FTIR used on biological specimens mounted on a slide provides a singular illumination pattern that highlights features of the specimen that otherwise would not be visible.
I took this image using a FTIR device that I developed. I call it Dimomo. It is a stack of 20 images.
Together with the FTIR image find the brightfield image.
Find full resolution image at the link: https://drive.google.com/file/d/14n-9r5na_fIIYEH3U8wbO0NVPqceXaVc/view?usp=sharing
B RGDS
Ramon
Silkworm 1 small.jpg
Silkworm bright small.jpg
Hi Ramon,
nice to meet you, i have some questions.
You are observing silkworms.
Normally, they are opaque.
You are using FTIR, or Fourier-transform infrared spectroscopy, a method for obtaining information about the chemical composition.
So you use the lighting from this FTIR device to take better photos and look inside the pupa?
Can't you see through a silkworm with a normal light microscope, or are the reflections too strong?
Or is it more of a kind of reflected light microscopy (epi-illumination)?
Best regards
Rudolf
Zitat von: rlu in Mai 22, 2026, 09:10:44 VORMITTAGFTIR, or Fourier-transform infrared spectroscopy
Bildschirmfoto 2026-05-22 um 14.07.12.jpg
I think these are two different methods ...
Zitat von: rlu in Mai 22, 2026, 09:10:44 VORMITTAGYou are using FTIR, or Fourier-transform infrared spectroscopy, a method for obtaining information about the chemical composition.
Hi Rudolf. FTIR stands for Frustrated Total Internal Reflection. As is pointed out by Peter.
Zitat von: rlu in Mai 22, 2026, 09:10:44 VORMITTAGSo you use the lighting from this FTIR device to take better photos and look inside the pupa?
Since, I think, is a new system of illumination. I am using it on all kinds of samples, be it transparent or opaque.
Zitat von: rlu in Mai 22, 2026, 09:10:44 VORMITTAGCan't you see through a silkworm with a normal light microscope, or are the reflections too strong?
As you can see in the brightfield image of the silkworm there is an area that it is opaque. With a trans illuminated microscope you can not see opaque samples. With the FTIR device I use you can see transparent and opaque samples. For example, I do metallography with my old biological microscope.
Zitat von: rlu in Mai 22, 2026, 09:10:44 VORMITTAGOr is it more of a kind of reflected light microscopy (epi-illumination)?
Its both epi and trans illumination. It depends on how you assemble it in the microscope.
You can find all the info on the FTIR device I use in the link: https://drive.google.com/drive/folders/17zpLael8OpDQ5c37_7FOTJiKIPID75xI?usp=sharing
I attach two photos, one is used as epi illumination and the other as trans illumination.
Be well
Ramon
Setup-Dimomo.jpgDimomo 1.jpg
@ Off-Topic an:
Abkürzungen können ihre Tücken haben.
Aus dem Medizin-Alltag:
HWI kann bedeuten
- Hinterwandinfarkt
- Harnwegsinfekt
- Häufig wechselnder Intimverkehr
und ist außerdem auch das Kennzeichen einer nord-ost-deutschen Hansestadt. ;D
@ Off-Topic aus.
Zitat von: Jürgen Boschert in Mai 22, 2026, 16:22:38 NACHMITTAGSAbbreviations can be tricky.
I developed this device years ago and just recently I learned that the principle it uses is FTIR. It was brought to my attention by an expert microscopist in Photomacrography.net forum.
I presented the device in Photomacrography.net forum few weeks ago. I was expecting that people there would be interested in exploring opaque samples with their biological microscopes. For example exploring the surface texture of metals. But contrary to my expectations people were more interested in its use in biological samples due its singular way of illumination.
I look forward to hear the comments of the community.
Ramon
PD Sorry that I dont speak or write german.
Hi Ramon,
very interesting topic!
Where do you see the main field of application?
Best
Peter
Hello Ramon,
Very interesting, indeed!
That's a valid link to "Photomacrography.net"from your device, right?
https://www.photomacrography.net/forum/viewtopic.php?p=312615#p312615
The link in your post here, only lead to the Main page...
Greetings Holger
Zitat von: Spectrum in Mai 22, 2026, 19:41:28 NACHMITTAGSThat's a valid link to "Photomacrography.net"from your device, right?
https://www.photomacrography.net/forum/viewtopic.php?p=312615#p312615 (https://www.photomacrography.net/forum/viewtopic.php?p=312615#p312615)
The link in your post here, only lead to the Main page...
Hi Holger. Yes, this is the device. In the following link you can find all the info:
https://drive.google.com/drive/folders/17zpLael8OpDQ5c37_7FOTJiKIPID75xI?usp=sharing
There is a lot to see, I have been working on this device for years. You honest opinion is warmly welcome.
Zitat von: Peter T. in Mai 22, 2026, 17:08:12 NACHMITTAGSvery interesting topic!
Where do you see the main field of application?
Well, it works wonders on metallographic samples. Because of the way it illuminates it provides more information than a metallographic microscope in brightfield. You can find all the info in the link.
I attach a photo so you can compare the results. Its C10 Steel Normalizad. The image on the left its made with a metallographic microscope. The image on the right its made with a biological microscope using the FTIR light guide. The different tones that you can see in the image on the right relate to the orientation of the crystal. It provides more information. You can find the full resolution images in the link.
But it may have other applications. The light hits the sample in very shallow angles that can be useful with some type of samples. If I am honest I was expecting that people in the community can help me find applications.
For example, I attach a photo of a Honey Bee Mouth mounted on slide. In this case its sort of a blend between bright and dark field.
