Pollen Fine Detail

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mrsonchus
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Pollen Fine Detail

#1 Post by mrsonchus » Tue Apr 21, 2020 11:53 pm

Hi all, I've been mounting pollen lately, fresh from the plant into fuchsin-infused glycerin-jelly, with coverslip. These make pretty good long-term but temporary mounts and are very simple to prepare.
Simply put, place a slide and coverslip (seperate from each other not one on top of the other) onto a heated surface such as a small hotplate or cup-warmer that is warm enough to melt the jelly but not hot enough to boil it - so about 60 deg C may be good.
Place a very small piece (perhaps 1 rice-grain in size for an 18mm square coverslip) of glycerin-jelly mountant onto the coverslip and let it melt (about 1-2 minutes). Shake pollen onto the molten jelly (or lightly dip a pollen-dusted anther, petal etc) very sparingly. Place warmed slide onto coverslip and let the mount cool to RT - about 2 minutes. Ready to be observed under the 'scope as follows....

I thought I'd show these images as I'm pretty impressed with the view of the truly tiny pores that are in the pollen's spine-butresses - usually I can't make these out very well with pollen mounted as part of a permanent resin-based slide of pollen-bearing plant tissue. Impressed because I expected less of the glycerin-jelly mounts at this level of magnification - even though the grains were effectively mounted against the coverslips. Also as an adjunct to a recent comment I made re the super-clarity/resolution of SEM images being an aid to the appreciation of just what we CAN see with 'ordinary' light microscopy rather than a source of envy!

Anyway, here's a SEM image open-sourced for the web of my subject - the pollen grains of the common garden Dandelion, AKA 'Taraxacum.officinale'...
This image shows these tiny pores quite well around the bases of the spines...
Image

Here's a std image of one my glycerin-jelly mounts with a 40x (Olympus Plan UIS2 objective on BX50) in what I may consider to be 'the usual quick state'. Quite good spine and exine (outer shell) 3D detail.
Image

This image has been scaled and measured with the super free 'micam 2.4' software to show the spacing of the spines is about 2-2.5µ for reference.
Image

This is an image with the 100x oil-immersion objective, the condenser is an aplanat/achromat 1.4 (used at the 1.25 n.a. of the objective) Olympus.
Extremely small depth of focus single image that begins to show those tiny pores. I used an Olympus IF550 green filter, shot in mono then edited the RAW image in PS in an attempt to optimise detail.
Image

A different grain, this time inverted, which very often seems to improve these pollen images quite considerably, sometimes...
Again, the pores are slightly visible.
Image

I think the inverted has better detail and a sense of the 3D shape and relationships of the spines - some are even bent!

Not of course anywhere comparable to the SEM image, but not bad for a bog-std 100x (in terms of the Olympus UIS2 objectives that is) objective and a glycerin'jelly mount I thought.

Hope you may find these interesting - I'm quite pleased with them.
John B

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Re: Pollen Fine Detail

#2 Post by Nanonaut » Wed Apr 22, 2020 12:45 am

Hi John,

Beautiful pictures! Very well done. I look forward to giving this a go.

PS. That SEM image is my image. :D

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Re: Pollen Fine Detail

#3 Post by MicroBob » Wed Apr 22, 2020 5:56 am

Hi John,
you've got some really impressive results here! These are probably the best pollen images I have seen so far.
What I didn't understand is how you applied the fuchsine - is this a two step process: First pollen into stained jelly, then into clear jelly?

Bob

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Re: Pollen Fine Detail

#4 Post by Hobbyst46 » Wed Apr 22, 2020 7:48 am

mrsonchus, thanks for the very nice images !
Would you say that such performance was not possible from your Orthoplan ? talking only about the image itself, not the scope mechanics ?
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Re: Pollen Fine Detail

#5 Post by MichaelG. » Wed Apr 22, 2020 7:53 am

Extraordinary work, John
... beautiful and very informative images

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Re: Pollen Fine Detail

#6 Post by daruosha » Wed Apr 22, 2020 8:06 am

I'll second Bob's opinion. Excellent images 👌.
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Re: Pollen Fine Detail

#7 Post by 75RR » Wed Apr 22, 2020 8:09 am

Well done! I see several members have accepted your challenge. Look forward to the results.
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Re: Pollen Fine Detail

#8 Post by MichaelG. » Wed Apr 22, 2020 8:14 am

mrsonchus wrote:
Tue Apr 21, 2020 11:53 pm


I thought I'd show these images […] Also as an adjunct to a recent comment I made re the super-clarity/resolution of SEM images being an aid to the appreciation of just what we CAN see with 'ordinary' light microscopy rather than a source of envy!
[…]
Hope you may find these interesting - I'm quite pleased with them.
Permit me please, John, to include a link to that very eloquent comment:
viewtopic.php?f=18&t=8925&p=76937

What you wrote there has been most admirably demonstrated here.

