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Sporangia, Sori and autofluorescence (2)
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WalterD



Joined: 06 Jul 2015
Posts: 412
Location: Rotterdam, the Netherlands

PostPosted: Thu Nov 01, 2018 2:39 pm    Post subject: Sporangia, Sori and autofluorescence (2) Reply with quote

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.


2.


3.


4.


5.


6.


That's it for today, more types in progress Smile

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

Regards,

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



Joined: 31 Jan 2011
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PostPosted: Thu Nov 01, 2018 2:50 pm    Post subject: Reply with quote

Very nice pictures Walter !
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JH



Joined: 09 Mar 2013
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PostPosted: Thu Nov 01, 2018 3:31 pm    Post subject: Reply with quote

Interesting pictures!
Best regards
Jörgen Hellberg
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Sumguy01



Joined: 28 Jan 2013
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PostPosted: Thu Nov 01, 2018 7:29 pm    Post subject: Reply with quote

Applause Very nice set.
Thanks for sharing.
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Marek Mis



Joined: 10 Jul 2010
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PostPosted: Thu Nov 01, 2018 9:53 pm    Post subject: Reply with quote

Great images Walter ! Number 4 and 5 are stunning !

Marek
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Jacek



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PostPosted: Thu Nov 01, 2018 10:56 pm    Post subject: Reply with quote

SUPER !
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Pau
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Joined: 20 Jan 2010
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Location: Valencia, Spain

PostPosted: Fri Nov 02, 2018 1:53 am    Post subject: Reply with quote

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
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lonepal



Joined: 28 Jan 2017
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PostPosted: Fri Nov 02, 2018 2:59 am    Post subject: Reply with quote

Nice work!
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Omer
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Adalbert



Joined: 30 Nov 2015
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PostPosted: Fri Nov 02, 2018 3:30 am    Post subject: Reply with quote

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



Joined: 06 Jul 2015
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Location: Rotterdam, the Netherlands

PostPosted: Fri Nov 02, 2018 4:35 am    Post subject: Reply with quote

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.



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.

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santiago



Joined: 25 Sep 2018
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PostPosted: Fri Nov 02, 2018 5:15 am    Post subject: Reply with quote

Beautiful pictures...
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Beatsy



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PostPosted: Fri Nov 02, 2018 6:16 am    Post subject: Reply with quote

Fantastic quality!
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Pau
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Joined: 20 Jan 2010
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PostPosted: Fri Nov 02, 2018 6:56 am    Post subject: Reply with quote

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.
Quote:
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/viewtopic.php?p=215041

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



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PostPosted: Sun Nov 04, 2018 8:59 am    Post subject: Reply with quote

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. Smile

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.


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.
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rjlittlefield
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PostPosted: Sun Nov 04, 2018 10:28 am    Post subject: Reply with quote

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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