Hi guys
Images are stacks of 5 photos each. W.B.5000K. Brightfield 5x and 10x and Fr-TIR 5x and 10x.
There is an interesting reddish reflection in the Fr-TIR images. Could it be Kramers-Kronig anomalous reflectance?
Chlorophyll absorbs at red (680nm) and blue (430nm). So, under normal light we have green. With Fr-TIR grazing angles we almost have no bulk absortion and backscattering. Therefore reflected light due K-K anomalous reflectance at 680nm-430nm will be visible. And will be the sum of red and blue.
Indeed we have some reddish reflection that could be red+blue. But there is also some green colour (in Fr-TIR), why?
Please guys, help me understand what we are seeing.
5x FrTIR
5x small.jpg
5x Brightfield
5x small.jpg
10x FrTIR
10x small.jpg
10x Brightfield
10x small.jpg
Best RGDS
Ramon
Hi Ramon,
I am not sure that there is something like Kramers-Krönig anomalous reflection. This might be the "Restrahlen" reflection. Generally, reflectivity is high where either the difference of the real and/ or imaginary part (extinction coefficient) of the index of refraction of the sample and the embedding medium is high. You then see either the usual absorption colour, if the colour impression is due to light transversing the sample, or the complementary colour, if it is due to light reflected from the surface.
I attach a picture of a streak of a red overhead marker. On a white paper, one mainly sees the red absorption colour, on a black background, the reflective Restrahlen colour.
Kindly
Florian
Hello Ramon,
Since I'm scratching my head for some while about the strange green phenomenon in some of your pictures, Florian explanation sounds reasonable.
As far as I can tell, all the pictures with this "green stuff" are already mounted slides.
All with a permanent mounting medium, right?
Greetings Holger
Hi Florian and Holger
I also have been scratching my head around this for some days :D . That is why I delayed my answer to Florian. I still dont have it 100% but I am getting close.
Florian introduced a very important point, the relation of the index of refraction of the sample and the embedding medium. This is a key factor I had overlooked.
Yes Holger, the "green stuff" we seeing is from commercial samples mounted on slide. Bought online in Amazon and the like.
Please guys, bear with me. I hope tomorrow I can present you with a proper answer.
As a spoiler I attach two plots
R(reflectivity) over n for different K values.
R_vs_n_asymmetry.jpg
R(reflectivity) over d (d=n-n0) for different K values.
R_vs_d-web.jpg
The mathematical expression key to this argument is the Fresnel equation:
R= (n-n0)^2+k^2/(n+n0)^2+k^2
Ramon
Hi Holger and Florian
The graphs of the post above explain the reddish color we see in the Mullein Verbascum. Not the "green stuff"!!!
Right now I am taking photos of the Fern Leaf and Inflamated Lung with Green and Blue light. This can confirm what was always suspected. That the "greeen stuff" is fluorescence.
Asap I have the results I will post them. I am having trouble matching what is recorded with the camera with what I see through the eyepiece.
Ramon
Hi Holger and Florian
Regarding the "green stuff" I attach images of the Fern Leaf and the Inflammated Lung under green and blue illumination.
I woul love to hear your comments.
Fern-Leaf-10x-Green-LED-500-525nm.jpg
Fern-Leaf-10x-Blue-LED-452-455nm.jpg
Inflammation-of-the-Lung-5x-Green-LED-500-525nm.jpg
Inflammation-of-Lung-5x-Blue-LED-452-455nm.jpg
I am not happy with the quality of the images. There was a considerable differece between what I saw through the eyepiece and what I photographed. This is the closest I can get to the image through the eyepiece (its pretty close).
Regarding the Mullein Verbascum branched leaf hairs I am still preparing the papeer. As soon as I have it I will post it.
Ramon
With covelline you can nicely see how refection colour changes with the index of refraction of the immersion medium:
https://www.mikroskopie-forum.de/index.php?topic=25232.msg188478#msg188478
Hi Florian
I dont understand why you need an immersion medium to photograph the covelline. What was your setup for the images you posted in https://www.microscope-forum.de/index.php?topic=25232.msg188478#msg188478?
Ramon
Hi Ramon,
in ore microscopy, you make use of the reflectivity change using an air objective or an oil objective to determine the minerals. In the link, I used an air objective, and an oil objetive with water, oil and bromo-naphthalene, because the change in reflection colour is spectacular. The index of refraction shows strong dispersion, so that with different media, the difference n_covelline(lambda) - n_medium(lambda) goes through 0 at different wavelength, where reflection becomes then minimal.
For some strange reason, I can see the four pictures on my smartphone, but on my computer, only two of the four are displayed.
Kind regards,
Florian
Hi Florian
Got it. I am thinking how would covelline look with the FrTIR device using different immersion media.
