Infinite Fluorescence

micro_pix · Post by **micro_pix** » Tue Jun 09, 2020 6:35 pm

Not very original as Fern sori in UV have been done so well before on the forum but I thought it would be a good way to test my modification to the BH2 RFL fluorescence attachment - so that I can use it with the infinity Olympus MS Plan objectives.

These are sori from ferns in my garden. One particular plant produced sori with really bright auto-fluorescence that hardly faded during a >100 photo stack. it's illuminated with blue excitation (400-490) by mercury lamp. The images are stacked in Zerene and post processed to remove stacking artifacts, the background has also been tidied.

(Edited: this was in Blue excitation not UV).

David

The sori are bursting out from the membrane that normally covers them on the underside of the leaf, the membrane is illuminated by the aotofluorescing sori inside.

Sumguy01 · Post by **Sumguy01** » Tue Jun 09, 2020 7:53 pm

Very nice.
Thanks for sharing.

Lou Jost · Post by **Lou Jost** » Tue Jun 09, 2020 8:40 pm

Really excellent! How do you keep them from dispersing their spores? I have trouble getting them to hold still.

Also, do you know what kind of fern this is?

Saul · Post by **Saul** » Tue Jun 09, 2020 11:21 pm

David, incredible stacks ! Any chance for the stereo ?

Pau · Post by **Pau** » Wed Jun 10, 2020 1:24 am

Very nice!

What's your modification?
I can guess removing both telan lenses of the epiilluminator and adding a tube lens or maybe just removing the lower telan lens (I've tested both approaches with my Zeiss but the second one didn't work well)

jmc · Post by **jmc** » Wed Jun 10, 2020 2:07 am

Beautiful, thank you for sharing them.

micro_pix · Post by **micro_pix** » Wed Jun 10, 2020 2:28 am

Thanks for the kind comments.

Lou Jost wrote:Really excellent! How do you keep them from dispersing their spores? I have trouble getting them to hold still.

Also, do you know what kind of fern this is?

I had a few stacks messed up by sori uncurling, obviously prompted by the UV. The fluorescence was so strong I was able to use the ND filter and still get ASA 100. With the filter in there was less activity and the odd one “popping off” while out of focus was easy to overcome in Zerene, as long as the whole subject didn’t jump around.

(edited) Having done some research I think this is Dryopteris filix-mas Interestingly though I tried two separate plants within two metres of each other and one gave very weak and mostly red fluorescence with hardly any yellow at all.

Saul wrote: incredible stacks ! Any chance for the stereo ?

It would make a good subject wouldn’t it. I’ll have a go.

Pau wrote: What’s your modification?

At the business end of the Olympus BH2 RFL, once you slide out the dichroic mirror, there’s only one lens assembly, at the bottom, that sits between the nosepiece and the dichroic mirror. I assumed that this was the diverging lens that compensates for the increased tube length in the finite system so I took it out. There are no other lenses, now it’s an empty tube so no Telan lens above the mirror (I bought a second RFL for this, the lens is easy to remove but not something you would want to keep doing).

Above the RFL I have a Raynox 5230, ThorLabs tubes, and the camera. I use Cam Ranger to my iPad as a monitor and to fire the camera. Without the RFL it’s a nice infinite system and works well with the later UIS 2 objectives. For these photos I used the Olympus MSPlans, they should get final chromatic compensation in the eyepiece/Photo eyepiece but it’s not really an issue in fluorescence because of the limited colours.

David

WalterD · Post by **WalterD** » Wed Jun 10, 2020 2:59 am

Hi David, excellent set and classic object for fluorescence microscopy.
Thanks for sharing.

Pau · Post by **Pau** » Wed Jun 10, 2020 3:27 am

Thanks, David.
So you use the microscope without head, do you?
I do the same with my few infinite objectives, in my case removing the Telan lenses.

micro_pix · Post by **micro_pix** » Wed Jun 10, 2020 3:41 am

Pau wrote: So you use the microscope without head, do you?
I do the same with my few infinite objectives, in my case removing the Telan lenses.

Yes, I have just used my horizontal macro rig - instead of the RMS adapter and objective at the end I put on a BH2 dovetail and that replaces the BH2 head. I quite like using the iPad and Cam Ranger for fluorescence, especially as it keeps me away from the UV. For standard epi (pol brightfield and darkfield) I use a BH2 UMA and standard head/photo-eyepiece set-up.

David

micro_pix · Post by **micro_pix** » Wed Jun 10, 2020 6:13 am

Lou Jost wrote:
Also, do you know what kind of fern this is?

I have done some research and I reasonably convinced that this is Dryopteris filix-mas and definitely not Bracken Fern. I’ll edit my earlier reply. Both of the plants sori I had looked at appear to be the same species but one had a much stronger auto-fluorescence - consistently on separate samples - otherwise the plants/leaves looked to be the same maturity.

David

Charles Krebs · Post by **Charles Krebs** » Wed Jun 10, 2020 11:09 pm

Beautiful images, from an obviously successful (and ambitious) modification.
Congratulations!

iconoclastica · Post by **iconoclastica** » Thu Jun 11, 2020 1:21 am

micro_pix wrote:
Lou Jost wrote:
Also, do you know what kind of fern this is?
I have done some research and I reasonably convinced that this is Dryopteris filix-mas and definitely not Bracken Fern. I’ll edit my earlier reply. Both of the plants sori I had looked at appear to be the same species but one had a much stronger auto-fluorescence - consistently on separate samples - otherwise the plants/leaves looked to be the same maturity.

David

If it was bracken you would know: their sori are in infinite strips in a fold of the leaf margin. The male fern has confined roundish sori, but I would have expected to see the indusium (a thin cover over the sorus). If you are in Europe, it is the most common species to pop up spontaneously in gardens though.

Did you make a preparation on a slide? If the sporangia are in water, they will not open. If it is filix-mas, it's still a bit early for them to shed the spores, although it won't be long now.

Marek Mis · Post by **Marek Mis** » Thu Jun 11, 2020 6:18 am

Great images !

micro_pix · Post by **micro_pix** » Thu Jun 11, 2020 8:15 am

Thanks for the kind comments all.

iconoclastica wrote:

The male fern has confined roundish sori, but I would have expected to see the indusium (a thin cover over the sorus). If you are in Europe, it is the most common species to pop up spontaneously in gardens though.

Did you make a preparation on a slide? If the sporangia are in water, they will not open. If it is filix-mas, it's still a bit early for them to shed the spores, although it won't be long now.

Hi Iconoclastica,

Yes, the sori were covered by round shaped indusium, between 3 and 12 of them on the underside of each leaf. The first image is of the sporangia on their own and in the second image, the membrane that the sporangia are sitting in, is the indusium.

Very few sori looked mature yet, the sporangia seem to go dark as they mature. I’ll have a look at the spores when I can. I’m pretty sure that it is filix-mas.

Here are the sori under the leaf. The second photo at the top of this post is a side-on view of a sorus that has been detached from the leaf and flipped over, the sporangia are protruding out from the area where it was attached to the leaf.