I really look forward that you guys find it interesting. And I would love to hear your opinions. I can provide the blueprints so that anyone can build it on their side. As I say in my introduction I applied for a patent for this device. Although I am not very confident that I will get it because is such a simple thing.
Well, I look forward to continue this conversation.
By the way, when I was younger I used to spend christmas in Germany. In Wolfenbuttel to be precise. I love the Harz Mountains!
Tecmicro-8-01_10X.jpg
Honey Bee Mouth 2.jpg
Hi Ramon,
in my view, the complementary colours you see in brightfield vs. your glazing incidence illumination are caused by the relation between absorption coefficient and reflection coefficient. In physics, these relations are dealt with by the so-called Kramers-Kronig relation: If you know the absorbance of a material over a broad wavelength range, you can calculate the reflection from that.
In consequence, this leads to the fact that a strong absorbance in a given wavelength range leads to a weak reflection in this range. This means e.g. that gold is yellow in reflection, but blue in transmission. If you have a very thin gold layer, this becomes easily visible. You can also try that out by using a blue pen and drawing a 5x5mm^2 square. If you look at this square from a small, glazing angle, the square will reflect in reddish colour.
In daily life, this phenomenon is easily overlooked, e.g. leaves are green in transmission, but also in reflection. But actually this presumed reflection is caused by scattering of the light within the leave, and not by "physical reflection" at the surface.
Jürgen
Zitat von: jcs in Mai 22, 2026, 21:27:46 NACHMITTAGSin my view, the complementary colours you see in brightfield vs. your glazing incidence illumination are caused by the relation between absorption coefficient and reflection coefficient. In physics, these relations are dealt with by the so-called Kramers-Kronig relation: If you know the absorbance of a material over a broad wavelength range, you can calculate the reflection from that.
In consequence, this leads to the fact that a strong absorbance in a given wavelength range leads to a weak reflection in this range. This means e.g. that gold is yellow in reflection, but blue in transmission. If you have a very thin gold layer, this becomes easily visible. You can also try that out by using a blue pen and drawing a 5x5mm^2 square. If you look at this square from a small, glazing angle, the square will reflect in reddish colour.
Hello Jurgen
There are a lot of physics packed in just this few sentences. Please allow me to review my notes on light-matter interactions. But let me first ask you:
Zitat von: jcs in Mai 22, 2026, 21:27:46 NACHMITTAGSin my view, the complementary colours you see in brightfield vs. your glazing incidence illumination
What images are you referring to? Regarding the image attached (Fern Leaf mounted on a slide), in the Photomacrography.net community we are having a discussion about the "green stuff" that we see in the right side image (taken with FTIR device) versus the left side image (bright field). Is that what you mean?
Fern Leave 01_10X.jpg
Zitat von: jcs in Mai 22, 2026, 21:27:46 NACHMITTAGSIn daily life, this phenomenon is easily overlooked, e.g. leaves are green in transmission, but also in reflection. But actually this presumed reflection is caused by scattering of the light within the leave, and not by "physical reflection" at the surface.
Again, let me dive into light-matter interactions to understand it and be able to comment.
For what I see so far what you mean is that in brightfield we see transmission and with the FTIR device we see reflection.
Thanks for your informed reply. I bet you have a physics degree. Or PhD!
Hello Ramon,
I think in order to understand, what's going on with the "green stuff" in your pictures, we have to sort out the different lighting techniques, involved.
In this picture:
IMG_20260523_175404.jpg
you use your device with the plate illuminated from the sides with LED's.
The slide was mounted on the plate via immersion oil.
No further lightening.
Right?
In the first picture:
IMG_20260523_175440.jpg
you illuminate from the bottom via the trans-illumination of your scope. Thus to speak the lightening in the bottom of the scope and brightfield condensor.
Correct?
Greetings Holger
Zitat von: Spectrum in Mai 23, 2026, 18:06:05 NACHMITTAGSThe slide was mounted on the plate via immersion oil. No further lightening.
Right?
Hi Holger. Yes, the slide is mounted on top of the light guide (collector). Between them I placed a drop of immersion oil to make optical contact (index-matching). In the draw below you can observe the setup.
light path bio.jpg
We have eight light sources but for clarity only one is active in the draw.
Yes, no further lighting. All light sources are at the perimeter of the collector.
Zitat von: Spectrum in Mai 23, 2026, 18:06:05 NACHMITTAGSyou Illuminaten from the bottom via the trans illumination of your scope, Thus to speak the lightening in the bottom of the scope and brightfield condensor.
Correct?
YES. It is a simple compound microsocpe. Not a Kohler. Just a light source and a brightfield condensor. Below you can find it. In this instance we have the Dimomo device assembled on it but the light am using is the standard brightfield.Microscopio Brighfield.jpg
Zitat von: Ramon in Mai 23, 2026, 20:45:28 NACHMITTAGSHi Holger. Yes, the slide is mounted on top of the light guide (collector). Between them I placed a drop of immersion oil to make optical contact (index-matching). In the draw below you can observe the setup.
light path bio.jpg
We have eight light sources but for clarity only one is active in the draw.
Yes, no further lighting. All light sources are at the perimeter of the collector.
Ok,
This resembles mostly some kind of darkfield illumination.
Most of the light raus are hitting the specimen from a flat angle from all around the sides.
Few of them reach the objektives entrance pupil, I would guess. (They are scattered more or less randomly at total reflection Level of the "plate", till they reach the oiled part.
But why does the fern show green colour?
Hm, the sporangium of ferns show strong fluorescence...
Especially when there is some UV or blue light present.
All "white" LED's have a very prominent content of blue light.
