MicrobeHunter.com Microscopy Forum

Hey guys, i hope you are doing fine.

Do you think a blue led light and a cheap yellow filter in a standart microscope is enought for seeing some autoflorescence in the samples? Wáter ponds or vegetal tissue... Or i should forget that?

Highly unlikely in a "standard" trans-illuminated microscope. Some internet sites have advocated sort of DIY epi-illumination arrangements on such microscopes, like a diagonal excitation LED beam (from above) combined with a long-pass filter on the eyepiece, etc. I am afraid that most of them are over-optimistic. Fluorescence microscopy requires a strong illuminating (say, blue) beam and a VERY efficient long pass filter that passes ONLY the (say, green, yellow, red) weak fluorescence. Usually a dichroic mirror at 45 degrees must be added to achieve such separation. It is explained in detail in the fluorescence sections of online microscopy sites of Nikon, Olympus, Zeiss etc.

Hobbyst46 wrote: ↑

Wed Nov 08, 2023 11:34 pm

Fluorescence microscopy requires a strong illuminating (say, blue) beam and a VERY efficient long pass filter that passes ONLY the (say, green, yellow, red) weak fluorescence.

Why would you be using a blue light, instead of a UV light? Not only would UV be easier to block, but wouldn't it also create excitation with less input power?

This approach works well at lower magnifications:
https://www.photomacrography.net/forum/ ... hp?t=33123

Results here:
https://www.photomacrography.net/forum/ ... hp?t=33112

But be careful, UV damage is real.

Topcode wrote: ↑

Thu Nov 09, 2023 12:48 am

Hobbyst46 wrote: ↑

Wed Nov 08, 2023 11:34 pm

Fluorescence microscopy requires a strong illuminating (say, blue) beam and a VERY efficient long pass filter that passes ONLY the (say, green, yellow, red) weak fluorescence.

Why would you be using a blue light, instead of a UV light? Not only would UV be easier to block, but wouldn't it also create excitation with less input power?

Blue was just an example. To trigger fluorescence at any wavelength, one must excite (illuminate) at a lower wavelength. The mere appearance of fluorescence depends on the wavelength. In addition, the brightness of the fluorescence grows as the intensity of the excitation grows. In other words: UV excitation (regardless of intensity) does not insure brighter fluorescence than blue excitation (regardless of intensity). And vice versa.
In practice, common inexpensive excitation lamps provide a fairly wide spectrum, so some violet-looking light may contain a near-UV component.

Some materials fluoresce when excited by UV, some when excited by blue, some when excited by green, it depends on the molecules involved. Plant auto-fluorescence is often due to chlorophylls (they shine red light), when excited by near UV or violet light.

If your purpose is to excite green plants or algae, violet (400-430nm) is an option. Still, using it on the "standard" trans-illumination system is controversial from the health aspect. The human eye is not well adapted to strong monochromatic light.

I would strongly suggest against using a UV lamp in what you describe as "standard" microscope, since UV light endangers human vision, and relying on a long pass filter in series as single protection would be risky. Even when UV illumination is not direct, such as in epi-illumination, precaution is a must.

The links provided by Viktor Nilsson above are examples of DIY "half-epi" illumination. Not "standard".

Here is a further example to the links provided by Viktor Nilsson, using a blue LED (450 nm) flashlight (torch) and a cheap acrylic orange filter to look at chlorophyll autofluorescence with a basic microscope https://sfmicrosociety.org/features/cli ... -challenge. The yellow filter from the same acrylic set blocked blue light but not sufficiently well to fully exclude the excitation light, so the background scattering dominated the image. To get high blocking with a yellow filter requires a research grade specifications. Note that the dichroic mirror in commercial fluorescence microscopes separates the excitation and emission beams, but the discrimination is not very high. It may let > 1% of the light leak through but to get good fluorescence signal/background you need to block to around optical density (OD) > 4 (<0.01% light breakthrough). This is why excitation and emission filters are used in conjunction with the dichroic mirror. The most sensitive of all fluorescence detection, which reaches the single molecule limit, is best achieved without a dichroic mirror. Instead excitation light is introduced by total internal reflection by illumination from the side of the specimen.

I dont want to use UV, too risky. So with a blue led still not enought.. i think i Will stay with bright fiel, darkfield, polarization, waveplate) and PContrast...

Take a look at the QBC Paralens Advance system…

Try looking at …

https://pubs.acs.org/doi/10.1021/acs.jchemed.5b00984

Inexpensive, Open Source Epifluorescence Microscopes

Chris Stewart and John Giannini

I did It. Using yellow filter from my PC kit with a blue led.
I watched some autoflorescence in some leaves. It was not a complete black background but you can tell where is the fluorescent stuff coming.

I cant expect proffesional quality or lots of thing yo see because its not a proffesional fluorescence microscope and i dont have the expensive dyes.

I have regular fluoresceine, so i can water a plant but nothing more. Do you know if i could mix methilene blue with fluorescein to have It attached to specific parts?