Hallo zusammen,
ich entwickle seit einiger Zeit einen einfachen Aufsatz für gewöhnliche Durchlichtmikroskope, der das Licht mittels frustrierter Totalreflexion (FrTIR) in den Objektträger einkoppelt — ich nenne es hier einfach das FrTIR-Gerät. Es beleuchtet das Präparat unter einem echten streifenden Winkel (nur wenige Grad zur Präparatebene) statt wie üblich von unten.
Zeigen möchte ich hier nicht das Gerät selbst, sondern eine Eigenschaft, die mich überrascht hat — ich würde sie multimodale Koregistrierung nennen: Da sich überhaupt nichts bewegt — Objektträger, Tisch und Objektiv bleiben fest, und ich schalte lediglich von einer Beleuchtung zur anderen um — kann ich dasselbe Bildfeld, durch dasselbe Objektiv, in drei verschiedenen Kontrastarten aufnehmen, die von Natur aus deckungsgleich (koregistriert) sind, Pixel für Pixel, ohne erneuten Ausrichtungsschritt.
Dimomo-assy-multimode.jpg
Die drei Kanäle sind:
Hellfeld (Absorption) — die normale Durchlichtbeleuchtung des Mikroskops.
Streifendes Weißlicht (Streuung) — das FrTIR-Gerät mit weißen LEDs; ein dunkelfeldartiges Bild, aber aus streifendem Einfall.
Streifende Fluoreszenz (Emission) — das FrTIR-Gerät mit Violett–UV-LEDs (~395–405 nm) plus einem gelben Langpass-Sperrfilter (~500 nm), sodass nur die Stokes-verschobene Emission das Objektiv erreicht.
Das Beispiel unten ist ein botanisches Fertigpräparat — ein Farn-Sporangium (die sporentragende Kapsel, mit dem Ring der Anulus-Zellen), Safranin-gefärbt, 10×-Objektiv, Moticam 10MP. Von links nach rechts: Hellfeld, streifendes Weißlicht, streifendes Violett + Filter. Man beachte, wie dasselbe Sporangium und dieselben Anulus-Zellen in allen drei Bildern wiederkehren — genau darum geht es: Die drei Modi überlagern sich exakt, weil es dasselbe Bildfeld ist, sodass sie zusammen eine multimodale, koregistrierte Ansicht des Präparats ergeben.
Fern leaf 2 small.jpg
Ein weiteres, auf die gleiche Weise untersuchtes Präparat ist ein Querschnitt eines Winterjasmin-Blattes. In diesem Fall ist das Anregungslicht blau.
Leaf of winter jasmin CS 12 small.jpg
Über eure Gedanken und Kritik — auch die skeptische — würde ich mich freuen.
Viele Grüße
Ramon
Moin, Moin,
gibt es jetzt Aprilscherze auch im Hochsommer? 😆
Fluoreszierende Grüße
Michael
Hallo -
besteht bei diesem Flachwinkel-Durchlicht-System ein Unterschied zur Verwendung von Epi-HD-Objektiven, bei denen ebenfalls Durchlicht und Dunkelfeld-Auflicht mit sehr flachem Einstrahlwinkel (allerdings von oben) möglich ist?
Der Hauptvorteil deiner Einrichtung ist, dass sie für geübte Bastler wesentlich billiger ist.
Gruß
Rolf
Ich frage mich, warum die Totalreflexion so frustriert ist.
Hi Michael,
first, an apology — I am afraid I do not speak German, so I hope you do not mind me replying in English.
And no April Fools', promise :) — I was skeptical myself at first.
The setup really is simple: LED light runs through a plastic/glass collector by total internal reflection, and a thin film of index-matched immersion oil (n ≈ 1.5) between the collector and the slide frustrates that TIR — so the light crosses into the slide and lights the specimen at a grazing angle. The key is that nothing moves between the three shots — only the light source is switched — which is why brightfield, scattering and fluorescence are pixel-perfectly aligned.
The fluorescence is what draws the most doubt, so let me start with the cleanest test I have. I imaged a pure chlorophyll extract (images below): green under white light, deep red under violet excitation + filter, and a matched alcohol-only blank stayed dark — so the red requires the chlorophyll, i.e. it is genuine fluorescence.
Chlorophyll test - three conditions.jpg
Chlorophyll extract, same field. Left: white light — the extract scatters green (chlorophyll is green). Middle: violet (~400 nm) + 500 nm filter — the same field emits deep red (~680 nm). Right: matched alcohol-only blank — dark. A green substance cannot reflect or scatter red, and the blank stays dark, so the red can only be Stokes-shifted fluorescence.
Find a short controlled write-up in the link:
https://drive.google.com/file/d/1Bt7OBpM8UmHdlkHUc4va9wCL74yyQ3SA/view?usp=sharing
And I am not the only one who has looked at this. A fellow microscopist — Pau, from the Photomacrography community — took the very same fern slide and imaged it on a proper research fluorescence microscope: a Leitz 16×/0.40 NPL Fluotar with real epifluorescence cubes (matched excitation, beamsplitter and emission filters) and a Zeiss darkfield condenser, six technique combinations on the same field. He could not reproduce my exact colours — different excitation/emission bands give different hues — but his conclusion was unambiguous: genuine fluorescence of the cell-wall components (cutin, cellulose) and chlorophyll or its derivatives, possibly the stain too. So the phenomenon is confirmed on a research fluorescence microscope; the FrTIR add-on simply makes it accessible on a standard one.
Pau independent fluorescence test - montage.jpg
Pau's independent test on a research fluorescence microscope (Leitz 16×/0.40): the same fern field in six excitation/filter combinations, all showing genuine fluorescence of the cell-wall components. The emission colour depends on the barrier filter, not the specimen.
You can find Pau´s Fern leaf test in the following link:
https://drive.google.com/file/d/1gnmHrEr31W6ndgvTaWAV-ckj8ddMNqvi/view?usp=sharing
On my own rig, a simple no-filter test makes the same point directly on tissue. Winter jasmine, blue excitation (images below): without the filter the blue light floods the whole field; with the Thorlabs 500 nm hard-coated long-pass the background collapses and only the structured green emission of the cell walls remains. The fern images in my post use violet (~400 nm) excitation rather than blue — even further from the 500 nm cut-on, so the background is darker still.
Control - Winter jasmine no-filter vs Thorlabs 500nm.jpg
Winter jasmine, cross-section, blue LED excitation — the same field, twice. Left: no barrier filter — scattered blue excitation floods the whole field. Right: with the Thorlabs 500 nm hard-coated long-pass — the scattered blue is blocked, the background collapses to dark, and only the structured green emission of the cell walls survives. A green that remains once the blue is filtered out can only be Stokes-shifted emission — genuine fluorescence, not scattered excitation.
For the wider picture of the method, there is a fuller technical overview here:
https://drive.google.com/file/d/1odlmWJy3f9dA-G-MNDF1EIwP7qsd7qhj/view?usp=sharing
Since you have a lot of experience with fluorescence and darkfield on the Polyvar and Jenamed, I would genuinely value your critical take — and honestly, on your optics this would look far better than on my rig. What control would convince you?
Fluorescent greetings back :)
Ramon
Hello Rolf,
Good question — and yes, there is a real difference. It comes down to the angle of incidence: FrTIR reaches true grazing (down to ~0° from the specimen plane, up to ~47.5°), while Epi-HD — like air-coupled reflected and darkfield systems generally — reaches a shallow oblique angle but never truly grazing. You're right that Epi-HD gives reflected dark-field at a shallow angle, and that it's the more expensive route; but that shallow-versus-grazing gap is exactly where the two part ways.
