Ultra-weak light 1. Delayed Fluorescence

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Lou Jost
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Ultra-weak light 1. Delayed Fluorescence

Post by Lou Jost »

The time constant of ordinary fluorescence is typically so short that it seems instantaneous; fluorescence starts the moment light is shone on the subject, and ends immediately when that light is turned off. There is no temporal separation between the excitation light and the fluorescence light. That means you can't easily use broad-spectrum light to excite fluorescence if the spectrum of the excitation light includes the wavelengths of the fluorescence light. The reflected excitation light will overwhelm the fluorescence.

But it turns out that a very weak fluorescence signal continues for a few tens of seconds after the excitation light is turned off. This is called "Delayed Fluorescence" or "DF". It is far too weak to be seen with our eyes. It more than 1000x weaker than regular fluorescnce.

For no good reason, I had to see this. So yesterday I built a contraption to photograph it. I had lots of unusual fast glass that has been gathering dust--- X-Ray lenses and the like. I also had some very fast f/1.2 Oly Pro lenses for MFT. After preliminary tests, I found my Olympus 17mm f/1.2 lens provided a good balance of speed and image quality. I took my cooled-sensor astrophotography camera and built a tower out of a lens hood, a focusing helicoid, the lens, and step-up and step-down rings, carefully chosen to provide the right flange distance with the astro camera, which does not support MFT lenses. I put the astro camera on top of the tower. The helicoid moves the tower up and down to focus on the subject.

The tower sits on a piece of glass (covered in aluminum foil and black flocking paper except for a small hole where the subject will be) which rests on a Cineo box light whose front panel can be removed and replaced with a "shutter" cut from black flocking paper. The idea is to briefly illuminate the subject by turning on the Cineo light, and the turning off the light and inserting the flocked "shutter" so that the afterglow of the LED lights in the Cineo will not overpower the very weak DF signal.

I then put a leaf fragment on the glass and put the tower on top of the glass+ leaf fragment. The key to success is heavy light baffling to prevent ambient light leaking into the target area. After the leaf and tower were in place I wrapped everything in aluminum foil and piled my dirty laundry on top of it.
_1210330.jpg
After some improvements to the lightproofing, it worked! I did about 50 on-off cycles and averaged the fifty photos (using Photoshop layers with the nth layer's opacity set to 1/n; this gives equal weight to all layers) to get this final photo. I colorized the original B&W photo using the normal color of the fluorescence of this particular species (red). Exposure was 7 seconds at maximum gain, aperture f/1.2.
comp2v4.jpg
I was taking continuous 7 second shots, and I could see that the DF had mostly faded after 14s, and was undetectable after 21 seconds.

I didn't need to cool the sensor, though that probably would have helped. If I had a faster lens I might be able to use an ordinary color camera. Stay tuned.

Some reading:
https://www.researchgate.net/publicatio ... uorescence

rjlittlefield
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Re: Ultra-weak light 1. Delayed Fluorescence

Post by rjlittlefield »

Very nice!

I was curious what is the difference between this effect and phosphorescence, so I went off to do some reading.

According to Wikipedia,
In a general or colloquial sense, there is no distinct boundary between the emission times of fluorescence and phosphorescence (i.e.: if a substance glows under a black light it is generally considered fluorescent, and if it glows in the dark it is often simply called phosphorescent).[1] In a modern, scientific sense, the phenomena can usually be classified by the three different mechanisms that produce the light, and the typical timescales during which those mechanisms emit light. Whereas fluorescent materials stop emitting light within nanoseconds (billionths of a second) after the excitation radiation is removed, phosphorescent materials may continue to emit an afterglow ranging from a few microseconds to many hours after the excitation is removed.[2]

There are two separate mechanisms that may produce phosphorescence, called triplet phosphorescence (or simply phosphorescence) and persistent phosphorescence (or persistent luminescence). Triplet phosphorescence occurs when an atom absorbs a high-energy photon, and the energy becomes locked in the spin multiplicity of the electrons, generally changing from a fluorescent "singlet state" to a slower emitting "triplet state". The slower timescales of the reemission are associated with "forbidden" energy state transitions in quantum mechanics. As these transitions occur relatively slowly in certain materials, absorbed radiation is reemitted at a lower intensity, ranging from a few microseconds to as much as one second after the excitation is removed.[3]
Reference [2] then adds that
IMMUNOASSAYS, TECHNIQUES | Luminescence Immunoassays
G. Gübitz, M.G. Schmid, in Encyclopedia of Analytical Science (Second Edition), 2005

Theory of Luminescence
Luminescence can be created by photoirradiation, which results in fluorescence, delayed fluorescence, and phosphorescence or by a chemical or biochemical reaction, which produces chemiluminescence and bioluminescence. Molecules can absorb energy, whereby electrons are excited from the ground state to a higher state. By returning of the electrons to the ground state, the absorbed energy can be turned to radiation as fluorescence. If the transition to the ground state occurs through a metastable triplet state, phosphorescence is observed. Some species, such as lanthanides, show delayed fluorescence, which is a result of two intersystem crossings, the first from the singlet to the triplet and the second from the triplet to the singlet state.
Putting all this together, the picture I get is that almost all of the excited electrons in your subject drop immediately to ground state, perhaps a matter of some nanoseconds, while a few of them get trapped in a metastable triplet state from which they decay with a much longer half-life of a few seconds.

I am curious to know if the spectrum of the delayed light is the same as that of the immediate fluorescence. Do you have any information about that?

--Rik

Lou Jost
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Re: Ultra-weak light 1. Delayed Fluorescence

Post by Lou Jost »

Rik, glad you found it interesting. I think one difference between plant DF and "phosphorescence" as it is usually understood is that plant DF can actually increase over time! There is often a local maximum a few seconds after the excitation light is turned off.