So, my guess...:
There is apart of the darkfield also some fluorescence happening. That, so my guess, is what causes the "green stuff".
Perhaps (no bet on that!) the green fluorescence is also amplified by some effect also used in a technique called "TIRf".
In TIRF illumination you use total reflection in order to illuminate only a narrow layer of the probe with the exiting wavelengh for fluorescence.
Look here:https://en.wikipedia.org/wiki/Total_internal_reflection_fluorescence_microscope
But it's very questionable if this is happening here.
Why?
Because common darkfield with the proper exitation wavelengh is causing fluo as well...
The frustrated total internainternal reflection is an effekt, that is similar with TIRF but with white light and with no fluorescence effects. It also only occurs very close to the layer facing the illuminated surface. And I mean
very close.
It is used for fingerprint sensors or touchscreens.
Look here:
https://www.asc.ohio-state.edu/schumacher.60/class/570/notes/p036.pdf
The first (left) picture looks like some common brightfield image.
Greetings Holger
Zitat von: Ramon in Mai 23, 2026, 16:29:01 NACHMITTAGSWhat images are you referring to? Regarding the image attached (Fern Leaf mounted on a slide), in the Photomacrography.net community we are having a discussion about the "green stuff" that we see in the right side image (taken with FTIR device) versus the left side image (bright field). Is that what you mean?
Again, let me dive into light-matter interactions to understand it and be able to comment.
For what I see so far what you mean is that in brightfield we see transmission and with the FTIR device we see reflection.
Hi Ramon,
yes, I was referring to your fern image, where you have red features in the brightfield image which turn into green in your FTIR/darkfield image. But you see a similar behaviour in your first posting (larvae), where reddish areas in brightfield appear blueish in FTIR/darkfield.
It's quite some time ago that I had to deal with solid state physics spectroscopy, that's why my reply will probably sound somehow clumsy, I'll try anyway:
The spectral profile of the reflection and absorption coefficient, respectively, can show very different peaks. Gold e.g. reflects green and red light very well, that's why gold is yellowish in reflection. In contrast, gold transmits blue light somehow well. If you take a thin gold layer and put it into the microscope, it will appear blue in transmission and golden in reflection (epi-illumination), as expected by the spectra of absorption and reflection coefficients.
If you take a non-metallic object (e.g. a green leaf), it will appear green in transmission, as expected from its absorption spectrum, see image below.
chlorophyl.jpg
The same leaf will also appear green in reflection, although the reflection coefficient might have a very different spectrum compared to the absorption coefficient. The reason for that is the fact that the reflection of such an object under vertical illummination is rather weak. Instead, the light penetrates the leaf and part of it get absorbed by the chlorophyyl. Some of that light gets scattered back, leaves the object and is observed as green light in reflected light microscopy.
When you illuminate non-metallich objects, the amount of reflected light depends highly on the illumination angle. Glass reflects only 4% when illuminated vertically, and this value goes up substantially when you switch to glazing illumination. That's why in glazing illumination the "real" reflection coefficient becomes dominant at some point and the color of the observed object might change.
This could explain the switch from reddish to greenish in your fern images (probably this is not the only explanation of the observations you made).
Regarding FTIR, Holger pointed out a very important aspect: The "F" in FTIR is related to the evanescent waves leaving the surface of the light guide. The depth of these evanescent waves is very small, typically less than 300nm. This means that only features which are in extreme proximity to your light guiding plate can "frustrate" the light. In your fern sample, most features of the fern are probably much further away from the surface, and this needs to be considered when discussing the phenomena taking place in your device.
Sounds complicated, and unfortunately these things are complicated, but hopefully it helps at least a little bit for your understanding.
Jurgen
Hello Ramon, Holger and Jürgen,
Interestingly, the question of these color-shift phenomena had already been intensively discussed at the beginning of the 20th century. Here, too, the issue concerned their misinterpretation as fluorescence phenomena. Organic structures stained with certain dyes such as methylene blue, fuchsin, or eosin appear orange, blue-green, and green under dark-field illumination. Natural pigments as well, such as those of Stentor coeruleus, appear blue-green in brightfield microscopy and pink in darkfield microscopy: Stentor coeruleus ein Porträt (https://www.mikroskopie-forum.de/index.php?topic=32350.msg239108#msg239108)
This phenomenon, referred to as selective diffraction, or later as anomalous dispersion, was described in detail by Berek already in 1921. It was of particular importance because researchers sought to understand the color phenomena observed in weakly stained bacteria under darkfield illumination.
Best Regards Stefan
Anomalous Dispersion in Ocean Optics (https://www.oceanopticsbook.info/view/theory-electromagnetism/level-2/anomalous-dispersion)
M. Berek, Über selektive Beugung im Dunkelfeld und farbige Dunkelfeldbeleuchtung, Z wiss Mik 38, S 237ff (https://www.biodiversitylibrary.org/item/23840#page/259/mode/1up).
Hello Stefan and Jürgen,
I agree, the colour shift pheenomenon is definetely a thing, worth considering, in this case.
But what brought me to flourescence, as (maybe only one of several) exlanations, of what we see here, is:
1. Why do we see the "green stuff" only at some, but not all, reddish areas?
2. Why do we find these odd colour prominently at exactly the parts, where you can observe the stongest fluorescence of fern specimen, under "conventional" Epi fluorescence?
...and the green resembles well with what we see with fern flou...
Lest to forget about the blueish background in this picture.🤔
I think a comparison picture, taken in "classic" blue light Epi Fluo could be very helpful.