Why FrTIR can graze where an air-coupled objective can't differs between transparent and opaque work:
Transparent specimens (like the fern you saw): to graze a slide-mounted specimen from an air-based source, the light must cross the air→glass boundary — and as the angle nears grazing, Fresnel reflection there rises toward total, so the light is turned away (reflected) before it reaches the specimen. FrTIR couples into the slide with immersion oil (n ≈ 1.5), removing that boundary, so it can skim the specimen inside the mountant.
Opaque specimens (your Epi-HD's shallow-angle domain): here there's no glass — the light travels through air onto the surface, and the limit is geometric. A reflected-darkfield objective delivers its oblique light through the collar around the objective, which reaches oblique but not truly grazing. FrTIR's disc sits flat on the surface (oil-coupled) and injects light by frustrated TIR at true grazing. On metals this reveals grain-orientation contrast — here is C10 steel (normalized, ferrite–pearlite) both ways
C10 steel normalized - reflected brightfield vs FrTIR.jpg
conventional reflected metallography (left) vs FrTIR (right), where each grain's crystallographic tilt reflects the grazing light differently into the objective (directional reflectance, grain mapping that usually needs polarized or interference contrast).
Full write-up: https://drive.google.com/file/d/1I4so0t1O9-_k6JhrxoHSDkIl6DmkBbvI/view?usp=sharing
And a third mode, Fluorescence: because the guided excitation stays trapped below the coverslip, a single long-pass filter — no filter cube — turns the same setup into a label-free autofluorescence mode.
And yes, as you say: it runs on any standard biological microscope with ordinary objectives, so for an experienced hobbyist it reaches this kind of contrast at a fraction of the cost of dedicated Epi-HD optics.
Regards,
Ramon
Was er meint ist mir noch unklar.
Es gibt wohl eine Art erzwungener Tatalreflexion in DLP-Video-Projektoren (Mikrospiegel), das funktioniert mit telezentrischer Optik (Parallelstrahlen); habe ich noch nicht verstanden.
https://de.wikipedia.org/wiki/Digital_Light_Processing#Digital_Micromirror_Device (https://de.wikipedia.org/wiki/Digital_Light_Processing#Digital_Micromirror_Device)
Das Prisma habe ich als PDF (wie PDF einfügen??)
Vielleicht meint er "Evanescent Wave Scattering"
Gruß - Werner
Hi Werner,
First, your practical question — to attach a PDF, expand "Anhänge und weitere Optionen" below the message box and add it there, just like an image.
thanks for digging into it — the name "frustrated TIR" does invite the evanescent picture, so let me clarify.
The FrTIR (Frustrated Total Internal Reflection) device is a disc-shaped acrylic/glass collector fitted with 8 SMD LEDs around its perimeter. Light from the LEDs enters the collector and propagates by Total Internal Reflection (TIR) toward the specimen-facing surface (geometric center). A thin film of immersion oil (n ≈ 1.5), identical in refractive index to the acrylic/glass, is applied between the collector and the specimen. This optical continuum frustrates the TIR condition: the whole guided beam, no longer reflected back into the collector, passes straight through the index-matched interface onward to the specimen surface.
The result is that light reaches the specimen at illumination angles ranging from approximately 0° to 47.5° relative to the specimen plane — true grazing-angle incidence — without the Fresnel reflection losses that limit conventional darkfield and oblique systems. From an optical standpoint, the oil creates a single continuous medium from the light sources to the specimen surface, so no refraction or interface loss occurs along the light path.
The device runs in two arrangements (see the sketch):
Dimomo parts 2.jpg
Figure. In the ray trace diagrams, for clarity, only one of the 8 available LEDs is active
Mode 1 (opaque specimens): the collector sits on top of the sample, oil-coupled to its surface, and the objective looks down through the disc's centre; the grazing light reflects off the surface — grain facets, thin films — into the objective (this is the metals work).
Mode 2 (transparent specimens): the collector sits below the slide, oil-coupled to it; the grazing light crosses up into the slide and skims a transparent section from below (this is the fern).
Either way it's a real, propagating beam at grazing incidence — not an evanescent field, and not tunneling across a gap. So it isn't "evanescent wave scattering" (that's the TIRF near-field). The DLP TIR prism is a cousin but does the opposite: it uses TIR to steer light onto the micromirrors; here we defeat TIR to let the grazing light out onto the specimen.
You can find more information in the following link: https://drive.google.com/file/d/1odlmWJy3f9dA-G-MNDF1EIwP7qsd7qhj/view?usp=sharing
I will be happy to clarify any doubt.
For transparency: there's a patent application on the device — I mention it only so the IP status is clear, not because anything's for sale. Forum admins where notified. I'm here to learn from your criticism and improve it, nothing more. Tough questions very welcome.
Regards,
Ramon
Da ich mir auf Englisch zwar eine Pizza bestellen kann, aber ansonsten doch etwas öfter mal ein Wörterbuch oder einen guten Freund fragen muss, habe ich meinen KI-Freund um eine Übersetzung des Titels von Ramons Paper gebeten.
Hier we go: "Echte streifende Beleuchtung durch verhinderte Totalreflexion"
Der in Titel des Threads genannte Begriff ,,Multimodale Koregistrierung" ist wohl auch ein üblicher. Er bezeichnet das passgenaue übereinanderlegen von Bilders einer Sache, die mit unterschiedlichen Methoden (Modi) aufgenommen wurden, und das kann man, wenn man's kann, mit ImageJ machen.
Alle, die es schon gewußt haben, Tschuldigung, ich bin das etwas hinterher. Dass das kein Aprilscherz ist, war mir aber schon klar.
Super Beitrag von Ramon! :)
Hallo zusammen,
in der medizinischen Bildgebung gibt es das schon ca. 20 Jahren, heißt dort Image Fusion. Es können CT, (f)MRT, Röntgenaufnahmen, Angiographien, Ultraschall un weitere b.B. deckungsgleich übereinandergelegt werden.
Hello Ramon,
I'm a bit sceptical about the fluorescence, at least in the case of the winter jasmine. One question is the wavelength of the excitation light. Is the blue wavelength around 463 nm? What dyes have been used to colour the plant cutting?
Hubert
Zitat von: Sourdough in August 21, 2026, 22:40:48 NACHMITTAGSDa ich mir auf Englisch zwar eine Pizza bestellen kann, aber ansonsten doch etwas öfter mal ein Wörterbuch oder einen guten Freund fragen muss, habe ich meinen KI-Freund um eine Übersetzung des Titels von Ramons Paper gebeten.
Hier we go: "Echte streifende Beleuchtung durch verhinderte Totalreflexion"
Der in Titel des Threads genannte Begriff ,,Multimodale Koregistrierung" ist wohl auch ein üblicher. Er bezeichnet das passgenaue übereinanderlegen von Bilders einer Sache, die mit unterschiedlichen Methoden (Modi) aufgenommen wurden, und das kann man, wenn man's kann, mit ImageJ machen.
Alle, die es schon gewußt haben, Tschuldigung, ich bin das etwas hinterher. Dass das kein Aprilscherz ist, war mir aber schon klar.