As far as I know, DF is the same color as ordinary chlorophyll fluorescence. I think there would not be any of the other colors normally seen in regular fluorescence ("prompt fluorescence").

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Re: Ultra-weak light 1. Delayed Fluorescence

Post by rjlittlefield »

Lou Jost wrote:
Mon Mar 28, 2022 6:10 pm
There is often a local maximum a few seconds after the excitation light is turned off.
How strange! Local maximum after a few seconds sounds like an interaction with some chemical process. Is the mechanism known in detail?

--Rik

Lou Jost
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Re: Ultra-weak light 1. Delayed Fluorescence

Post by Lou Jost »

Not well known. Check out the introductory review I cited in the post, but much of it is beyond my chemical knowledge. The second peak seems to depend very much on the chemical environment in the cells.

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Re: Ultra-weak light 1. Delayed Fluorescence

Post by rjlittlefield »

Lou Jost wrote:
Mon Mar 28, 2022 6:24 pm
Check out the introductory review I cited in the post
How embarrassing... I thought that I could not access that paper without a ResearchGate membership, which they have not been fast about approving. But I finally realized, that particular paper is publicly accessible even without membership.

Yes, the paper tells exactly what I wanted to know. Thanks for the reminder!

--Rik

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Re: Ultra-weak light 1. Delayed Fluorescence

Post by Lou Jost »

I wonder if the website is designed to make someone think they need to join....

Lou Jost
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Re: Ultra-weak light 1. Delayed Fluorescence

Post by Lou Jost »

Going back to your earlier question about the difference between DF and phosphorescence, phosphorescence decays steadily over time after the excitation light is turned off, but DF drops almost instantaneously to a low value after the excitation light turns off, and then stays more or less steady for some seconds. So it is a more complex process than phosphorescence.

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Re: Ultra-weak light 1. Delayed Fluorescence

Post by ray_parkhurst »

LED and fluorescent light phosphors have a fairly long time constant and a visible decay. I'm reminded of glow balls that my dogs love to chase. Very, very long time constants. Lots of stored energy.

Lou Jost
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Re: Ultra-weak light 1. Delayed Fluorescence

Post by Lou Jost »

Yes, that's why it is important to use a shutter in front of any light used to excite delayed fluorescence. As a precaution, to make sure that I am not recording that afterglow and misinterpreting it as DF, I place the light behind the subject. Any afterglow will make the background brighter than the leaf fragment. Because the backgrounds are black in my pictures, I know I am recording DF rather than afterglow.

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Re: Ultra-weak light 1. Delayed Fluorescence

Post by rjlittlefield »

rjlittlefield wrote:
Mon Mar 28, 2022 5:59 pm
Putting all this together, the picture I get is that almost all of the excited electrons in your subject drop immediately to ground state, perhaps a matter of some nanoseconds, while a few of them get trapped in a metastable triplet state from which they decay with a much longer half-life of a few seconds.
That seemed like a reasonable inference, but it's not even close to what actually happens.

According to the document that Lou linked:
The difference between the more widely known prompt chlorophyll fluorescence and delayed fluorescence is in the origin of the excited single state of the emitting pigment molecule. Delayed fluorescence originates from the repopulation of excited states of chlorophyll from the stored energy after charge separation, whereas prompt fluorescence reflects the radiative de-excitation of excited chlorophyll molecules before charge separation. This is why delayed and prompt fluorescence contain information about different fundamental processes of the photosynthetic apparatus.
...
Delayed fluorescence is affected by many chemical and physical variables, such as ATP, proton gradient in the thylakoids, chill stress, different xenobiotics, excitation light spectral and intensity characteristics, cell culture growth stage, to name only some of them.
So yeah, it's way way more complicated than described in the Wikipedia article and its reference.

I am now interested to see that Google searches on wikipedia "delayed fluorescence" chlorophyll and site:wikipedia.org "delayed fluorescence" chlorophyll find almost nothing. There is one sentence at https://en.wikipedia.org/wiki/Govindjee (a biographical article) that "Govindjee has not only provided the first comprehensive theory of thermoluminescence in algae and plants, but has shown how prompt and delayed fluorescence of Chlorophyll a can be used as a signature of various reactions in photosynthesis." Searches within Wikipedia find nothing more

So, delayed fluorescence in photosynthetic systems seems to be a concept that Wikipedia has essentially missed!

--Rik

Lou Jost
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Re: Ultra-weak light 1. Delayed Fluorescence

Post by Lou Jost »

So, delayed fluorescence in photosynthetic systems seems to be a concept that Wikipedia has essentially missed!
Photographers also seem to have missed it. There are very few delayed fluorescence images on the internet....The image in this thread seems to the only one on any photography website.

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Re: Ultra-weak light 1. Delayed Fluorescence

Post by Scarodactyl »

For such a strange and marginal phenomenon, not yo mention the practical challenge, it amazes me that such a nice image is possible. I'm impressed!

Lou Jost
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Re: Ultra-weak light 1. Delayed Fluorescence

Post by Lou Jost »

Yes, I was surprised I didn't have to cool the sensor. But it did take summing (not stacking) fifty images as in astrophotography.

dickb
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Re: Ultra-weak light 1. Delayed Fluorescence

Post by dickb »

Nice to see other people also using an overdose of stepping rings to connect things that weren't intended to be connected together.

You mention using an astro camera here - does the short register allow you to mount your fast X ray lenses in a way that results in infinity focus? Mine can't on my Sony E mount cameras as for most their rear element housings are too wide to be mounted deep enough into the E mount. And the few that can (TV Heligon 42/0.75 I think) need to be mounted so close to the sensor that they hit the mechanical shutter. Since the QHY163 is electronic shutter only that shouldn't be a problem.

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