Zitat von: jcs in Mai 24, 2026, 15:07:16 NACHMITTAGSZitat von: Ramon in Mai 23, 2026, 16:29:01 NACHMITTAGSSounds complicated, and unfortunately these things are complicated, but hopefully it helps at least a little bit for your understanding.
Jurgen
I agree!
But very interesting topic, isn't it.
Greetings Holger
Hi Stefan,
very interesting paper, thanks for digging that out! Nowadays, physicists use the complex refractive index to describe various optical phenomena. From this complex refractive index, absorption and reflection can be derived. That's why I had to struggle with Berek's language, and I am not sure if I understodd his key points correctly.
Reading the key message on page 245 "... sondern die Farbwirkungen resultieren lediglich nach Maßgabe der Brechungsindizes und Absorptionsindizes an den Grenzflächen der beugenden Elemente gegen die Umgebung" he uses refractive index and absorption coefficient as material constants. When instead using reflective and absorption coefficient as material constants, you can come to similar (or even the same) conclusions.
But the important conclusion from this paper is, that you need neither frustrated total internal reflection nor fluorescence to explain these color changes that can be observed in darkfield microscopy. At least that's how I understand it at the moment.
Jurgen
Hello Holger,
i agree with your concerns, too. So eating the pudding will be the proof of the concept: but if the reddish stain is eosine, than classical Fluorescence will show us bright green everywhere, cause unfortunately eosine shows fluorescence as well as anomalous dispersion. And dispersion of the stains is depending on the biological stained matrices as well, too.
A conventional darkfield would be of interest, may be it looks different to Ramons technique, may be not.
After that an unstained fern preparation in Ramons technique, classical darkfield and of course fluorescence epi will show the real situation.
Hello Jürgen,
fortunately Berek was a physicist and not a medical doctor of these days, so his language was not so pretentious and cloudy ;)
best regards Stefan
Hello Jürgen and Holger
Zitat von: Spectrum in Mai 23, 2026, 22:04:29 NACHMITTAGSThis resembles mostly some kind of darkfield illumination.
Most of the light raus are hitting the specimen from a flat angle from all around the sides.
It is indeed a kind of Darkfield but in this FTIR device we have index-matching (immersion oil) so that grazing rays that would be mostly reflected by the slide in standard Darkfield in FTIR, since it is index-matched, we have no reflection and all the light will hit the specimen.
Zitat von: Spectrum in Mai 23, 2026, 22:04:29 NACHMITTAGSBut why does the fern show green colour?
Hm, the sporangium of ferns show strong fluorescence...
Hello Holger,
Yes, the fluorescence hypotheses was proposed by some people at the Photomacrography.net community. We were lucky that one of the moderators has an epi fluorescence microscope and lives not far from my place. I sent him the fern leaf sample and he took the images with the epi fluorescence microscope. You can find the images at:
https://www.photomacrography.net/forum/viewtopic.php?f=14&t=49734
His conclusion is that we have a lot of fluorescence in this sample.
Zitat von: Spectrum in Mai 23, 2026, 22:04:29 NACHMITTAGSThere is apart of the darkfield also some fluorescence happening. That, so my guess, is what causes the "green stuff".
I think you are right. As you read, we have proof that fluorescence is happening here.
Zitat von: Spectrum in Mai 23, 2026, 22:04:29 NACHMITTAGSPerhaps (no bet on that!) the green fluorescence is also amplified by some effect also used in a technique called "TIRf".
In TIRF illumination you use total reflection in order to illuminate only a narrow layer of the probe with the exiting wavelengh for fluorescence.
I don't know about that. In TIRf light does not get through, only the evanescent wave.
Zitat von: Spectrum in Mai 23, 2026, 22:04:29 NACHMITTAGSThe frustrated total internainternal reflection is an effekt, that similar with TIRF but with white light and with no fluorescence effects. It also only occurs very close to the layer facing the illuminated surface. And I mean very close.
It is used for fingerprint sensors or touchscreens.
I took the idea for this device from backlight panels for screens. FTIR is also used in the rain sensors of cars. However, I found out that lots of FTIR research papers don't use any "connecting media" (I use immersion oil as connecting media) but instead they use mechanical pressure.
Zitat von: Spectrum in Mai 23, 2026, 22:04:29 NACHMITTAGSThe first (left) picture looks like some common brightfield image.
It is indeed Brightfield. I use an old biological microscope with brightfield condenser (not a Kohler).
Hello Jürgen,
Zitat von: jcs in Mai 24, 2026, 15:07:16 NACHMITTAGSThe same leaf will also appear green in reflection, although the reflection coefficient might have a very different spectrum compared to the absorption coefficient. The reason for that is the fact that the reflection of such an object under vertical illummination is rather weak. Instead, the light penetrates the leaf and part of it get absorbed by the chlorophyyl. Some of that light gets scattered back, leaves the object and is observed as green light in reflected light microscopy.
The AI (Claude) agrees with you. I prompted it with you initial comment: "in my view, the complementary colours you see in brightfield vs. your glazing incidence illumination are caused by the relation between absorption coefficient and reflection coefficient. In physics, these relations are dealt with by the so-called Kramers-Kronig relation: If you know the absorbance of a material over a broad wavelength range, you can calculate the reflection from that."
Claude added a consideration that I think is appropriate. The stain used in most commercial slides, Safranin. This dye has a strong absorbance at 500-510 nm. Below see absorbance graph for different concentrations of Safranin T.
Safranin T Absorbance coeficient.jpg
According to the AI what is happening is as follows:
Safranin is highly selective when binding to different tissues. It binds specially to lignified and cutinized cell walls. Xylem, sclerenchyma, annulus cells, epidermis/cuticle (lignified or cutinized walls).
