Multimodale Koregistrierung an einem Standardmikroskop

[Lupus]

Hi Ramon,

Regarding 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).

... 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

[Ramon]

Hi Hubert

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).

Thanks, I will do the red light test

is worth noting that the issue is not the small wavelength difference itself, but specifically the insufficient narrowing of the excitation wavelength.

Noted. Thanks!

Ramon

[reblaus]

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

[Ramon]

Hello Rolf

My 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?



Thks!
Ramon

[reblaus]

Hi Ramon -

sloppy prep.- what else should that be ?

Rolf

[Spectrum]

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

[Ramon]

Hi Rolf and Holger

sloppy prep

In my blissful ignorance I didnt spot that the sample was defective. Thanks for pointing it out.

what 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.

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.

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