Super Beitrag von Ramon! :)
Original in english AI translated
Vielen Dank, und Hut ab für die Übersetzung — ,,Echte streifende Beleuchtung durch verhinderte Totalreflexion" trifft den Kern genau. (Und keine Sorge wegen des Englischen: mein Deutsch reicht auch gerade so für die Pizza, den Rest erledigt bei mir ebenfalls ein KI-Freund. 😉)
Ein schöner Zusatz zu deiner Bemerkung über ImageJ: Du hast völlig recht, dass man Bilder aus verschiedenen Modi normalerweise nachträglich passgenau übereinanderlegen muss. Das Besondere beim FrTIR-Aufsatz ist, dass diese Koregistrierung quasi schon ,,eingebaut" ist — Absorption (Hellfeld), Streuung/Reflexion und Fluoreszenz entstehen alle durch dasselbe Objektiv, im selben Bildfeld, ohne die Probe zu bewegen. Die Modi sind also von Natur aus weitgehend deckungsgleich; der ImageJ-Schritt entfällt meist oder wird zur reinen Feinkorrektur.
Und nein, kein Aprilscherz. 🙂 Danke fürs freundliche Wort — das motiviert wirklich!
Zitat von: Jürgen Boschert in August 21, 2026, 22:54:56 NACHMITTAGSHallo zusammen,
in der medizinischen Bildgebung gibt es das schon ca. 20 Jahren, heißt dort Image Fusion. Es können CT, (f)MRT, Röntgenaufnahmen, Angiographien, Ultraschall un weitere b.B. deckungsgleich übereinandergelegt werden.
Hallo Jürgen, (translation from english original)
danke für den Hinweis — der Vergleich ist absolut treffend. Die ,,Image Fusion" ist in der medizinischen Bildgebung tatsächlich seit rund zwei Jahrzehnten etabliert, und das Ziel ist dasselbe: Aufnahmen unterschiedlicher Modalitäten deckungsgleich übereinanderzulegen.
Der interessante Unterschied liegt darin, wie die Deckungsgleichheit zustande kommt. In der Radiologie stammen CT, (f)MRT, Ultraschall usw. aus verschiedenen Geräten und Zeitpunkten, sodass die Bilder nachträglich per Software (Landmarken, Registrierungsalgorithmen) zur Deckung gebracht werden müssen. Selbst moderne Hybridgeräte (PET/CT, PET/MRT), die einen Teil davon schon ,,hardwareseitig" lösen, nehmen die Modalitäten noch mit getrennten Detektoren auf.
Beim FrTIR-Aufsatz entstehen die verschiedenen Kontrast-Modi (Absorption/Hellfeld, Streuung/Reflexion, Fluoreszenz) dagegen durch dasselbe Objektiv, im selben Bildfeld, an derselben Probe, ohne diese zu bewegen. Die Koregistrierung ist damit nicht das Ergebnis einer nachträglichen Berechnung, sondern von vornherein ,,eingebaut" — Pixel für Pixel deckungsgleich per Konstruktion; der Fusions-/ImageJ-Schritt entfällt weitgehend.
Also dasselbe Ziel wie die medizinische Image Fusion, aber auf optischem Weg und ohne nachträgliche Registrierung erreicht. Danke für den treffenden Begriff!
Zitat von: Lupus in August 21, 2026, 23:05:08 NACHMITTAGSHello Ramon,
I'm a bit sceptical about the fluorescence, at least in the case of the winter jasmine. One question is the wavelength of the excitation light. Is the blue wavelength around 463 nm? What dyes have been used to colour the plant cutting?
Hi Hubert,
first off — I was also skeptical about the Jasmine. I went back to the bench to check it properly.
That image used blue LEDs (~452–455 nm) with a cheap long-pass filter, and my blue strip has a green spectral leak. I checked with a specimen-free blank (same optics, no sample; only slide/alcohol/cover slip): under blue excitation the empty field already shows a green background — so part of the green in that jasmine image is scattered excitation, not fluorescence. I would not defend that particular image as clean fluorescence; your scepticism was justified.
To separate the two, I re-imaged the same section with violet–UV excitation (~395–405 nm) and a proper 500 nm long-pass filter. Here the excitation sits entirely below the filter cut-off, so no excitation light can reach the camera — and indeed the specimen-free blank (only slide/alcohol/cover slip) is essentially black. Yet the section itself glows green at roughly 6× the blank. That green cannot be scattered excitation (there is almost none to scatter), so it reads as genuine emission: cell-wall autofluorescence (lignin/cutin), well documented in plant sections. However, looking at the image I sill remain skeptical. Something is off in this image.
Jasmine - Violet-UV + 500nm (autofluorescence).jpg
Jasmine - Violet-UV + 500nm LPF (autofluorescence?)Jasmine - Violet-UV blank control (near-black).jpg
Violet-UV blank + LPF control (near-black)Jasmine - Blue blank (green leak).jpg
Blue LEDs blank + LPF(green leak)The slide is a commercial prep, so I cannot exclude a stain contributing to the reddish signal in the vascular bundle — but the green cell-wall autofluorescence stands on its own. If you or others see a hole in even that, I am all ears; I genuinely want to get this right.
Thanks again, Hubert.
Hello Ramon,
I'm very curious about your device, too.
Concerning the proof of the fluorecence capabilities (as we already discussed via private messages):
Try to find some freshwater with algea in it!
Best would be some algea mat, swimming on top, with filamentous algea in it, like spirella. But any green algea film on the bottom or the walls, or even the green biofilm on a humid planting pot will do the job.
Scrape some of the green sludge of the surface, disperse it in water and take some pictures. Use your violet/blue lights and your emission filter.
Just like in this description:
http://www.microscopy-uk.org.uk/mag/indexmag.html?http://www.microscopy-uk.org.uk/mag/artmar09/dw-fluor1.html
If the chloroplasts of the algea will show up in red ....
That will be a distinctive proof of fluorescence.
As you remember, you already showed me some pictures, with such water mounted fresh specimen.
I'm pretty shure that you can already see the well known and distinctive red fluorescence of chlorophyll in this footage.
Greetings Holger
Zitat von: Spectrum in August 22, 2026, 21:08:26 NACHMITTAGSHello Ramon,
I'm very curious about your device, too.
Hi Holger!
I was waiting for you to contact me. Remember that we agreed upon having a video call so that I can show you the nuances of the gadget.
Look forward hearing from you soon.
Ramon
Hello Ramon,
the problem is that it is not entirely sufficient for the cut-off filter to cover the entire emission wavelength range of the excitation LED. Its intensity must also be significantly reduced, particularly when the fluorescence yield is low. This is because cameras typically have a significant overlap in the spectral sensitivity of the RGB channels, meaning that blue light can still excite the G and to a lesser extent the R channel.
In the image below, the top left shows the original photo with UV-violet excitation, whilst on the right the blue channel has been strongly amplified. However, you can see that it has also been exposed. At the bottom left, the red channel has also been amplified. It is also worth noting, however, that the camera's JPG conversion can also artificially create small amounts of colour in an unexposed colour channel if neighbouring pixels of a different colour are more heavily exposed.
And at the bottom right is the RG colour separation, but with the green channel attenuated and the red channel amplified. The red areas could be typical chlorophyll fluorescence. The light green areas are more likely to be scattered light. The red and dark green areas can be seen in the same colour in your brightfield image.