In Brightfield light passes through the specimen from below. Safranin has a strong absorption peak at approximately 510-530 nm (green wavelengths). The tissue absorbs green and some blue. What arrives at the objective is white light minus green, which appears pink/red. This is a pure transmission color - the color that was not absorbed by the stain.
For safranin-stained tissue: k (absorbance coefficient) is large at green wavelengths (510-530 nm). By Kramers-Kronig, n also changes anomalously near green wavelengths. The result is that the stained tissue behaves in a more metallic manner at green wavelengths - and metals reflect strongly. This is sometimes called anomalous surface reflection.
Light hits internal structures (annulus cell walls, xylem) from the side. Some is scattered upward into the objective. The scattered light carries the surface optical signature of those structures. Safranin strongly absorbs green (510-530 nm). By Kramers-Kronig, n changes anomalously at green wavelengths, causing preferential surface scattering of green (anomalous reflection). Green-enriched scattered light reaches the objective and forms the image.
I hope this helps.
You can view all the conversation with the AI at this link:
https://drive.google.com/file/d/1KICGh9jXW6ozjlgVIAi6bT5kQPfrVOVl/view?usp=sharing
Zitat von: jcs in Mai 24, 2026, 15:07:16 NACHMITTAGSRegarding FTIR, Holger pointed out a very important aspect: The "F" in FTIR is related to the evanescent waves leaving the surface of the light guide. The depth of these evanescent waves is very small, typically less than 300nm. This means that only features which are in extreme proximity to your light guiding plate can "frustrate" the light. In your fern sample, most features of the fern are probably much further away from the surface, and this needs to be considered when discussing the phenomena taking place in your device.
Aren't you referring here to TIRF rather than FTIR? In this device all the surface of the specimen is contacted to the light guide by a film of immersion oil. We have no evanescence wave at the contact point because we index-matched the interface.
Finally. With opaque inorganic specimens we also have color differences when using this FTIR little device. Below see an Integrated Circuit lithography imaged with Epi illumination, FTIR and "Wrap Around" Diffuser. The band that in FTIR is blue in the other techniques has a different color.
IC 3 ASSY 1 5x.jpg
Is it that the surface of the lithography acts like a diffraction grating? Or is it the there is a thin film on top of the specimen? Or is it something else?
Pls., allow me to gather all the info on this issue and I will bring it to you. I hope you find it interesting.
Cya
Ramon
Zitat von: Ramon in Mai 24, 2026, 21:43:17 NACHMITTAGSFor safranin-stained tissue: k (absorbance coefficient) is large at green wavelengths (510-530 nm). By Kramers-Kronig, n also changes anomalously near green wavelengths. The result is that the stained tissue behaves in a more metallic manner at green wavelengths - and metals reflect strongly. This is sometimes called anomalous surface reflection.
Light hits internal structures (annulus cell walls, xylem) from the side. Some is scattered upward into the objective. The scattered light carries the surface optical signature of those structures. Safranin strongly absorbs green (510-530 nm). By Kramers-Kronig, n changes anomalously at green wavelengths, causing preferential surface scattering of green (anomalous reflection). Green-enriched scattered light reaches the objective and forms the image.
As mentioned, things are complex in this field. But in my understanding the Berek-paper, Claude and me are somehow saying the same thing, albeit in different language. Assuming that "normal dispersion" refers to the dispersion of glass (gradual decrease of n from blue to red), "anomalous dispersion" refers to materials with differing spectral dispersion. Stains exhibit such behaviour, with very pronounced absorption peaks at energies (wavelengths) where electronic transitions happen. According to Kramers-Kronig, these absorption peaks influence refractive index/reflection coefficient, which in consequence becomes visible under glazing illumination.
To discriminate this phenomenon from fluorescence, you could illuminate your fern sample with green or yellow light. If the green color is still there, fluorescence can be ruled out: Green fluorescence can only be triggered by shorter wavelengths (Stokes shift), e.g. blue or UV.
Zitat von: Ramon in Mai 24, 2026, 21:43:17 NACHMITTAGSAren't you referring here to TIRF rather than FTIR? In this device all the surface of the specimen is contacted to the light guide by a film of immersion oil. We have no evanescence wave at the contact point because we index-matched the interface.
I understood that you are using immersion oil. But I guess your fern-sample is still covered with a cover glass?
Jurgen
Hi Jurgen
Zitat von: jcs in Mai 24, 2026, 22:26:50 NACHMITTAGSAs mentioned, things are complex in this field. But in my understanding the Berek-paper, Claude and me are somehow saying the same thing, albeit in different language. Assuming that "normal dispersion" refers to the dispersion of glass (gradual decrease of n from blue to red), "anomalous dispersion" refers to materials with differing spectral dispersion. Stains exhibit such behaviour, with very pronounced absorption peaks at energies (wavelengths) where electronic transitions happen. According to Kramers-Kronig, these absorption peaks influence refractive index/reflection coefficient, which in consequence becomes visible under glazing illumination.
There are many new terms for me here. I never went so deep in the understanding of light-matter interactions. Need time to study it.
Zitat von: jcs in Mai 24, 2026, 22:26:50 NACHMITTAGSTo discriminate this phenomenon from fluorescence, you could illuminate your fern sample with green or yellow light. If the green color is still there, fluorescence can be ruled out: Green fluorescence can only be triggered by shorter wavelengths (Stokes shift), e.g. blue or UV.
That can be done easiy.