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Re: Pollen Fine Detail

#9 Post by Roldorf » Wed Apr 22, 2020 9:08 am

Hi John I will definitely have to try that. I have been looking at all these beautiful dandelions over the last few days.
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Re: Pollen Fine Detail

#10 Post by mrsonchus » Wed Apr 22, 2020 9:57 am

Good morning all - a lovely sunny morning here in the U.K. - wildflowers sprouting-up everywhere in our gardens, and all-day to study them! :D

Many thanks for all your kind comments, it's always great when others share an adventure.

MicroBob,
The fuchsine (probably the de-facto stain for pollen, at least when used alone I think) comes pre-mixed in the glycerin-jelly, as a small pot.
The pot I bought when I started (which incidentally was with pollen - easy and available...) of this jelly is still about half-full and going strong - admittedly I seldom use it. It has I'd say some form of steriliser added as it has absolutely no signs of 'alien invasion' after 5 years!

Here's an image of said pot, bought from Brunel Microscopes.
fuchsin glycerin jelly.jpg
fuchsin glycerin jelly.jpg (109.02 KiB) Viewed 2575 times
It's easy enough to make some (just add a couple of drops of 'Listerine' or similar to prevent mould etc) to some glycerin jelly with a tiny amount of fuchsin added during it's making. I made some way-back as inspired by the Late Walter Dioni's articles, but the pot is so very cheap, available and convenient that I don't make my own now.

Here's an image or three of similarly prepared pollen from the 'Honesty' plant - Lunaria.annua
WS_lunaria annua pollen (2).jpg
WS_lunaria annua pollen (2).jpg (40.73 KiB) Viewed 2575 times
WS_lunaria annua pollen (1).jpg
WS_lunaria annua pollen (1).jpg (129.24 KiB) Viewed 2575 times
and inversion compared,
WS_lunaria annua pollen.jpg
WS_lunaria annua pollen.jpg (122.69 KiB) Viewed 2575 times


Hobbyst46,
Hmm, all I can say for sure is that the Olympus outperforms the Orthoplan in whatever one may label 'image quality' within the context of my use for Botanical work. The kit I had with my Orthoplan was, after a lot of searching, of top quality, and did perform excellently. I'd probably guess (based upon a lot of experience with both 'scopes) that with this particular application that the Orthoplan would come very close indeed if not the equal-of these images. The ever-decreasing-returns does, at this amateur level, definitely apply I think, especially with this particular method of preparation and the necessarily-limited ancillary equipment of my 'lab'....

The biggest (relative to the Olympus BX40 & 50) weakness of the Orthoplan I found was the illumination - namely the perpetual struggles with uniformity and alignment that finally prompted me to dig rather deeply into my wallet to move to Olympus.
John B

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Re: Pollen Fine Detail

#11 Post by mrsonchus » Wed Apr 22, 2020 10:13 am

Roldorf wrote:
Wed Apr 22, 2020 9:08 am
Hi John I will definitely have to try that. I have been looking at all these beautiful dandelions over the last few days.
Go get-em Roldorf. The tap root of the Dandelion is very easy to hand section - dig-up the whole root (usually about 6 inches deep) for some very nice images - stain with just about anything, but safranin (weakly) is always good, and Toluidine-blue particularly-so.

Perhaps also try the pollen dry on the slide - I havent looked at that but it may be interesting also.
John B

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Re: Pollen Fine Detail

#12 Post by tgss » Wed Apr 22, 2020 11:59 am

Excellent, excellent, excellent! Wonderful images. And you are absolutely correct - there's no need to be envious of super resolution SEM images when we can be envious of mrsonchus' LM images :mrgreen:
Tom W
P.S. The dandelions in my garden have no need to worry... yet, since they haven't yet appeared. There's snow on the ground this morning!