Fluoreszenz Violett Farbauszüge.jpg
Hubert
Hello Ramon,
I addressed a similar topic in this article about condenser-free illumination:
https://www.mikroskopie-forum.de/index.php?topic=54614.0
Using a blue excitation LED in simple dark-field illumination from below and a much more rudimentary red film filter, I attempted to demonstrate chlorophyll fluorescence on a fresh, unstained plant section. It is important to use fresh sections rather than ready-made slides, as chlorophyll degrades.
Here is a section (parsley) under white transmitted light, followed by two fluorescence images. Green should be scattered light.
38 Petersilie HF.jpg
39 Petersilie Autofluoreszenz.jpg
40 Petersilie Autofluoreszenz.jpg
Hubert
Hi Hubert,
Yesterday I posted regarding Photo Bleaching as a method for testing for fluorescence in the Jaasmine sample. I had to put it down because there were some errors. I will correct it and put it back in this afternoon/evening.
Zitat von: Lupus in August 23, 2026, 08:53:52 VORMITTAGI addressed a similar topic in this article about condenser-free illumination:
https://www.microscope-forum.de/index.php?topic=54614.0 (https://www.mikroskopie-forum.de/index.php?topic=54614.0)
Using a blue excitation LED in simple dark-field illumination from below and a much more rudimentary red film filter, I attempted to demonstrate chlorophyll fluorescence on a fresh, unstained plant section. It is important to use fresh sections rather than ready-made slides, as chlorophyll degrades.
Here is a section (parsley) under white transmitted light, followed by two fluorescence images. Green should be scattered light.
Those are very good images for such a simple setup. And quite humbling because for months now I have been trying to get similar results with the FrTIR gadget. Without success sadly. I imaged mainly moss and algae and the results have been no where near your results. I am going to try again, this time with a fresh stem similar than the one you used.
Ramon
Hello Ramon,
Chlorophyll is not only bleached by light; there is also biochemical degradation following cutting.
A fundamental problem lies in the spectral sensitivity distribution of a typical camera sensor. The image shows my situation with blue LED excitation. The dotted line illustrates the typical sensitivity curve of an RGB sensor (which may vary significantly in individual cases). My simple red cut-off filter is shown schematically in dark red; the residual transmission is not negligible in the case of blue light. On the far right is the red fluorescence from chlorophyll.
You can see
1. that unblocked residual blue light can also excite the green and less the red pixels, and
2. that the chlorophyll fluorescence lies very far into the long-wavelength red region, where the camera sensor may already be relatively insensitive.
This means that the contrast ratio between the excitation light and the fluorescence light may become very unfavourable. This makes it all the more important to use a fresh plant sample with maximum fluorescence yield, and a good cut-off filter. The Thorlabs 500 nm filter, for example, has a transmission of 0.05 per cent in the blue spectral range – which is not bad but not zero either.
And, of course, minimising stray light from the dark-field illumination is also part of the concept to suppress the excitation light as much as possible.
Spektrum Kamera LED Sperrfilter Chlorophyll.jpg
Hubert
Hi Hubert
In fairness I should add: the reason I can keep up with your points and reply quickly is that I work through them with an AI assistant. The experiments and the judgement are mine, but it helps me analyse the data and put my thoughts together at a pace I could not manage alone.
thank you — the spectral diagram is really useful, and both your points are well taken. I want to be honest about my motive here: I have no stake in the "fluorescence" answer. I'd rather end up with a correct "no" than a comfortable "yes."
On biochemical degradation: you are right however this section is a commercial fixed/mounted slide rather than fresh tissue (so little active biochemistry), and the signal that fades most is the green cell-wall autofluorescence (lignin/cutin), which is structural and photobleaches rather than degrades.
On the camera/filter spectral point: Your diagram is blue excitation (~460 nm) with a >600 nm cut-off aimed at chlorophyll red (~680 nm) — which, as you show, lands where the sensor is already insensitive. My run used violet excitation (~400 nm) with a 500 nm long-pass, and the signal I am actually relying on is the green cell-wall autofluorescence (~510–560 nm). That green sits at the sensor's peak sensitivity and just above the filter cut-off, and it is far from a violet excitation the Thorlabs filter blocks hard (~0.05% in the blue, less at 400 nm). So your "far-red, sensor-insensitive" concern really applies to the red/chlorophyll channel — which is exactly the channel I am now treating as unconfirmed.
Thanks again — this genuinely sharpened the work.
Now let me present you the latest version of the Photo Bleaching test:
Photobleaching test — winter jasmine
Frustrated-TIR grazing illumination · violet-UV (~395–405 nm) bleaching · 500 nm long-pass · imaged with the device's own (stable) UVA LEDs. REV3, 24 August 2026.
Purpose
To test whether the emission from the winter-jasmine section is genuine fluorescence or scattered/reflected excitation. Photobleaching distinguishes the two: under a constant excitation, fluorescence fades as the fluorophores are destroyed, whereas scattered or reflected light does not. The same field was bleached in steps and re-imaged, and the region intensities were compared. Excitation is violet-UV (~395–405 nm, UVA SMD5050 LEDs); the emission filter is a 500 nm long-pass.
Set-up and method
This test uses two SEPARATE light sources — this distinction is essential to reading the result:
Photobleaching - table 1 setup.jpg
Image series
The same field imaged at increasing cumulative violet BLEACHING exposure (each panel labelled with total exposure time; each captured under the stable imaging LEDs):
Photobleaching - image series montage.jpg
Quantitative analysis
Mean pixel intensity (0–255) in four fixed regions across all six frames: the diffuse background halo, the bright green parenchyma cells, the reddish vascular bundle, and a dark background area far from the specimen.
Photobleaching - table 2 region data.jpg
Plotted against cumulative bleaching dose, the fade is approximately linear (dashed fits):
Photobleaching - fade vs cumulative dose (linear fit).jpg
Interpretation
The imaging excitation was constant. Because each photo was taken with the FrTIR device's own imaging LEDs — ON for only a few seconds, with the bleaching LED off — the light exciting the specimen at every capture was the same.
Under a constant excitation, scattered or reflected light stays constant (it cannot bleach); only fluorescence fades. The signal fell by roughly 25–34%, so the fading part of the signal is genuine fluorescence, not scattered light. The fade is approximately linear with cumulative dose (slope ≈ −0.64 units/min), consistent with photobleaching. Every region fades — including the diffuse halo — so the halo cannot be purely scattered light either.
I wonder if the answer is that we are having fluorescence plus scatter.
I hope to keep up this lively conversation.
Ramon
Hello Ramon,
I don't actually identify green autofluorescence.
1. The bleaching experiment assumes that areas outside the fluorescence emission spectrum have the same intensity when exposed to the same light, or at least that the measurements are normalised accordingly to this 'background brightness'. However, the green halo also changes in intensity, in a similar ratio to the cell area.
2. Autofluorescence, e.g. from lignin, is brighter in the region of the cell walls or corresponding structures; here, however, the opposite is true, in that the cell interior glows more brightly.
This paper demonstrates lignin fluorescence very clearly (Fig. 5) https://pmc.ncbi.nlm.nih.gov/articles/PMC7288016/?utm_source=chatgpt.com
Hubert
Hi Hubert
Zitat von: Lupus in August 24, 2026, 20:14:18 NACHMITTAGSI don't actually identify green autofluorescence.
1. The bleaching experiment assumes that areas outside the fluorescence emission spectrum have the same intensity when exposed to the same light, or at least that the measurements are normalised accordingly to this 'background brightness'. However, the green halo also changes in intensity, in a similar ratio to the cell area.