Have you seen the post I linked? https://www.photomacrography.net/forum/viewtopic.php?f=14&t=49734
Zitat von: jcs in Mai 24, 2026, 22:26:50 NACHMITTAGSI understood that you are using immersion oil. But I guess your fern-sample is still covered with a cover glass?
Yes.
Ramon
Hello Ramon,
Thank's for pointing me to this link:
https://www.photomacrography.net/forum/viewtopic.php?f=14&t=49734
At first glance that looks just exactly like the proof of the pudding Stefan was pointing at.
But the more I watch the fluorescence pictures from Pau, the more I'm getting nonplussed...
Yes, the emittance in his first picture shows light emission exactly at the regions, we see the odd "green stuff"
But something is off here...🤔
Ramon, do you have any spectral data of the LED's you use?
Best regards Holger
Alo Holger
Zitat von: Spectrum in Mai 25, 2026, 00:58:42 VORMITTAGAt first glance that looks just exactly like the proof of the pudding Stefan was pointing at.
But the more I watch the fluorescence pictures from Pau, the more I'm getting nonplussed...
Yes, the emittance in his first picture shows light emission exactly at the regions, we see the odd "green stuff"
But something is off here...🤔
Pls., share with us what do you think that does not fit.
Zitat von: Spectrum in Mai 25, 2026, 00:58:42 VORMITTAGRamon, do you have any spectral data of the LED's you use?
I am using: Cree® XLamp® XP-E High-Efficiency White LEDs. Find the PDF at the following link:
https://drive.google.com/file/d/1qmXnVD3tPFycoHtnLBwbxbapkC0WwldK/view?usp=sharing
Ramon
Hallo Ramos,
Zitat von: Ramon in Mai 25, 2026, 16:13:46 NACHMITTAGSAlo Holger
Zitat von: Spectrum in Mai 25, 2026, 00:58:42 VORMITTAGYes, the emittance in his first picture shows light emission exactly at the regions, we see the odd "green stuff"
But something is off here...🤔
Pls., share with us what do you think that does not fit.
Hello,
The odd green is very bright in relation to the overall brightness of the picture. Much more than you would expect when you compare it with the Epi Fluo pictures from Pau.
And it's not only the "relative brightness" colour that puzzles me.
It's also the colour itself.
There is nearly no UV present in the light of your LED's.
So the exitation is mostly done by blue light, which is quite dominant in "white light" emitters:
IMG_20260525_233524.jpg
That' more or less the spektral content of such a LED. Your data sheet looks similar.
So most of your exitation is done by this blue wavelenghs.
The pictures where Pau uses blue light in classic epi fluorescence or Darkfield fluorescence with blue light just didn't blend with the "green stuff" we see in your picture with your device.
So fluorescence: yes, present. But....
The kind of fluorescence that would explain the bright green at it's given location, with no other green around: no
At least not with fluorescence as the only explanation.
And last but not least the other picture with some "odd green" that you posted, this time a "moss slide":
10x_03-Dimomo.jpg
Here the brightfield equivalent:
10x_03-Brightfield.jpg
That's definetely more than odd, to my eyes...
Don't know what's going on there...
Very strange!
How thick is the acrilic layer you are taking the photos through?
Greetings Holger
Zitat von: Spectrum in Mai 26, 2026, 00:10:08 VORMITTAGThat's definetely more than odd, to my eyes...
Don't know what's going on there...
Very strange!
I am quite loss here. I dont have the proper knowledge to interpret all this. I am trying to catch up on all the theory that you guys are proposing. Like Anomalous Dispersion that Stefan comented few posts up.
Zitat von: Spectrum in Mai 26, 2026, 00:10:08 VORMITTAGHow thick is the acrilic layer you are taking the photos through?
The acrylic layer is 2.8mm thick in the center. BUT! In this photos there is not acrylic layer between the specimen and the objective lens. The light guide is mounted below the specimen slide. I attach a draw:
light path bio.jpg
When the device is used with opaque samples it has to be mounted on top of the specimen. Between specimen and objective lens. When used with transparent samples you can use both configurations (top and bottom). But this photos were taken with the slide on top of the acrylic light guide.
It seems that misteries keep piling up.
Ramon
Hello guys,
I feed the AI with the info on the Frustrated TIR device and this conversation. If you want to review it is in the link:
https://drive.google.com/file/d/1DGK37YTaPS1SzuFDgNePUBLLeKO4N8pG/view?usp=sharing
Be well
Ramon
Hello Ramon,
I'm still very interested and curious about some of the results you are showing us here.
Especially the weird "green stuff" and the appearance of the "blue band" in your pictures of an opaque even specimen catched my eye...
The monochromaticity of these phenomenon, is what confuses me the most.
So I also went through all the discussions on the photomacrography forum.
Besides the summery of all the suggestions made till now, and witch are now liened up in the AI text you linked above, I found the conversation with Rik about the ball bearing experiment much more enlightening to potentially understand what's going on:
https://www.photomacrography.net/forum/viewtopic.php?f=14&t=49681&hilit=Dimomo&start=15
At one point Rik suspected, let me quote:
"It could be that the effect is some combination of surface texture to redirect the light and thin film interference to filter it. Offhand I do not know how to distinguish this possibility from diffraction alone."
That's shurely a point worth looking at.
So what about following experiments (for the upright "biological" setup):
1.Use only a perfectly clean slide with a perfectly clean cover slip with a drop of destilled water.
watch it dry out.
At some point the cover glass narrows down to the surface of the cover slip during evaporation.
2.Repeat this with some tap water. The minerals in it will cristallize and bring in some imperfections.
3. If you have a surface coated half transparent mirror at hand it would be interesting what happens if such a surface narrows down to the cover slip during evaporation. (Silvered surface facing your device.)
