Sequoia gigantensis cross section in Epi-Fluorescence

[MicroBob]

Hi together,
In the 1860s Kaiser Wilhelm ordered a pound of Sequoia gigantensis seeds, fitting to his general mind set. He didn't know that these big trees have tiny seeds and he got a lot of them. They were distributed to different forrest wardens and plants were grown from them. From this time we have a couple of hundered Sequoia gigantensis trees in Germany.
We visited a couple and brought some material from the ground in front of these trees. Growing plants from them didn't work unfortunately - they would have made nice presents: "Here, I have a beautiful tree for your little back garden!" .
Some needles and twigs of different size were stored in cleaning ethanol "Brennspiritus 96%". They became quite hard and part of the material was moved to a mixture of equal parts of glycerol, acetic acid and ethanol. From this needle material I made free hand cuts (Just a mood) which were a nice even 50µ. The first stain I tested was Acriflavin. I mounted over Isopropanol into LOCA TP 2500, an UV curing smartphone display repair glue. It is important to choose a mountant with no or little autofluoresecence, so Euparal didn't work.

Bob

[75RR]

In the 1860s Kaiser Wilhelm ordered a pound of Sequoia gigantensis seeds, fitting to his general mind set. He didn't know that these big trees have tiny seeds and he got a lot of them. They were distributed to different forrest wardens and plants were grown from them. From this time we have a couple of hundered Sequoia gigantensis trees in Germany.

Wonderful trees. Do they propagate naturally? Have heard that they don't tend to outside of their natural habitat.

[MicroBob]

They live quite happily in Europe and it is possible to grow them from seeds but it is not easy. Our experiments with cuttlings and seeds didn't fruit. Others have managed to grow plants, there are some tricks to optimize the results, as always.

[MicroBob]

I did some more testing and optimized my lighting unit.
The next stain I tested was rhodamin B in combination with acriflavin.

[Hobbyst46]

This one is better, shows more specific staining.
Acriflavine is excited at 416-430nm, depending on the solvent (416 in water). and the fluorescence peak is at 54-540nm, again depending on the medium. So violet-blue excitation and green-yellow fluorescence. The dark orange in the image is rhodamine.
Great progress, Bob! you jumped into the cold deep water!

BTW there is a fluorescence-free (almost) mounting medium, easy to use (no organic solvents), although not inexpensive - it is called Fluoromount. Sold by Sigma-Merck.

Another small tip about propagation of plants from cuttings: it might depend on the season. During the correct time of the year (that may be 1-2 months only) the cutting will grow roots. I have witnessed such behavior (not with Sequia, just a common garden plant .

[MicroBob]

Hi Doron,
one of my sons is often picking up plants or makes cuttlings and grows plants from them. This usually works quite well, better than with the Sequoia cuttlings. There are som specific tricks like applying willow bark solution (a tree that grows in very wet spots, e.g. next to river beds) and simulate forrest fires... Our material was not cut freshly but was blown out of the trees (3m diameter).
I still have a lot of seeds but my first try didn't work out. Maybe they are just not ripe.

Here is a picture of a stoma in Acriflavin stain and a complete section in Rhodamine-Acriflavine combination.

It is really fun to experiment with Epi-Fluorescence. I had to repair my lighting unit. The eyepiece/collector seat in the 3D-printed illuminator heated up and got off course. So I had to install an intermediate aluminium disc to keep the heat a bit better away and provide a new seat. My exposure times at ISO 160 are around 1 second. I hope the slides with LOCA as mountant will survive for a long time.

Bob

[PeteM]

Great images (and story), Bob. Thanks.

[MicroBob]

Hi together,
one more picture.
In bright field the slide didn't look too good so I think it might be a promising approach to optimize staining for brith field and then check the view in epi-fluorescence.
This was just a quick test to get an idea of th usability of the epi-fluorescence condenser for my purposes. Quick hand cut, crude staining, experimental mountant and intermedium - there is room for further improvement.
What I'm happy about:
- The comparatively cheap epi-fluorescence condenser has proove functional and in good / usable condition
- By changeing the excitation and emission filters in one of the ports of the head I got to the effect I was looking for for acceptable money
- The quality of the images is already as good as I would have expected after a lot more work
- I learned quite a bit in the process in a very agreeable way!

Bob

[mintakax]

Wow ! All beautiful pics !

[mrsonchus]

Must agree!
Startlingly-good images!

Have you any at a higher-mag? Great to peer-into them a little closer for some of the superb details.
Excellent post.

[MicroBob]

Hi John,
here two quick images with the Zeiss Jena 40:1 0,95 Apo.
The sectioning and staining was just a quick first try to see whether this has potential, so you can expect better results with more precise technique. Maybe you can make a stained section and I take epi-fluprescence images from it?