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Re: Pollen Fine Detail

#13 Post by MichaelG. » Wed Apr 22, 2020 2:09 pm

mrsonchus wrote:
Wed Apr 22, 2020 9:57 am
Here's an image or three of similarly prepared pollen from the 'Honesty' plant - Lunaria.annua
That’s a fascinating comparison, John ... and demonstrates a significant point about our visual perception:

The strong highlight on the normal image jumps forward, and seems quite intrusive
... Invert the image and it recedes inoffensively into the ‘shadows’

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Re: Pollen Fine Detail

#14 Post by MicroBob » Wed Apr 22, 2020 3:52 pm

Hi John,
thank you for the explanations!
I mixed a glycerin jelly like this:

0,7g ordinary household gelatine
4,2g dem. water
5,0g glycerin
0,01g thymol
a trace of fuchsin

and mixed it on my heater/stirrer. It became a solid jelly after cooling down.
Making slides was easy and the results are really nice. My stained dandelion pollens look different than yours: Mine came out yellow/pink. There might be a difference between our stained jellys: I used pure fuchsine, but I also found a recipe for use of phenolic fuchsine.

Here a single shot, 100:1 Leitz NPL Fluotar with Zeiss DIC "alt". A stack is running through Picolay right now.

This really is a nice joyful microscopy topic in this unfriendly time.

Bob
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Pollen Löwenzahn Fuchsin Glyceringelatine 1024 DSC_0120.JPG
Pollen Löwenzahn Fuchsin Glyceringelatine 1024 DSC_0120.JPG (171.86 KiB) Viewed 2509 times

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Re: Pollen Fine Detail

#15 Post by mrsonchus » Wed Apr 22, 2020 5:06 pm

Looks like a sterling-start to me Bob! There's a little detail peeking out of the spines I can see.

You may find stacking to be sub-optimal with these subjects - the tendency is great to 'overstack' beyond what is effectively a 'top surface' - albeit a 3D one. If this happens, as is very common with translucent transmission brightfield imaging, the results may look from stunning to awful, but be essentially false.

My first advice would be to keep it simple, with well-chosen single images. For example, attempts to stack a focus down the edge of a spine will almost certainly bring 'ghost details' from elsewhere that aren't actually visible without the transparency of the subject. The harder edges seen for example in an insect's exoskeleton, together with a greater measure of opacity, give a stacking routine far more to 'bite into' as it were, when constructing a meaningful image-stack.
This is the reason I most-often use photoshop's 'unsharp mask' sharpening rather than the far more capable (in 'usual stacking') 'smart-sharpen', edge-hunting stack routines.....

It's great to see others having a go! A superb start Bob - more! :D
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Re: Pollen Fine Detail

#16 Post by mrsonchus » Wed Apr 22, 2020 5:12 pm

Looks like a sterling-start to me Bob! There's a little detail peeking out of the spines I can see.

You may find stacking to be sub-optimal with these subjects - the tendency is great to 'overstack' beyond what is effectively a 'top surface' - albeit a 3D one. If this happens, as is very common with translucent transmission brightfield imaging, the results may look from stunning to awful, but be essentially false.

My first advice would be to keep it simple, with well-chosen single images. For example, attempts to stack a focus down the edge of a spine will almost certainly bring 'ghost details' from elsewhere that aren't actually visible without the transparency of the subject. The harder edges seen for example in an insect's exoskeleton, together with a greater measure of opacity, give a stacking routine far more to 'bite into' as it were, when constructing a meaningful image-stack.
This is the reason I most-often use photoshop's 'unsharp mask' sharpening rather than the far more capable (in 'usual stacking') 'smart-sharpen', edge-hunting stack routines.....

It's great to see others having a go! A superb start Bob - more! :D

Here's a link to my post a while back re the making of glycerin jelly...
John B

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Re: Pollen Fine Detail

#17 Post by daruosha » Wed Apr 22, 2020 10:40 pm

It's shame I couldn't reproduce a result not even close to your fantastic pictures. It's just garbage. Do you have any suggestion for me?

Image
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Re: Pollen Fine Detail

#18 Post by MicroBob » Thu Apr 23, 2020 1:40 am

Hi Daruosh,
the visibility of my pollen came mainly from the yellow original colour and the pink fuchsin stain. Did you use fuchsin? It seems to attatch well to the pollen. I added a tiny amount to my glycerin jelly until it looked like John's.

Bob

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Re: Pollen Fine Detail

#19 Post by PeteM » Thu Apr 23, 2020 3:44 am

Very ingenuous, great subject, wonderful images. Thanks.