2. Autofluorescence, e.g. from lignin, is brighter in the region of the cell walls or corresponding structures; here, however, the opposite is true, in that the cell interior glows more brightly.
This paper demonstrates lignin fluorescence very clearly (Fig. 5) https://pmc.ncbi.nlm.nih.gov/articles/PMC7288016/?utm_source=chatgpt.com (https://pmc.ncbi.nlm.nih.gov/articles/PMC7288016/?utm_source=chatgpt.com)
Give me a day or two to go through this.
Zitat von: Lupus in August 23, 2026, 08:53:52 VORMITTAGI addressed a similar topic in this article about condenser-free illumination:
https://www.mikroskopie-forum.de/index.php?topic=54614.0 (https://www.mikroskopie-forum.de/index.php?topic=54614.0)
Using a blue excitation LED in simple dark-field illumination from below and a much more rudimentary red film filter, I attempted to demonstrate chlorophyll fluorescence on a fresh, unstained plant section. It is important to use fresh sections rather than ready-made slides, as chlorophyll degrades.
Here is a section (parsley) under white transmitted light, followed by two fluorescence images. Green should be scattered light.
I dont know what I am doing wrong.
Sample: section of stem of a fresh plant
Excitation: cheap blue LED with a green tail (leaks green)
Barrier filter: Throlabs 500nm LPF
Illuminated through the FrTIR device (true grazing angles because we do away with the air/glass interface)
Fresh stem 5x 2.jpg
No sign of red emission from chlorophyll
Ramon
Hello Ramon,
ZitatExcitation: cheap blue LED with a green tail (leaks green)
What exactly does that mean? Do you have the emission spectrum from the data sheet?
If the spectral distribution is similar to that in this graph (the yellow line corresponds to your block filter), then B, G and R will, of course, be relatively strongly illuminated by the excitation light, compared with the chlorophyll fluorescence at the less sensitive end of the of the R channel of the camera sensor.
Spektrum Kamera LED blau Sperrfilter gelb rot Chlorophyll.jpg
How long was the exposure time for this photograph?
You can see from the histogram of your image (left) that the blue component is also very high. On the right, I've created a green-red colour separation and brightened the red component. So you can already see the chlorophyll fluorescence.
Is the plant section not a cross-section, but rather a stem cut from the side?
GR Farbauszug R verstärkt.jpg
Hubert
Es wäre schon schöner, wenn in einem deutschen Forum auf deutsch kommuniziert würde.
Ich bekomme zwar auf englisch mein Essen, aber mit diesen Texten sind sicher manche überfordert. Jedes mal mit einem Übersetzungsprogramm übersetzen, ist halt auch nicht ganz zielführend.
Peter Mikropit
Hallo Peter,
das sind eigentlich Ausnahmefälle, hier ist es ein spanischer Forumsteilnehmer.
Es ist doch ganz einfach: Rechte Maustaste - "Übersetzen in Deutsch" anklicken. Dann erscheint der ganze Thread auf Deutsch.
Hubert
Hallo, Hubert, diese Möglichkeit kannte ich nicht. Vielen Dank. Ja das ist zumutbar. Nur die Diagramme werden nicht übersetzt.
Peter Mikropit
Zitat von: mikropit in August 25, 2026, 11:43:33 VORMITTAGEs wäre schon schöner, wenn in einem deutschen Forum auf deutsch kommuniziert würde.
Ich bekomme zwar auf englisch mein Essen, aber mit diesen Texten sind sicher manche überfordert. Jedes mal mit einem Übersetzungsprogramm übersetzen, ist halt auch nicht ganz zielführend.
Peter Mikropit
Hallo Peter,
du hast völlig recht — aber leider spreche ich kein Deutsch. Kurioserweise ist mein bester Freund seit 30 Jahren Deutscher, und ich habe viele Weihnachtsferien in der schönen Stadt Wolfenbüttel verbracht — ich habe also eine echte Zuneigung zur Sprache und zum Land, auch wenn davon nie wirklich etwas auf meinen Wortschatz abgefärbt hat!
Der eingebaute Übersetzer des Forums ist allerdings wirklich gut. Ich verlasse mich selbst darauf, um jede deutsche Antwort in diesem Thread zu lesen, und ich finde ihn zuverlässig genug, um der Diskussion recht genau zu folgen — einschließlich Huberts detaillierter technischer Punkte, die nicht einfach sind.
Ganz allgemein: Ich bin seit fünf Monaten im Mikroskopie-Forum und auf Photomacrography.net aktiv, und ohne das Feedback aus diesen Gemeinschaften — unter anderem von Hubert, Spektrum und Jürgen — wäre dieses Projekt noch in der Steinzeit. Ich bin zutiefst dankbar und hoffe, dass ich hier weiter dazulernen darf.
Wenn es dir lieber ist, übersetze ich gerne alle meine Beiträge ins Deutsche, bevor ich sie poste. Sag mir einfach Bescheid.
Viele Grüße,
Ramon
Zitat von: Lupus in August 25, 2026, 11:54:08 VORMITTAGdas sind eigentlich Ausnahmefälle, hier ist es ein spanischer Forumsteilnehmer.
Es ist doch ganz einfach: Rechte Maustaste - "Übersetzen in Deutsch" anklicken. Dann erscheint der ganze Thread auf Deutsch.
Vielen Dank, Hubert, dass du ein gutes Wort für mich eingelegt hast. Ich gehe gerade unseren Austausch zur Winterjasmin-Probe noch einmal durch. Ich muss dir sagen: Die KI stimmt deinem letzten Beitrag zum Jasmin inzwischen zu 100 % zu. Gib mir bitte etwas Zeit, dann antworte ich ausführlich.
Ramon
Zitat von: Lupus in August 25, 2026, 09:22:01 VORMITTAGWhat exactly does that mean? Do you have the emission spectrum from the data sheet?
Hi Hubert,
It means that my blue LEDs emmit unwanted green light.
I dont have datasheet for the blue LEDs. I bought them on Amazon and not much info was available. All the info I have on them is what the AI was able to dig out by the reference. That info being: Wavelength 460 nm (nominal, blue), LED package SMD 2835 (single-die, monochromatic blue — no phosphor).
You're right about the geometry — it's a stem cut from the side (longitudinal), not a transverse cross-section.
Zitat von: Lupus in August 25, 2026, 09:22:01 VORMITTAGHow long was the exposure time for this photograph?
The exposure was long — the software's Exposure setting was ~4550, near its maximum (~5100).
Your spectral point is well taken: with the 500 nm long-pass, the passband opens right where the green channel is most sensitive (~530 nm), so anything in the 500–560 band dominates, while the chlorophyll fluorescence at ~680 nm is faint and sits where the sensor is already going insensitive. The red is there, but swamped.
I always knew that my blue LEDs had a green tail (they emmit green). Putting together the green leak with the passband openning right where the green channel is most sensitive plus a chlorophyll fluorescence at ~680 nm where the sensor is already going insensitive...Well, its easy to understand why I dont see red.
Regarding the "green stuff" in the Jasmine. Please bear with me, I am still working on it. I just discovered that my Violet-UV LEDs are also leaking green light. I am building a DIY spectrometer to confirm it.
Just one question regarding the Jasmine. On the green halo, how could it be scattered light if there are no cell structures there that can scatter?
Ramon
Hello Ramon,
ZitatIt means that my blue LEDs emmit unwanted green light.
