrared (the Wood Effect) was due to fluorescence. This was new to me, and I have not been able to confirm it. In the Wikipedia article where the image you cite is used, it says:

“… the "Wood Effect," an effect mainly caused by foliage (such as tree leaves and grass) strongly reflecting in the same way visible light is reflected from snow.[1] There is a small contribution from chlorophyll fluorescence, but this is marginal and is not the real cause of the brightness seen in infrared photographs.”

Robert was going to look into this, but I thought I would nudge you guys a little. Below is the photo you refer to. My understanding is that maybe one or two percent of the NIR radiating from sun lit foliage is due to fluorescence and the rest is just reflection. Lots of NIR is also transmitted through thin leaves.

Chris

Phew - Ok, talked with Dave about it to confirm about the reflectance contribution piece, here's my response:

First, yes I think it is misleading to say this picture is describing in any significant way chlorophyll fluorescence, so I'll stop doing that (sorry)! As you said, most of the IR seen in leaves here is just old fashioned reflectance and dispersion on the leaves. That IR by itself doesn't really tell us anything about photosynthesis. I'll replace that image with an image from here in the lab of chlorophyll fluorescence of plants in the chambers which is just the fluorescence response.

Just to clarify, what we are measuring with the MultispeQ is the portion of the IR which the plant is fluorescing (a very small portion of the effect seen above). We can pull out this small portion by pulsing light on the surface of the plant, and using electronic filtering to remove any background IR from the environment. In addition, any reflectance based IR caused by our pulsing LEDs has been minimized (by choosing LEDs with minimal IR signature) and accounted for (by using some fancy calibration schemes).

To confuse this even further is the NDVI measurement, which also has little to do with either things described above. In it, you take an image using a 650nm wavelength bandpass filter (so filter everything but 650nm). This wavelength corresponds to greenness of the leaf (which relates to N and chlorophyll content). In addition, take an image using a 940nm bandpass filter which is a kind of measure of canopy density or leaf thickness, but it's really just acting just as a calibration factor. The 650 bandpass is doing the real work.

Thanks Chris for pushing to resolve this, it's an important point. If there's anything else you think is off, please post and I'll see if I can give or find and answer or fix it if we're wrong!

Thanks Greg, that helps a lot. I'm just getting a feeling for how complicated the fluorescence measurements are. It looks like fluorescence will be key to your measure of "Photosystem II and Photosystem I efficiency" and also maybe "Photosynthetic regulation" and the combined photosynthesis measure. I could not figure out at what wavelength you will be measuring fluorescence -- will it be the same for all of the different fluorescence measurements?

I assume you are measuring fluorescence in the infrared. In the FAQ you state that "Fluorescence, in the case of photosynthesis, is infrared light emitted by the sample in response to being hit with some other wavelength of light." It seems that most of the fluorescence of chlorophyll is not quite infrared, but just long red, with peaks at 690 nm and 740 nm. Below is one example for chlorophyll a in methanol.
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(Source) .
I remember vividly in the first biology class I ever took a demonstration of chlorophyll fluorescence under UV illumination which looked exactly like this:
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Chlorophyll in alcohol (top) and illuminated by UV light (bottom). Source
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The red in the flask above is from that peak at 690 nm. The professor in that biology class explained that the photons emitted from the excited atoms were lower energy (longer wavelength) than the absorbed UV photons due to the second law of thermodynamics (some energy was lost in the transfer). I went up after class and reminded him that the laws of thermodynamics did not apply at the atomic level (I guess I haven't changed much). I wonder if he remembers that moment as well as I do 35 years later (Me: "I guess I impressed him." Him: "Wise-ass kid better watch his step.").