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Re: Pollen Fine Detail

#20 Post by MicroBob » Thu Apr 23, 2020 9:13 am

Hi Daroush,
I did a bit of image editing on your bright field image - how do you like it?
And a single bright field image from my slide.

Bob
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Einzelbild Hellfeld DSC_0017.jpg
Einzelbild Hellfeld DSC_0017.jpg (202.75 KiB) Viewed 2421 times
Daroush.jpg
Daroush.jpg (60.37 KiB) Viewed 2421 times

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Re: Pollen Fine Detail

#21 Post by daruosha » Thu Apr 23, 2020 10:13 am

Thanks Bob, my picture turned out to be a tad better with your PP techniques.

I have two problems: The image in the eyepieces is much much better than what camera has captured (both cell phone and DSLR) and the second problem is the detail is not there, no matter how much post process I do, I cannot reveal something that doesn't exist in the first place.

I'll try fuchsin stain and come back to the topic.
Daruosh.

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Re: Pollen Fine Detail

#22 Post by MicroBob » Thu Apr 23, 2020 12:07 pm

A couple more single bright field images, Leitz NPL Fluotar 50:1 1,0 and Leitz Fluorescenz 63:1 1,3.
Attachments
Pollen Löwenzahn Fuchsin Glyceringelantine DSC_0262 1024.JPG
Pollen Löwenzahn Fuchsin Glyceringelantine DSC_0262 1024.JPG (127.46 KiB) Viewed 2385 times
Pollen Löwenzahn Fuchsin Glyceringelantine DSC_0255 1024.JPG
Pollen Löwenzahn Fuchsin Glyceringelantine DSC_0255 1024.JPG (166.64 KiB) Viewed 2385 times

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Re: Pollen Fine Detail

#23 Post by daruosha » Thu Apr 23, 2020 1:52 pm

Very nice images Bob. Thumbs up.
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Re: Pollen Fine Detail

#24 Post by mrsonchus » Thu Apr 23, 2020 7:35 pm

Definitely getting better Bob - more detail is definitely emerging to my eyes. It would be good if the (yellow) pollenkit were removed - as in my images.
This happened 'all on it's own' I must say - just a bit of luck I think but maybe related to the pollen itself or time of collection, in terms of clock-time or plant-development time/stage. Perhaps the formula of jelly, or it's temperature etc....

Good stuff Bob!
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Re: Pollen Fine Detail

#25 Post by daruosha » Thu Apr 23, 2020 8:23 pm

I found this in my archive:

Image
100x Oil Immersion Plan Achro NA 1.25

Am I cheating by phase contrast?
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Re: Pollen Fine Detail

#26 Post by mrsonchus » Thu Apr 23, 2020 9:37 pm

Cheating! Not in any way old chap!
Phase is great for giving a peek at the nuclei of pollen grains, as can be seen in your image. There is taxonomic variation in the number of nuclei in a pollen grain at various times during it's development. Ultimately all angiosperms, as far as I know, have three nuclei in the mature pollen grain - which is actually a germinated full-blown yet minute gametophyte.
The pollen grain at it's 'birth' (in the anther's interior) is a microspore, which then matures, after divisions of nuclei to three, into a gametophyte - the gamete-producing form of the angiosperms - the 'main plant' as we know it being the sporophyte generation - search for 'alternation of generations' in plants for info.

The number of nuclei may be two when the pollen is released, the generative (sperm) and increase to three during the nuclei passage through the pollen tube towards the egg-cell, or may as in some Sonchus I think, be three before being shed - I think that's how it goes anyway.... Please correct if I'm wrong.
So, the number of nuclei present in a pollen grain is variable and taxonomically useful!
This is a great use for phase as it often gives in my experience a peek at the pollen's innards not visible unambiguously with a stained brightfield grain.

In fact I did apply phase to some Lily-of-the-valley, as far as I can remember - I was in full 'pollen overload mode' at the time (Convallaria.majalis) grains at the time of making the GJ slides, with as in your image, a green IF550 filter for increased resolution and contrast (theoretically at least...).

