Pollen autofluorescence

Pau · Post by **Pau** » Sat May 16, 2020 11:59 am

Some big pollen grains from garden flowers.

All taken with a Nikon M Plan Apo 40/0.80 (Extreme Short Working Distance

)
Epi fluorescence with UV 380nm LED, Zeiss WL, direct projection

1. Hibiscus

2. Lilium, white cultivar

3. Amarillis

Olympusman · Post by **Olympusman** » Sat May 16, 2020 1:32 pm

Very nice pollen images. Dry mounts?

Mike

viktor j nilsson · Post by **viktor j nilsson** » Sat May 16, 2020 2:19 pm

Very nice set, Pau.

Figured this could be a good time to tell you that I've been greatly inspired by your LED fluorescence setup. In fact, over the last year or so I've been toying with the idea of 3D printing a Kohler-capable LED-based epi fluorescence illuminator for my old Wild M20, using various optics and irises that I've had lying in my drawers. I've tried out many different combinations on my rudimentary optics bench (holding optical comments with play-doh!) and come up with a solution that should work pretty well. It definitely taught me a lot about optics and illumination systems.

This week, however, I was able to pick up a Zeiss fluoro condenser relatively cheap, so I'll probably end up modifying that for my needs instead. After realizing how finicky it can be to construct a good and compact epi illuminator, I'm sure I'll appreciate having such a robust unit to build upon.

At my typical speed, it'll probably take a year or so to get it going, but I'll post it here if I ever do.

Ken Ramos · Post by **Ken Ramos** » Sat May 16, 2020 4:22 pm

These are all very detailed and I like those pastel colors but that first one, though really good, reminds me of that stupid virus that is going around right now.

Lou Jost · Post by **Lou Jost** » Sat May 16, 2020 6:51 pm

These are very beautiful images.

sebba28 · Post by **sebba28** » Sat May 16, 2020 7:07 pm

Very,very nice.

WalterD · Post by **WalterD** » Sun May 17, 2020 12:57 am

Excellent images, the Hibiscus pollen are the most spectacular, I think.

Pau · Post by **Pau** » Sun May 17, 2020 1:39 am

Thanks all you so much for your very kind comments!

- Mike, yes they are dry, just placed on the glass slide and arranged to be flat enough to allow being imaged with this 0.7mm WD objective (I've needed to clean pollen from the objective tip twice during the Hibiscus session)

- Ken, in fact it seems closer to the the actual coronavirus morphology than many of its representations shown at the media.

- Viktor, I'm glad that you found my setup ideas useful. Now it has been improved (some new LEDs, a new protective shield...) I plan to update the post when I deal with my desktop mess

Will be very interesting to see your setup when done

jmc · Post by **jmc** » Sun May 17, 2020 6:26 am

Fab pictures. Great to see strong UV induced fluorescence from 380nm. Was worried that shorter wavelengths would be needed.

Adalbert · Post by **Adalbert** » Sun May 17, 2020 6:30 am

very nice

Sumguy01 · Post by **Sumguy01** » Sun May 17, 2020 7:41 am

Very nice.
Thanks for sharing.

viktor j nilsson · Post by **viktor j nilsson** » Mon May 18, 2020 4:02 am

Pau wrote: - Viktor, I'm glad that you found my setup ideas useful. Now it has been improved (some new LEDs, a new protective shield...) I plan to update the post when I deal with my desktop mess
Will be very interesting to see your setup when done

Looking forward to seeing your updates! May I ask how you updated the protective shield? I have a large sheet of orange acrylic on hand that I plan to use. It's from a crafts store, but from what I've gathered, any orange acrylic should absorb virtually all UV radiation.

Pau · Post by **Pau** » Wed May 20, 2020 2:46 am

Thanks Jonathan, Adalbert and Sumguy for your kind comments

Pau · Post by **Pau** » Wed May 20, 2020 2:49 am

viktor j nilsson wrote:May I ask how you updated the protective shield? I have a large sheet of orange acrylic on hand that I plan to use. It's from a crafts store, but from what I've gathered, any orange acrylic should absorb virtually all UV radiation.

Not sure about this, Acrylic by itself does not absorb UV (Polycarbonate does it very effectively although I've not found orange or red tinted polycarbonate). Maybe orange tinted could do but I would test it before to be sure.

What I use are dedicated microscope UV shields, both bought at Ebay from Nikon and Leica microscopes. The Nikon orange one I already had is pretty effective but does not cut all UV wile the new red one* I've adapted to shield the LEDs cuts all it at least within my primitive qualitative testing ability**

*from an inverted Leica DMIRB microscope
**I test them with a Nichia 365nm in a Convoy torch shinning to the wash machine darkend room -lots of highly fluorescent stuff in there!
I wear UV protection eyeglasses, of course made of polycarbonate and formerly tested.

Here you have a poor quick phone photo showing both shields. The red one is to prevent the potential damage when a LED is illuminated while placed outside the right position inside the illuminator, here I've switched on the green one out of place to show the effect

viktor j nilsson · Post by **viktor j nilsson** » Wed May 20, 2020 3:55 pm

Thanks, Pau!
Nice looking upgrades. I've been a little worried about those unshielded LEDs. Now I can finally sleep.

I will for sure test my orange acrylic in a spectrophotometer at work before I do anything stupid (and I will wear proper polycarbonate eyewear). But I do believe that it will turn out to work well as I don't think that there is anything really special about microscope protective shields. I base this on for example this discussion:
https://openbuilds.com/threads/laser-pr ... post-68282

And this paper:
https://www.nature.com/articles/bdjopen201714

And various graphs like figure 23 here:
https://www.plasticonline.com.au/plexig ... tallation/

But as I said, I will test it first. And I don't encourage anyone to take any chances.