4.Repeat the dry out with some "textured" transparent probe-> Diatoms.
5.Repeat this with some fine sized fluorescend particles->pollen and/or tumeric powder
6. Use small glass beads suspendet in water. They are available as an additive for paint. You can get them as solide spheres and also as hollow ones. If you want, I could send you a small amount of each of them, as well as some strew diatom material, mentioning above.
Examine each of the setups during dryout and look for this weird monochrome colour phenomenon. Let us know if it appears again.
Check visual observation against your camera view for artifacts happening in your cam.
Video footage showing colour evolving during the evaporation process will be most informative.
Greetings Holger
Hello Holgen & community
Zitat von: Spectrum in Mai 29, 2026, 09:10:51 VORMITTAGI'm still very interested and curious about some of the results you are showing us here.
Especially the weird "green stuff" and the appearance of the "blue band" in your pictures of an opaque even specimen catched my eye...
The monochromaticity of these phenomenon, is what confuses me the most.
I expected you´d be interested so I prepared an information pack for you. Find all the info at the link:
https://drive.google.com/drive/folders/1faspwqt1k8589w5ruHvqHRN-5L77jKd2?usp=sharing
There you will find the discussion (PDF file) with some AI contributions plus the images at full resolution.
Zitat von: Spectrum in Mai 29, 2026, 09:10:51 VORMITTAGSo I also went through all the discussions on the photomacrography forum.
Besides the summery of all the suggestions made till now, and witch are now liened up in the AI text you linked above, I found the conversation with Rik about the ball bearing experiment much more enlightening to potentially understand what's going on:
Yes, Rik has been a great help. I am reproducing the Ball Bearing experiment with a more stable setup. As soon as I finish I will let you know. This link will give you access to the current Ball Bearing experiment so far:
https://drive.google.com/drive/folders/1_5blfCbccGn-hfR2ubEcUaUO_Az0sCVv?usp=sharing
Zitat von: Spectrum in Mai 29, 2026, 09:10:51 VORMITTAGAt one point Rik suspected, let me quote:
"It could be that the effect is some combination of surface texture to redirect the light and thin film interference to filter it. Offhand I do not know how to distinguish this possibility from diffraction alone."
Rik spotted the need for diffraction at onces and so far this is the best explanation.
Zitat von: Spectrum in Mai 29, 2026, 09:10:51 VORMITTAGSo what about following experiments (for the upright "biological" setup):
1.Use only a perfectly clean slide with a perfectly clean cover slip with a drop of destilled water.
watch it dry out.
At some point the cover glass narrows down to the surface of the cover slip during evaporation.
2.Repeat this with some tap water. The minerals in it will cristallize and bring in some imperfections.
3. If you have a surface coated half transparent mirror at hand it would be interesting what happens if such a surface narrows down to the cover slip during evaporation. (Silvered surface facing your device.)
4.Repeat the dry out with some "textured" transparent probe-> Diatoms.
5.Repeat this with some fine sized fluorescend particles->pollen and/or tumeric powder
6. Use small glass beads suspendet in water. They are available as an additive for paint. You can get them as solide spheres and also as hollow ones. If you want, I could send you a small amount of each of them, as well as some strew diatom material, mentioning above.
Examine each of the setups during dryout and look for this weird monochrome colour phenomenon. Let us know if it appears again.
Check visual observation against your camera view for artifacts happening in your cam.
Video footage showing colour evolving during the evaporation process will be most informative.
Perfect! I will perform the experiments you suggest. Right now I am doing the directional incident light experiment. I will post the results as soon as I finish.
I am going to have a good time with all this new experiments :)
Be well
Ramon
Hello Holger
At the link you will find all the info regarding the collectors I used for the IC lithography and the Ball Bearing Experiments.
https://drive.google.com/drive/folders/1CO_C6v82t1PbbE7yMzV_2dXxnT1Q2GIJ?usp=sharing
Ramon
Hi Holger
Zitat von: Spectrum in Mai 29, 2026, 09:10:51 VORMITTAG3. If you have a surface coated half transparent mirror at hand it would be interesting what happens if such a surface narrows down to the cover slip during evaporation. (Silvered surface facing your device.)
Let me know if this is what you mean.
surface coated half transparent mirror.jpg
Zitat von: Spectrum in Mai 29, 2026, 09:10:51 VORMITTAGRepeat the dry out with some "textured" transparent probe-> Diatoms.
Can you let me know where I can buy this "textured" Diatom specimens?
Zitat von: Spectrum in Mai 29, 2026, 09:10:51 VORMITTAG5.Repeat this with some fine sized fluorescend particles->pollen and/or tumeric powder
Regarding the Pollen; can I use pollen collected from the enviroment or we need Pollen prepared on a slide?
Zitat von: Spectrum in Mai 29, 2026, 09:10:51 VORMITTAG6. Use small glass beads suspendet in water.
Let me know if this will be OK for the experiment. Regarding the glass beads; how should I mount them? Should I use the immersion oil or water? If in oil or water the refraction index of the media and the glass beads will be pretty similar. We may have no reflection or reffraction. Will we see anything?
Glass Beads.jpg
Zitat von: Spectrum in Mai 29, 2026, 09:10:51 VORMITTAGIf you want, I could send you a small amount of each of them, as well as some strew diatom material, mentioning above.
Lets see if I can get all the material we need. Regarding the specimens on slides; when I use the small version of the FrTIR device (Diameter 86mm) I have to cut the slide for it to fit on top of the acrylic light guide.
If I cannot get the proper material I will ask you.