Here are a few of those phase images,
WS_lily of the valley pollen.jpg
WS_lily of the valley pollen.jpg (139.6 KiB) Viewed 2321 times
and
WS_lily of the valley pollen (1).jpg
WS_lily of the valley pollen (1).jpg (53.66 KiB) Viewed 2321 times
A rather poor but quick attempt I made with the darkfield position of my BX50's phase condenser - could be worse though I suppose.
WS_lily of the valley pollen (3).jpg
WS_lily of the valley pollen (3).jpg (64.4 KiB) Viewed 2321 times
All the different techniques are complementary when trying to glean as much information from just about any subject I suspect - certainly true for plant tissue and here pollen. The BF images are great for the (also taxonomically useful) character of the grain exine's ornamentation and often nuclei-number, whilst with the phase the internals including the nuclei and cytoplasm (enclosing plasma-membrane/s included often) may be studied quite closely with luck....

Oh-my now you've got me looking through my archive also! Here are a few images of my pollen slides - different as these are sectioned pollen permanently-mounted in resin. My particular favourite is the image of Sonchus pollen with three nuclei visible while still in the anther - as mentioned above....

Here are a couple of examples of fresh-mounted grains I made a while back now, with some quite nice exine detail, images from my Orthoplan I think - a good demo of the ability of the Orthoplan to more-or-less equal my current Olympus BX50 'scope with these subjects....
WS_fresh pollen.jpg
WS_fresh pollen.jpg (76.98 KiB) Viewed 2321 times

Here are three images of permanently-mounted sectioned slides I made, also a while back now - quite different again to the full-grain jelly mounts! I love the image at the bottom - showing the trinucleate Sonchus grains....
All three images are of pollen grains in their respective anthers and sectioned, stained and mounted in resinous mountant ('NuMount' I think back then - I now use 'Histomount').
The Lily pollen seems to have two nuclei whilst it's in the anther, the Sonchus.oleraceus appears to have three nuclei - the two sperm nuclei are clearly visible as the two thin curved nuclei - a common shape for pollen sperm nuclei I believe.
The middle image is just me trying-out different PP techniques to try to reveal extra features - I'm still doing this today, and enjoying it every-bit as much as back then! :D
WS_sectioned pollen mounts.jpg
WS_sectioned pollen mounts.jpg (131.87 KiB) Viewed 2321 times
Anyway - I got a bit carried away! Pollen is my first microscopy subject and still possibly my favourite after nearly 5 years now since I started. No more pollen images - I promise! :D :D :D
John B

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Re: Pollen Fine Detail

#27 Post by daruosha » Thu Apr 23, 2020 10:01 pm

mrsonchus wrote:
Thu Apr 23, 2020 9:37 pm
Pollen is my first microscopy subject and still possibly my favourite after nearly 5 years now since I started. No more pollen images - I promise!
You become my pollen micro photography hero. Please do more, by all means.
Daruosh.

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Re: Pollen Fine Detail

#28 Post by MicroBob » Fri Apr 24, 2020 9:37 am

mrsonchus wrote:
Thu Apr 23, 2020 9:37 pm
No more pollen images - I promise! :D :D :D
Please don't keep this promise! You las post was again very interesting for me especially since it covered som any other aspects.

Here a stacked image made with the Zeiss 40:1 0,85 oil achromat in DIC:

Bob
Attachments
Pollen Löwenzahn 40er Öl DIC gestackt V2 Hochpass Werte 1024.jpg
Pollen Löwenzahn 40er Öl DIC gestackt V2 Hochpass Werte 1024.jpg (119.97 KiB) Viewed 2286 times

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Re: Pollen Fine Detail

#29 Post by mrsonchus » Fri Apr 24, 2020 10:29 am

Lovely colours to this one! There's definitely a hint of 'peering into the pores' as it were with your excellent images, please give us more, they're coming-on nicely.
Thanks again for your kind comments Bob. :D

Many thanks Daruosh, great to know others are enjoying the mysteries of pollen! :D
John B

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Re: Pollen Fine Detail

#30 Post by Roldorf » Sat Apr 25, 2020 3:49 pm

Eventually got round to collecting some dandelion pollen:

10x
I never knew they were such a delicacy:
"The entire plant, including the leaves, stems, flowers, and roots, is edible and nutritious. "Wikipedia"
Or so useful:
"Dandelions secrete latex when the tissues are cut or broken, yet in the wild type, the latex content is low and varies greatly. Using modern cultivation methods and optimization techniques, scientists in the Fraunhofer Institute for Molecular Biology and Applied Ecology (IME) in Germany developed a cultivar that is suitable for commercial production of natural rubber." "Wikipedia"
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Cropped Dandelion Pollen.jpg
Cropped Dandelion Pollen.jpg (82.55 KiB) Viewed 2241 times
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