Sporangia, Sori and autofluorescence (2)

Images made through a microscope. All subject types.

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WalterD
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Sporangia, Sori and autofluorescence (2)

Post by WalterD »

In my previous thread I mentioned my search for unopened sporangia. (Sporangia are the structures that contain spores and can be found on ferns and other plants. Sori are clusters of sporangia)
I found them at the end of my holiday in a tropical country. The climate there allows development of sporangi allthrough the year. Ferns were abundant over there, and I was able to find many different variants. Back home it was fun to observe them with a fluorescence microscope.
Many differences in size, colour of radiated light, size of sori and how populated on the leafs. E.g. the sporangi in photo 6 were exclusively covering the outer edge of the soft finger shaped leafs.

Photo 1 to 3 show a not yet identified plant with long flat yucca-ish leafs. The sporangia were on the backside in a square pattern.
Photo 1 and 2 with (approximately) 7x objective and respectively the green and blue filter block. (merged from 2 stacks) Photo 3 the same but with 9x objective. The remaining pictures were also taken with the 9x. Photo 4 and 5 are from one of the ferns, you can see some sporangia cracked open. Photo 6 as previously mentioned, radiation colour more purple and blue. Excitation for all was ultraviolet.

1.
Image

2.
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3.
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4.
Image

5.
Image

6.
Image

That's it for today, more types in progress :)

Note: (In case you didn't know, the microscope I used for this thread is descibed here: http://www.photomacrography.net/forum/v ... hp?t=36484)

Regards,

Walter
Last edited by WalterD on Sun Nov 11, 2018 1:21 pm, edited 3 times in total.

Saul
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Post by Saul »

Very nice pictures Walter !

JH
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Post by JH »

Interesting pictures!
Best regards
Jörgen Hellberg
Jörgen Hellberg, my webbsite www.hellberg.photo

Sumguy01
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Post by Sumguy01 »

=D> Very nice set.
Thanks for sharing.

Marek Mis
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Post by Marek Mis »

Great images Walter ! Number 4 and 5 are stunning !

Marek

Jacek
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Post by Jacek »

SUPER !

Pau
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Post by Pau »

Walter, very nice pictures, again improved over older ones.

Fern sporangia are also one of my favorite subjects. I want some info about your pictures, in particular #4 and #6:
Could you tell me the exposure time and ISO settings for these pictures and your objective NA?
The issue I have is the fast photobleaching of chlorophyll both with UV and blue excitation that prevents to have well exposed leaf and sporangia together in the same stack...likely my excitation intensity is too high.

Your images #5, #2 and #3 seem to show this photobleaching
Pau

lonepal
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Post by lonepal »

Nice work!
Regards.
Omer

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Post by Adalbert »

Hello Walter,
Really amazing pictures!
The composition of the nr. 5 is great, I like it :-)
BR, ADi

WalterD
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Post by WalterD »

Saul, Jorgen, Sumguy, Marek, Jacek, Pau, Omer and Adi; thanks for your positive words!

Pau, it is indeed a race against the clock. Photobleaching occurs with any botanical object as far as I've experienced, and probably also with animal and nonorganic material.
With the fern in photo 6 there was something peculiar which looked like reverse photobleaching. the intensity of the red increased while taking the pictures. See below thumbnails as evidence.

Image

This might be because the radiation "just started up". (?)
I've been thinking about using/tested with thicker excitation filters combined with longer exposure but not sure if this reduces the photo bleaching.
Both pictures are 100 iso/ 1 sec shutter speed. Higher iso creates more grainy images during postprocessing, I noted. The 9x objective has a 0,21 nummeric aperture.

Besides photobleaching the spores were literally flying around my ears, depending the object. Another concern is keeping the leafs fresh, storing some in fixative might be a solution.
Picture 7 was actually taken before 6, I think it shows a bit more detail (e.g. detail of the spores kept inside).
7.
Image

santiago
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Post by santiago »

Beautiful pictures...
Santiago
Flickr

Beatsy
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Post by Beatsy »

Fantastic quality!

Pau
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Post by Pau »

Walter, thank you for your answers. Your exposure times are not so different from mine, a bit shorter I think, if your fern and filters behave close to mine you must have stacked really fast.
With the fern in photo 6 there was something peculiar which looked like reverse photobleaching. the intensity of the red increased while taking the pictures. See below thumbnails as evidence.
This is really weird and interesting, nothing I would had expected with chlorophyll. I've seen this kind of effect with pollen grains and I was unable to find info about the phenomenon https://www.photomacrography.net/forum/ ... p?p=215041

The spores and other structures are much more resistant to photobleaching.
Pau

WalterD
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Post by WalterD »

Thanks Santiago and Steve.
@Pau: What a coincidence you experienced the same! Still not sure what causes this..Due to my white point setting the colours became warmer anyhow.
By the way these lilly pollen pictures you posted are very interesting and pleasing the eye, picture 2 is my favourite. So after sporangia I'll maybe try pollen. :)

The next challenge was to work with higher magnification. Lomo manufactured a standard set of M27 epi objectives with 9x, 21x, 40x and 95x magnification. Besides that there's the "old" set of M27 epi objectives that are longer and haven't got the magnification engraved, but the Na and the focal length. Because somehow the 21x objective did not provide the expected detail, I switched over to the F 8,2 / Na 0,37 objective. Based on the Na the expected magnification would be 18x, when compared with a calibration slide it appears to be 22,5x. This was better than the 21x objective, see below picture


8.
Image

Note: I've kept a couple of interesting samples in FAA (Formalin-Alcohol-Acetic Acid), so they can be stored for years. By killing in fixative the sporangi opening reflex (activated by light) is eliminated.

rjlittlefield
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Post by rjlittlefield »

WalterD wrote:With the fern in photo 6 there was something peculiar which looked like reverse photobleaching. the intensity of the red increased while taking the pictures. See below thumbnails as evidence.
Looking at histograms of the thumbnails, what I see is that all colors RGB are fading (becoming less intense), but green is fading faster than the others. The result is that the hue shifts to red, even though the intensity of red drops.

--Rik

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