You can find the four images in the link:
https://drive.google.com/drive/folders/17zpLael8OpDQ5c37_7FOTJiKIPID75xI?usp=sharing
RGDS!
Hi Ramon,
you seriously confused me! I #7, you changed the link address form "www.mikroskopie-forum.de" to "www.microscope-forum.de". Took me quite some time to work out why the link wasn't functional.
Kind regards
Florian
Hi Florian
Big sorry!!! I copy/paste from post #6 and I didnt double check.
I will be more careful.
B RGDS
Ramon
Hi guys
Regarding the Anomalous Reflected Colours under Frustrated TIR in the Mullein (Verbascum):
From the Kramers-Kronig relations and the Fresnel equation we can predict a specific optical effect: every material with a strong absorption band also has an ENHANCED SURFACE REFLECTANCE at wavelengths close to that band. The reflected colour is therefore governed by the absorption band, and is approximately complementary to the transmission colour.
The optical response of a material is described by the complex refractive index:
ñ(λ) = n(λ) + i·k(λ)
• n(λ) - the real part: refraction and DISPERSION (how much light is slowed and bent).
• k(λ) - the imaginary part: ABSORPTION (how strongly light is extinguished).
Reflectance at an interface is set by the contrast in COMPLEX index between the material and its surrounding (embedding) medium - mountant or immersion oil, not air:
R(λ) = [ (n(λ) − n₀(λ))² + k(λ)² ] / [ (n(λ) + n₀(λ))² + k(λ)² ]
Two things follow. First, at fixed n, R increases with k - so absorption alone raises reflectance. Second, even at PERFECT index match (n = n₀) the reflectance is not zero:
R(n = n₀) = k² / (4n₀² + k²)
So a strong absorber embedded in an index-matched medium still reflects (k = 1 gives 10%, k = 3 gives 50%). The reflection is also strongly ASYMMETRIC in the index contrast d = n − n₀: R rises steeply when n < n₀ (reaching 1 at n = 0), but only very slowly when n > n₀. This explains why under Fr-TIR air bubbles in the sample or in the immersion oil film are so reflexive. Air bubbles in the immersion oil have ruined me many photos. This part was advanced by Florian when he comented about the difference in refractive indexes between sample and mounting medium.
This is the same criterion that produces the Reststrahlen band in ionic crystals and 'bronzing' in strong dyes.
R_vs_d.jpg
Image: R versus the signed index contrast d = n − n₀ (full range left, zoom right). Note the non-zero intercept at d = 0 (absorption alone reflects) and the strong asymmetry between the rarer and denser sides.
The anomalous reflection is ALWAYS present near the absorption maximum. Then, why brightfield cannot show it? The effect is real but WEAK, and in brightfield it has to compete with a far stronger signal. In normal transmitted-light microscopy what reaches the objective is dominated by: BULK ABSORPTION and VOLUME BACKSCATTER. Both are orders of magnitude stronger than the thin surface-reflected component. For a typical stained specimen under brightfield the transmitted/backscattered light is roughly 50 TIMES stronger than the anomalous surface reflection.
Fr-TIR suppresses the dominant transmitted signal by ~50x and leaves the anomalous reflection standing alone.
The mullein case. Mullein (Verbascum) branched leaf hairs appear uniformly green in brightfield (consistent with fast green staining of the cellulose walls, absorption maximum ≈ 625 nm) and show a REDDISH rim reflection under Fr-TIR. The chromophore absorbs in the red, so its enhanced surface reflectance lies in the red, and the rim-localised (surface, not volume) appearance is what a surface reflection should look like (this part of the Mullein sample is yet to be demonstrated).
RGDS
Ramon
This is how it is, Ramon.
Just one point:
"The anomalous reflection is ALWAYS present near the absorption maximum. Then, why brightfield cannot show it? The effect is real but WEAK, and in brightfield it has to compete with a far stronger signal. In normal transmitted-light microscopy what reaches the objective is dominated by: BULK ABSORPTION and VOLUME BACKSCATTER. Both are orders of magnitude stronger than the thin surface-reflected component. For a typical stained specimen under brightfield the transmitted/backscattered light is roughly 50 TIMES stronger than the anomalous surface reflection."
With polished absorbing samples, as used in ore microscopy, the effect is visible in brightfield illumination and can even be measured quantitatively.
Florian
Hi Florian
Zitat von: Florian D. in Juli 20, 2026, 08:43:18 VORMITTAGThis is how it is, Ramon.
I am glad at last I figured it out. Thanks!
Zitat von: Florian D. in Juli 20, 2026, 08:43:18 VORMITTAGWith polished absorbing samples, as used in ore microscopy, the effect is visible in brightfield illumination and can even be measured quantitatively.
I wish I could photograph ore samples with the Fr-TIR device. Can you let me know how/where can I get hold of these type of samples?
RGDS
Ramon