Ramon
Zitat von: Ramon in Mai 30, 2026, 19:36:07 NACHMITTAGSHi Holger
Zitat von: Spectrum in Mai 29, 2026, 09:10:51 VORMITTAG3. If you have a surface coated half transparent mirror at hand it would be interesting what happens if such a surface narrows down to the cover slip during evaporation. (Silvered surface facing your device.)
Let me know if this is what you mean.
surface coated half transparent mirror.jpg
No, I mean a small semitransparent mirror made of thin glass.
They are used as beamsplitters in optical instruments.
You can buy them cheap online (send you a PM about that)
Zitat von: Ramon in Mai 30, 2026, 19:36:07 NACHMITTAGSZitat von: Spectrum in Mai 29, 2026, 09:10:51 VORMITTAGRepeat the dry out with some "textured" transparent probe-> Diatoms.
Can you let me know where I can buy this "textured" Diatom specimens?
Best is to ask the diatom community here in the forum.
You need a solution of them.
Best would be some plain marine probe with some big flat species with a grid pattern like Cosconidiscus, or similar. I hasn't got to be something fancy, but it must be
unmounted and still diluted in water!I have some selfmade freshwater species in water here, that will also do the job.
(I hope so)
Zitat von: Ramon in Mai 30, 2026, 19:36:07 NACHMITTAGSRegarding the Pollen; can I use pollen collected from the enviroment or we need Pollen prepared on a slide?
Yes.
Some plain fresh collected pollen from a flower will do the job.
Dandelion or Lily, whatever.
Separate the pollen grains and dilute them in a drop of water on a slide, cover glass...done.
Try tumeric powder (kitchen) as well.
This test specimen should show fluorescence under the blue light included in the spektrum of your LED's. If there is some TIRF fluorescence going on with your device, it should show up here.
(And in a much more standardized way, than in your fixed mounted fern...)
Zitat von: Spectrum in Mai 29, 2026, 09:10:51 VORMITTAG6. Use small glass beads suspendet in water.
Let me know if this will be OK for the experiment.
Glass Beads.jpg
[/quote]
No.
You need so called "microsphere" glass beads-> PM you about that.
Zitat von: Ramon in Mai 30, 2026, 19:36:07 NACHMITTAGSRegarding the glass beads; how should I mount them? Should I use the immersion oil or water? If in oil or water the refraction index of the media and the glass beads will be pretty similar. We may have no reflection or reffraction. Will we see anything?
For
all test specimen just use a clean plain glass cover slip.
The specimen (pollen, micro beads, diatoms) should be mixed with one drop of destilled water and covered with a cover slip.
That's all.
Mount this cover slip via immersion oil (under the cover slip) on your device and record what you see.
The water will evaporate after some time.
Then the gap between the cover glass and the cover slip will be getting thinner and thinner...
Pressing the specimen more and more on the slide.
If there is some "Frustrated Total Internal Reflection" happening, it will show up.
Wait till all the water is evaporated and show your results...
(For the first experiment just use water, and for the half silvered mirror test, use the mirror instead of the cover glass)
Got it?
Greetings Holger
Hello Ramon,
Found a very informative article about a very similar device, based on the same principle:
https://journals.plos.org/plosone/article?id=10.1371/journal.pone.0058344
Definetely worth a look.
A lot of your questions concerning the properties of using this kind of lighning for biological specimen are answered there.
Greetings Holger
Alo Holger
I saw this device before. Guys from Photomacrography.net showed it to me. Also, see similar devices made by amateur microscopists:
IMG_1712-web600x800U100.jpg
dark1sc.jpg
Thanks for pointing it out! Indeed this device works by the same principle than the Frustrated TIR device I developed. However, there are differences. In the FrTIR device the light guide is designed at will to provide the proper spatial distribution of light. And another difference is that with the with the FrTIR device you can investigate transparent and opaque samples.
I am going through your last post about the IC lithografies. Let me study it and I will come back to you.
Be well Holger, it is a pleasure to engage in these discussions
Ramon
Zitat von: jcs in Mai 22, 2026, 21:27:46 NACHMITTAGSin my view, the complementary colours you see in brightfield vs. your glazing incidence illumination are caused by the relation between absorption coefficient and reflection coefficient. In physics, these relations are dealt with by the so-called Kramers-Kronig relation: If you know the absorbance of a material over a broad wavelength range, you can calculate the reflection from that.
In consequence, this leads to the fact that a strong absorbance in a given wavelength range leads to a weak reflection in this range. This means e.g. that gold is yellow in reflection, but blue in transmission. If you have a very thin gold layer, this becomes easily visible. You can also try that out by using a blue pen and drawing a 5x5mm^2 square. If you look at this square from a small, glazing angle, the square will reflect in reddish colour.
In daily life, this phenomenon is easily overlooked, e.g. leaves are green in transmission, but also in reflection. But actually this presumed reflection is caused by scattering of the light within the leave, and not by "physical reflection" at the surface.
Jürgen
Hello Jurgen
I think I finally figured it out. Below find a summary of all that is happening in four scenarios. Fresh leaf under ambient and FrTIR light and Fearn leaf under ambient and FrTIR light.
Fresh leaf vs Fearn leaf light interactions V01 small.jpg
Be well
Ramon
Hello Jurgen and all
I made a mistake. Pls., let me correct it. In Fresh Leaf/Extremely oblique lighting/ Energy balance says; Colour stays Green. It should say; if wax cuticle surface layer is not removed colour stays green
Sorry about that. I corrected it in the draw below:
Fresh leaf vs Fearn leaf light interactions V01 small.jpg
Ramon