It is clear that a blue LED also emits longer-wavelength light tending towards the green. My graph shows the emission spectrum from the datasheet of my Nichia LED, which has a peak wavelength of 463 nm; this applies similarly to any LED. As the fluorescence efficiency is often only 1 per cent, or even less, unless special fluorescent dyes are used, any external light of this magnitude masks the fluorescence.
The UV LED also has a similar emission spectrum of comparable relative width, but I think this is barely relevant in the long-wavelength range >500 nm when the emission peak lies 100 nm away.
My question about the exposure time refers to the fact that a very long exposure time could be affected by stray light from the room lighting. Your lighting setup also picks up ambient light; if the room isn't darkened, this can also become a factor. I would carry out a blank test without LED lighting using the same exposure time.
ZitatJust one question regarding the Jasmine. On the green halo, how could it be scattered light if there are no cell structures there that can scatter?
It's clear that this is scattered light just because there are no fluorescent plant structures there. Perhaps there are scattering particles in the embedding medium of the specimen. These could be illuminated by emissions scattered horizontally from the nearby specimen. I consider this commercial specimen unsuitable for conducting fluorescence experiments. Your own plant section also shows – as far as can be seen in the image – no scattered light outside the section.
Regarding your fresh plant section: One disadvantage of this longitudinal, surface-cut section of the plant, compared with a cross-section, is that the chlorophyll autofluorescence is overlapped by the green scattered light from the non-chlorophyll plant cells lying behind it.
Hubert
Hello Hubert
Zitat von: Lupus in August 28, 2026, 10:35:26 VORMITTAGIt is clear that a blue LED also emits longer-wavelength light tending towards the green. My graph shows the emission spectrum from the datasheet of my Nichia LED, which has a peak wavelength of 463 nm; this applies similarly to any LED. As the fluorescence efficiency is often only 1 per cent, or even less, unless special fluorescent dyes are used, any external light of this magnitude masks the fluorescence.
The UV LED also has a similar emission spectrum of comparable relative width, but I think this is barely relevant in the long-wavelength range >500 nm when the emission peak lies 100 nm away.
Duly noted. This green leakage alters many of my interpretations of different samples under FrTIR fluorescence.
Zitat von: Lupus in August 28, 2026, 10:35:26 VORMITTAGMy question about the exposure time refers to the fact that a very long exposure time could be affected by stray light from the room lighting. Your lighting setup also picks up ambient light; if the room isn't darkened, this can also become a factor. I would carry out a blank test without LED lighting using the same exposure time.
I carried the blank test, it came out negative (see below) but I think that your point is fair. I live on the sunny Mediterranean coast, where we get a lot of bright days.
Blank under high exposure.jpg
The shading in the light guide comes from the fact that these guides are designed for light entering as a collimated or convergent beam, which requires discrete LEDs and lenses. For me it's much cheaper to use LED strips (a wide beam), which allow for simpler light guides. But with a wide beam, some of the light breaks the TIR condition before reaching the sample and ruins the contrast — hence the shading.
Bear in mind that these light guides are CNC-machined, and the cost rises sharply with complex geometries, such as the recesses needed to fit the lenses. Below is a photo of the two types of light guide I use: on the left, the discrete-LEDs-and-lenses type; on the right, the LED-strip type. It's also worth mentioning that, for proper contrast, the discrete-LEDs + lenses type needs to have its perimeter blackened.
Two types of light guides small.jpg
Zitat von: Lupus in August 28, 2026, 10:35:26 VORMITTAGIt's clear that this is scattered light just because there are no fluorescent plant structures there. Perhaps there are scattering particles in the embedding medium of the specimen. These could be illuminated by emissions scattered horizontally from the nearby specimen. I consider this commercial specimen unsuitable for conducting fluorescence experiments. Your own plant section also shows – as far as can be seen in the image – no scattered light outside the section.
I agree. Over the past two days I've been thinking about all kinds of possible explanations. One was that the mounting medium was fluorescing (mounting resins are known to fluoresce yellow-green), but I ran a test with Canada balsam and its fluorescence was far too faint to account for the signal.
Please let me know whether you agree with the following interpretation of the image below (Marchantia section with antheridia at 5× from a prepared sample, under brightfield, FrTIR/White, and FrTIR/Blue + Yellow LPF): what we're seeing in the FrTIR/B+Y LPF image is the green leaked from the blue LEDs and scattered by the cell structures (possibly with some added yellow-green from the yellow long-pass filter itself, which is a cheap one that can self-fluoresce) — except, perhaps, for some genuine fluorescence in the greener structure on the right side of the image.
Marchantia sec with artheridia 5x.jpg
And thank you, sincerely, for the time you spend on these answers — they're consistently informative, and precisely the kind of feedback I was hoping to find when I joined this community.
Ramon, from sunny Catalonia Spain :)
Hello Ramon,
As has already been discussed, combining blue light excitation with a 500 nm cut-off filter – which still allows a significant proportion of the longer-wavelength excitation light to pass through – is not suitable for a clear demonstration of fluorescence.
In the image below, I have provided a comparison between dark-field illumination using white light (left) and blue light excitation (right). You can see individual structures that glow significantly brighter on the left and are therefore exhibiting fluorescence.
The image intensity has been adjusted in some pictures through image processing.
Marchantia DF Weißlicht Blaue Anregung.jpg
I can barely detect any difference in fluorescence between green and red emissions, except in small areas at the bottom right. I am not a biologist; with the relevant knowledge, one could explain which areas should theoretically exhibit fluorescence due to the plant's structure. There is a good reason why one always uses a suitable set of narrow-band excitation and cut-off filters in fluorescence microscopy. This is also because fluorescence often occurs relatively close to the excitation wavelength.
In order to distinguish between red chlorophyll fluorescence and the green fluorescence of other cellular components, one would need to take one image using a blue LED and a red cut-off filter, and another using a UV LED and a green bandpass filter.
Hubert
Hi Hubert,
Well, I think you explained it all. I am comfortable with all you said.
Following up on your point that residual excitation still registers in the G and (less) the R channel — and, bigger still, the green the LEDs leak straight into the filter's passband — I've asked the AI to check, pixel by pixel, whether the red channel is simply a weighted combination of the green and blue channels. I've now put a number on it, on the images already in this thread (the winter-jasmine and the Marchantia) plus one clean control I ran to isolate the effect. Short version: the "red" in these images, under blue/violet excitation with no red in the illumination, is a camera artifact — the red channel is almost entirely reconstructable from the green and blue channels.
A word on the notation, since it recurs below. For each pixel I write its red value as a weighted combination of its own green and blue values plus a constant — R = a·G + b·B + c — and let a least-squares fit choose the weights (a, b) and the offset (c) that best reproduce the actual red across the whole image. The R² that comes with it scores the match: 0 means no relation, 1 means the red is reproduced perfectly. If the red is nothing but crosstalk from green and blue, this simple formula fits it well and R² sits near 1. (The offset c is small, so I drop it from the fits quoted below and report only the weights a and b.)
1. The winter-jasmine. The fit on my blue + yellow-LPF capture: R ≈ 0.26·G + 0.89·B (R² = 0.89) — ~90% of the red reconstructed from green and blue. So the "red areas" that looked like they might be chlorophyll are crosstalk.
2. The Marchantia. Same result, and at the same strength as the jasmine. On a full-RGB capture of the section: R ≈ 0.28·G + 0.66·B (R² = 0.87) under blue excitation, and R² = 0.91 under UV. Your DF separation already showed the red tracking the green; with the blue channel available too, the full two-term fit confirms it — the red is a combination of green and blue, nothing more.