Bob

[mrsonchus]

Excellent! The layers 'S1,S2 and S3' of the fiber secondary cell-walls are easily distinguishable.
There's a whole-lot more to this technique also, I would say at a first look.
There are degrees of subtlety to texture and tone that add to the information content to my eye.

It woulld be interesting to see the technique applied to mounted sections - I have a few fluorescent dyes (I think) including acridine orange, acriflavin, aniline-blue....

Leave it with my my friend and I'll see what wax-block I have left in my containers....

I'd love to see some more - it seems that cell-walls may be a fertile testing-ground, especially longitudinal, where a good expanse of wall may be exposed.

A really interesting thread, makes me want to get a U.V. lamp to test for Iincident) autofluorescence.....

[MicroBob]

Hi John,
the pictures with colours in the yellow-green range are stained with acriflavin.
The ones that include red are stained with acriflavin and rhodamine B, but the staining was not done without any precision, just as a test if there will be fluorescence.
So far I only know these two stains that do well in blue excitation. There will for sure be more, but since so few people use this technique there is little information available.
With blue excitation blue fluorescence is ruled out so only green yellow and red are to be expected. Probably a nice mix of acriflavin and rhodamine B will offer this colour range in a nice way.
I would expect that a section that is stained perfectly for bright field will also look good in fluorescent light. I have two filter sets available:
1. Blue excitation (everything between 400nm and 460nm or so), 510nm dichroitic mirror, emission filter that lets through 520 upwards. (The setup I find very promising)
2. Green excitation, red emission filter (which allows red-black images, nothing else)

For acridine-orange and aniline-blue I don't know where they would connect to and with which excitation and emission range they would work.

Bob

[Hobbyst46]

Aniline blue fluoreces when it is inside a hydrophobic cavity. If there is any such region in the plant, it will emit blue (470-480nm) when excited at 380-390nm. Perhaps your 400nm has some intensity below that.
Acridine orange peaks are similar to those of fluorescein, ~500 and ~510-520nm, exc and em, but it can be blue-shifted when attached to other molecules (RNA).
These data are from the web.

[mrsonchus]

Hi, very interesting indeed - I just sectioned a dozen Lily-leaf TS slides as I'm experimenting with what used to be called 'reimbibition' would you believe! Basically placing a roughed wax block (roughed to expose tissue cut-face) into water or similar as a way of mitigating the toughness of some tissue, and the consequent high-compression during sectioning. The dozen from tonight, 10µ, are those cut straight from roughing, the wax-block whence they came is now in water 'til tomorrow, when I'll be taking some more sections for a simple comparison. If any of those sections are half-decent I'll stain afew of them with the rhodamine-B / acriflavin combo....

I've quite a few slides that I stained with the 'wacker-like' combination (serially-applied as it happens) of rhodamine-B, acriflavin and alcian-blue. I'll sort-out a few of thos and send them off to you for your tests.... I'll let you know what I come up with soon.

Keep up the good work, very interesting indeed.

[MicroBob]

@Doron: When looking at these specifications I think my setup is far away from what is promising for these two dyes. My LED in use is a warm white version with little punch in the low 400ereds.
The cold white leds have twice the power in the blue peak but only half in the blu-green valley. So I hesitate to install a different LED. My mislead LEDs have appeared again!

@John:: The Wacker stain is known to be good in epi-fluorescence so this will work very well. Whether the aclian blue adds something to the fluorescence image - I don't know. I did some tests with mountants and many have a lot of autofluorescence with blue excitation - I showed a comparison here. What do you use as a mountant? So far the favourite from my tests would be dammar resin in xylene taken from an artists bottle of dammar varnish that originally contained a differnt solvent. The Euparal I normally use for plant sections has a light green autofluorescence, my canadabalsam too. Both unusable for this reason. There are mountants that show no autofluorescence like Entellan and Eukitt which have their drawbacks for general use, and I don't have them.
Perhaps it might be easiest if i would apply the mountant myself to make sure it shows little autofluorescence? My Dammar is now in xylene and should be consistently to apply to a xylene wet specimen.
I think this is a very promising collaboration project! The fluorescence from the stains is quite strong over a black background and this is probably much more attractive with very thin sections.
BTW: After our group meeting yesterday we were talking about a climate change-related topic series - and paraffine sectioning! One of our members actually has a box of Wacker's own blocks.

Bob

[mrsonchus]

Hi, the resin-based mountant I use is 'Omnimount' at this time.

Wacker's own blocks - great!

National Diagnostics is an Omnimount supplier Here's the link to it's page...

I'm probably going to switch over to Histomount sometime soon however. I still prefer Histoclear 1 to Histoclear 2, so don't really need the compatibility of Omnimount with Histoclear 2...

[MicroBob]

Hi John,
they write "low fluorescence" so this will do just fine.
In my slides the specimens sit in a green light puddle - this is probably Acriflavin leaking out into the mountant.

Bob

[Sabatini]

Very impressive.
Thanks for all that good information.