3. A clean control (new) — a moss section. The moss is the cleanest of the three: green and blue are both strongly present, so the two terms separate well and the fit is tightest:
R ≈ 0.39·G + 0.63·B (R² = 0.96)
96% of the red channel reconstructed from green and blue — the cleanest demonstration that the red here is simply a recipe of the other two, not a record of red light.
Moss LS of arteridial cluster 5x.jpg
Channel separation:
Moss LS - channel separation (FrTIR-W, B+YLPF, UV+YLPF).jpg
The three fits together — every one of them says the red channel is a linear combination of green and blue:
Camera crosstalk fits table.jpg
A methodological corollary, following your own point. Because the red is reconstructable as R ≈ a·G + b·B, amplifying the red channel to look for chlorophyll doesn't really test for it: brightening the red just brightens a scaled copy of green and blue, so "red areas" appear whether or not any chlorophyll is present — and the amplification makes that artifact look like real structure. The honest test isn't the amplified red image but whether any red survives above the a·G + b·B prediction (the "excess"). In these images almost none does — which is exactly what the R² approaching unity reflects.
Honest caveat / next step. The real fix is upstream — an excitation band-pass filter to strip the LEDs' green (and residual blue) at the source, which is what the FrTIR gadget still lacks. You had already flagged this gap yourself.
I've been sourcing excitation filters to place over the LEDs, and the Rosco and LEE lighting gels look like the most affordable fit. The one caution I've had is that such gels cut the excitation intensity a lot — which, with an already faint fluorescence, could leave the signal too dim to capture.
Thank you again — this exchange has been highly informative.
Ramon
Hi Ramon -
The fluorescence phenomena you're anaylzing are not only due to a mixture of single components but by their interaction in a very complex photosynthetic system. That makes it very difficult to interpret your observations.
Around 50 years ago we have been studying delayed fluorescence of plant leaves caused by inhibitors of photosynthesis like draught or herbicides - e.g. Simazin which inhibits the transfer of electrons from the excited chlorophyll molecules. The unused energy is then returned by a delayed red fluorescense (sort of luminescence).
In this context it would be interesting to know whether the photosynthetic system in your fresh preps is still intact.
See the following article in Wikipedia:
https://en.wikipedia.org/wiki/Chlorophyll_fluorescence
Good luck with your illumination device!
Rolf
Hello Rolf
Zitat von: reblaus in September 03, 2026, 12:30:33 NACHMITTAGSThe fluorescence phenomena you're anaylzing are not only due to a mixture of single components but by their interaction in a very complex photosynthetic system. That makes it very difficult to interpret your observations.
Around 50 years ago we have been studying delayed fluorescence of plant leaves caused by inhibitors of photosynthesis like draught or herbicides - e.g. Simazin which inhibits the transfer of electrons from the excited chlorophyll molecules. The unused energy is then returned by a delayed red fluorescense (sort of luminescence).
In this context it would be interesting to know whether the photosynthetic system in your fresh preps is still intact.
See the following article in Wikipedia:
https://en.wikipedia.org/wiki/Chlorophyll_fluorescence (https://en.wikipedia.org/wiki/Chlorophyll_fluorescence)
Good luck with your illumination device!
I am starting to find out that fluorescence mikroscopy is not a joke. The more I learn the more I realize that there is so much that I dont know that I feel humbled. I find it beautiful and fascinating. Few weeks ago I knew nothing about fluorescence. I just aim at being able to do some kind of amateurish fluorescence that can interest entry level microscopists. I will keep posting my journey into the interesting field of fluorescence mikroscopy and other applications of the gadget.
Thanks a lot for your warm ending words :)
Ramon
Hello Ramon,
Here is a short post on autofluorescence, which includes two good examples of simultaneous green fluorescence from lignified cell regions and red chlorophyll fluorescence.
https://www.mikroskopie-bonn.de/bibliothek/botanik/151.html
One of the authors has also posted a few threads on this topic here in the forum, here is an example:
https://www.mikroskopie-forum.de/index.php?topic=35600.msg260410;topicseen#msg260410
However, he always used high-quality Zeiss filter sets, which allow the excitation and emission wavelengths to be closer together.
And the plant sections were freshly prepared in each case.
Hubert
Hello Hubert and Rolf,
Finally the LEE excitation filters arrived. I repeated the test using an excitation filter to remove the green leak of the blue LEDs. And the results are not what I expected.
In our earlier exchange we concluded that the green "halo" around the jasmine section was most likely scattered green leaking from my blue LEDs, rather than fluorescence. I have since built a simple DVD-grating spectroscope and run a test that disputes what we thought was clear.
The LED spectrum and the gel (Fig. 1 — qualitative)
• White LED: full continuous spectrum — reference.
• My blue LED: a dominant blue peak, but with a clear green tail (leak) and a faint red — so the LED does leak green (and a little red), as suspected.
• Blue LED + a LEE "Deep Blue" 120 gel over the source: the green and red are gone; essentially clean blue reaches the sample.
This is a DIY spectroscope + phone camera, so please treat it as qualitative, not calibrated — but the leak and its removal by the gel are unambiguous. LEE's own published transmission curve for this gel (Fig. 1b) confirms it quantitatively: it passes ~38% at my 450 nm excitation line but drops the green-leak band to <0.3%. (Its quoted Y = 1.1–2.1% is luminous, i.e. eye-weighted, transmission — which is why the gel looks almost opaque yet still lets the blue excitation through.)
Spectrometer - White vs Blue vs Blue+DeepBlue120 (montage) small.jpg
Fig. 1 — DIY DVD-grating spectroscope. White LED (full spectrum), blue LED (blue peak + green/red leak), blue LED + Deep Blue 120 (leak removed). Qualitative; both blue panels brightened equally.
LEE 120 Deep Blue - spectral transmission (from LEE Filters).jpg
Fig. 1b — LEE 120 Deep Blue spectral transmission, plotted from LEE Filters' published data. ~38% at 450 nm; green-leak band <0.3%.
The prediction.
If the halo is scattered leaked-green, then filtering that green out at the source (Deep Blue 120) should make the halo disappear.
The result (Fig. 2).
It does not. Same section, same blue excitation, same Thorlabs 500 nm emission filter — the only change is the Deep Blue 120 gel on the source. The green halo is still clearly there. A rough halo-to-tissue green ratio (which cancels the exposure difference between the two frames — they were matched by eye at the eyepiece, not metered) is essentially unchanged: ~0.40 without the gel, ~0.42 with it.
Jasmine halo - Blue vs Blue+DeepBlue120 (montage).jpg
Fig. 2 — Same jasmine section, blue excitation, Thorlabs 500 nm emission. Only the Deep Blue 120 excitation gel differs. The green halo persists.
Removing the green leak does not remove the halo, so "scattered leaked green" explanation must be put on hold.
Honestly guys, this is as far as I can go. I have no explanation. Help is very welcome here! :)
Ramon
Hi Ramon,
I'm not familiar with plant sections, but if I understand correctly, you are using an older, commercially available, and stained section of jasmine. In my opinion, that is an unsuitable starting point; the strong scattered light between the cell walls is also a clue.
My fresh cutting in water (post #17) was also excited by a blue LED. The green here is not fluorescence, it is also blue excitation of the green color channel - but mainly on the cell walls. I doubt that green fluorescence can be observed with blue excitation using such simple tools.
Hubert
Hi Ramon,
I've got quite a number of stem cuttings embedded in Euparal showing similar effects. I think they are due to diffusion of stains since it wasn' present in fresh preps but increased during time. I suppose some of the compounds are soluble in the Euparal solvent (Isoprop ?)
Rolf
Zitat von: Lupus in September 10, 2026, 10:22:28 VORMITTAGMy fresh cutting in water (post #17) was also excited by a blue LED. The green here is not fluorescence, it is also blue excitation of the green color channel - but mainly on the cell walls. I doubt that green fluorescence can be observed with blue excitation using such simple tools.
Hi Hubert
On the jasmine — you're right. It's a commercial slide bought online (unknown staining and mounting), a poor starting point exactly as you say. Let's forget about it.
Moving on, your opinion regarding the green fluorescence on the FrTIR gadget leaves me in a quite uncomfortable spot. Let me explain.
I my post #4 regarding the green fluorescence on the fern leaf I explain that this very sample was sent to Pau (Photomacrography.net) — whose expertise I trust — he imaged it on a professional epifluorescence microscope and read it as genuine green fluorescence, and the FrTIR gadget reproduces the same green cell-wall pattern. So I am holding two expert readings (you and Pau) that point in opposite directions, and I don't feel I can honestly claim fluorescence until I understand why. I'd genuinely like to come clear on this with you: how do you read the fern result?
Regards
Ramon
Hi Ramon,
I don't see any contradiction in the opinions; my doubt concerned the winter jasmine. This specimen apparently exhibits strong scattered light, whereas the fern leaf does not. The fern was excited with violet light (400 nm). My most recent doubt ("blue excitation using simple means") referred to the close proximity of the blue excitation spectrum - without a blocking filter - to the 500 nm long-pass filter.
Hubert
Zitat von: reblaus in September 10, 2026, 15:06:29 NACHMITTAGSI've got quite a number of stem cuttings embedded in Euparal showing similar effects. I think they are due to diffusion of stains since it wasn' present in fresh preps but increased during time. I suppose some of the compounds are soluble in the Euparal solvent (Isoprop ?)
Hi Rolf
That makes a lot of sense. Do you think that these stains are fluorescing or scattering?
Ramon
Hi Ramon -
It might be both. Looking for a wellknown substance contained in the cuttings (stilbene, blue fluorescence) I had no time to worry about side effects which didn'disturb at that time.
But I'll see whether I can find the old slides.
Rolf
Zitat von: Lupus in September 10, 2026, 22:43:17 NACHMITTAGSI don't see any contradiction in the opinions; my doubt concerned the winter jasmine. This specimen apparently exhibits strong scattered light, whereas the fern leaf does not. The fern was excited with violet light (400 nm). My most recent doubt ("blue excitation using simple means") referred to the close proximity of the blue excitation spectrum - without a blocking filter - to the 500 nm long-pass filter.
Hi Hubert. My apologies, I miss read your post. And honestly, I'm relieved! It's good to know you and Pau are essentially of the same mind on the fern.
On the optics: a 450 nm excitation with a 500 nm long-pass sits too close together — too small a separation for a clean test of green fluorescence. A 400 nm excitation with the same 500 nm cutoff is the cleaner pairing. Now I get it.
Regarding the green halo on the Jasmine. Could it be scattering and fluorescence as Rolf suggest?
Ramon
Zitat von: reblaus in September 11, 2026, 12:45:00 NACHMITTAGSIt might be both. Looking for a wellknown substance contained in the cuttings (stilbene, blue fluorescence) I had no time to worry about side effects which didn'disturb at that time.
But I'll see whether I can find the old slides.
Hi Rolf. Perfect!
Big thanks
Ramon
Hi Ramon,
ZitatRegarding the green halo on the Jasmine. Could it be scattering and fluorescence as Rolf suggest?
I do not know, it might be one or the other. But perhaps fluorescence can be ruled out by using long-wavelength lighting (red LED).
Zitat... too small a separation for a clean test of green fluorescence
It is worth noting that the issue is not the small wavelength difference itself, but specifically the insufficient narrowing of the excitation wavelength.
Hubert
Hi Hubert
Zitat von: Lupus in September 12, 2026, 08:42:42 VORMITTAGI do not know, it might be one or the other. But perhaps fluorescence can be ruled out by using long-wavelength lighting (red LED).
Thanks, I will do the red light test
Zitat von: Lupus in September 12, 2026, 08:42:42 VORMITTAGis worth noting that the issue is not the small wavelength difference itself, but specifically the insufficient narrowing of the excitation wavelength.
Noted. Thanks!
Ramon
Hi Ramon -
just to show problems with fluorescence:
0-3: photos from 2015 (section of leaf of Casuarina, unstained, in Euparal)
4: photo from the the same section 11 years later
0 transmitted light
1 epifluorescence 365 nm Zeiss (Filter 01: 365 395 397)
2 450-490 (Filter 09: 450-490 510 520)
3 510-560 (Filter 14: 510-560 580 590)
4 like 2 but from 2026. Low magn. to show the halo
My conclusion: from 2015 to 2026 components of the unstained section diffused into the drying Euparal forming a halo.
Rolf
Hello Rolf
Zitat von: reblaus in September 14, 2026, 12:27:20 NACHMITTAGSMy conclusion: from 2015 to 2026 components of the unstained section diffused into the drying Euparal forming a halo.
I think your images prove your point. The halo is diffussion over time of the sample endogenous tissues to the mounting media.
Problem solved! :)
I would like to know your opinion on this other sample; Moss LS of arteridial cluster from a comercial collection. Can I claim that the green smudge is fluorescence?
Moss---multimodal-montage-(BF,-White,-Blue,-UV,-DeepBlue-gel).jpg
Thks!
Ramon
Hi Ramon -
sloppy prep.- what else should that be ?
Rolf
Hi Ramon,
Once again, please show us some alive and water mounted specimen.
Not already mounted ones.
Choose some algea or some moss leaf instead!
That would be of much higher value, to jugde what's going on here.
Greetings Holger
Hi Rolf and Holger
Zitat von: reblaus in September 16, 2026, 18:16:26 NACHMITTAGSsloppy prep
In my blissful ignorance I didnt spot that the sample was defective. Thanks for pointing it out.
Zitat von: reblaus in September 16, 2026, 18:16:26 NACHMITTAGSwhat else should that be ?
Let me explain why I keep double-checking things that must look obvious to you. I know my knowledge is limited, and when someone in this forum raises questions I take them very seriously. When this thread started the idea met a fair bit of skepticism — there were even jokes. So I thought that I was overclaiming, luckily the specimen in question (Fern leaf) was imaged with a proper Fluorescence rig and I could back my claims.
There's also a concrete reason for the caution: I have a patent application pending on the illuminatio device that I use, so I genuinely cannot afford to overclaim — anything I put out has to be something I can stand behind if I'm challenged.
Zitat von: Spectrum in September 16, 2026, 19:45:47 NACHMITTAGSOnce again, please show us some alive and water mounted specimen.
Not already mounted ones.
Choose some algea or some moss leaf instead!
That would be of much higher value, to jugde what's going on here.
Yes Holger. I am on it. Remember that my blue LEDs had a green leak so I ordered some gel filters from LEE. Now I have a gel filter Deep Blue 120 that blocks the green leak pretty well (I checked it with a DIY spectrometer). I will be imaging algae and fresh stems pretty soon.
Ramon