I assume they are myelinated? If not they still show up at least under light and electron microscopy.

I assume they are myelinated? If not they still show up at least under light and electron microscopy.

Dear Laura Paletto ,

I only would like to point you to images which can be found at/in :

Article No Obvious Abnormality in Mice Deficient in Receptor Protein...

Article Wfs1-deficient rats develop primary symptoms of Wolfram synd...

Article Axonal Protection by Ripasudil, a Rho Kinase Inhibitor, via ...

If you want serious explanation it would be of great help to have at least two or more images from your X-Ray-microtomographs....

Naturally I know that the images achieved by X-Ray-microtomography might NOT meet the "quality" and clearness you'll get by either conventional light microscopy (1 µm thick, stained resin sections) or special LM-techniques/IF, multiphoton, ....etc..., or, as you can see in those three articles (non exhaustive references!) I mentioned - by Transmission Electron Microscopy (30-90 nm stained resin sections).

And yes, I forgot to point you to:

https://www.cardiff.ac.uk/people/view/38254-albon-julie:

📷

Dr Julie Albon

Senior Lecturer, Deputy Director of Research, Optic Nerve Head Group Leader, Person designate, Cathays Park HTA satellite licence

School of Optometry and Vision Sciences

[email protected] +44 29208 75427 +44 (0)29 2087 4859

She is also on RG: Julie Albon

QUOTE:

"Research Overview

My research focuses on investigating the human optic nerve head, in particular the lamina cribrosa and its insertion region into the sclera, with an overall aim to i) understand how ONH structure and connective tissue micro- and nano-organisation influences a) its biomechanics and b) its susceptibility to the development of glaucomatous optic neuropathy. In the Optic Nerve Head Group, we use a number in ex vivo and in vivo techniques to analyse the cells and connective tissue (collagen, elastin and proteoglycans) and overall architecture of the 3D optic nerve head (non-linear microscopy, xray microtomography, OCT) alters in ageing and glaucoma. In addition, we are investigating the regional micro- and nano-organisation of connective tissue fibres, collagen and elastin (SAXS, SALS, second harmonic imaging, two photon excited fluorescence) in the ONH, and how they influence the biomechanical behaviour of the human ONH.

PhD position

A funded PhD position, is available in my lab.

Please click the link below for more information:

Optic nerve microstructure: a significant indicator of disease severity"

(END OF QUOTE)

For some µCT-micrographs, you might have a look into:

http://orca.cf.ac.uk/102705/1/i1552-5783-58-4-2070.pdf :

Feola AJ, Coudrillier B, Mul-vihill J, et al. Deformation of the lamina cribrosa and optic nerve due to changes in cerebrospinal fluid pres-sure. Invest Ophthalmol. Vis Sci. 2017;58:2070–2078. DOI:10.1167/iovs.16-21393

and perhaps other scientific articles....like:

Article Visualization of porcine eye anatomy by X-ray microtomography

by Leszczyński B. et al, Experimental Eye Research, Volume 167, February 2018, Pages 51-55

Visualization of porcine eye anatomy by X-ray microtomography

[==> https__://doi.org/10.1016/j.exer.2017.11.004 ...NB: .delete underscore character before linking to URL in your browser!) :

(QUOTE:) Highlights

• Application of micro-CT for porcine eye globe anatomy visualization.

• Investigation of staining procedure for most accurate contrast enhancement in micro-CT eye images.

• Eye globe anatomical details identification based on 3D volume rendering models of the samples. (END OF QUOTE)

Nice discussion and images display by RG colleagues.

Dear Wolfgang H. Muss

thank you for your suggestions.

However, I have to tell you that Dr, Julie Albon is my PI, I've been working with her in the last three years...

I was just looking for the effect of osmium onto axons...I will have a look at the articles you suggested.

Thanks again

Dear Laura Paletto ,

yes, I thought Prof. Dr. Albon (whom I found when I searched for references on ‚µCT – images‘) possibly might be your PI, but wasn’t quite sure…

Theoretically (and only theoretically!) I know a bit from literature: ~ 20 - 25 years ago (and 3-DIM-reconstruction of rat brains was the (and MY) task for my dissertation in the late 1970ies) but using or even purchasing such a machine in the late 80ies or 90ies or millenium years was out of any sight as well as (in the first place) of financial power) should / could help you in the interpretation.

Unfortunately I worked in an era of / which used (definition by the former head of my institution) 'ancient /ancillary instruments'.... and I - Egad! - am a bit envious of the possibilities researchers can use these days...

So:

I could - naturally - tell you - or describe for you - or provide perhaps an image of "only osmicated" (unmyelinated / myelinated neurons / axons... sectioned at 1µm thickness) if you are in need of such an information.

If you would provide (as said before) one or two images of your µCT-preps, we could do an interpretation of your images (Fortunately, I no more am a competitor for your research work...).

The reason why I - for the present – guess / doubt that images of your µCT-ed optic nerve axons can be compared to already osmicated axon preparations (be it as a "whole mount" or in a stained semithin -1µm resin section) are as follows:

1. ‚whole mount‘ optic nerve ( or only 'optic nerve head' , i. e. microdissected from surrounding eye tissue) as a 'whole specimen' - in a way you might use also for SEM=scanning electron microscopy = which allows for more/less surface imaging - only will look like a black 'micro-dog", depending on the way of preparation / microdissection with more/less rough, kind of knotty or, eventually also smooth surface: but you won't see anything (no structures) under the surface.

2. a) semithin 1µm resin sections from that region imply tough specimen preparation for TEM proper, We do not know at present how you were

i) preparing your optical nerve specs,

ii) how you osmicated these specimens,

iii) how these pieces were postprocessed, so finally,

iv) we only can guess about the mode you were analyzing your specimen(s) by use of a µCT-‚machine’ or µCT-‚instrument‘ (which one?, conditions?, specimen thickness?, etc).

2. b) UNSTAINED (i. e.: not stained with basic dye solutions as is shown in: cf. the stained image above in my RE [003] ) 1.0 µm - 2.5µm thick semithin sections out from for subsequent TEM-analysis routinely osmicated specimens don't have much 'contrast' (to discriminate "structures" contained in such a section) due to the low 'mass' of the section / contained tissue and the bound / already precipitated „OsO2“ .... such sections in transmitted light microscopy show ONLY .... faint-faintest... brownish (if at all) colouring. In case one had added para-phenylene-diamine after the osmication in the 70%EtOH-dehydration step - due to a 'mordanting' effect on otherwise during higher alcohol dehydration eluted Os-binding sites and therefore LOST substrate in the specimens - at least myelinated axons would exhibit substantially increased brown to sometimes also black colouring (that effect of increased color density depends on the section thickness).

2. c) depending on the pre-processing and processing conditions, a "morphological equivalent image" (as formerly we said in the pre-modern LM-Imaging era) naturally will change accordingly.

If you want to see images of osmium-treated tissue (in your case ‚optic nerve head‘ ) one can assume that such images at present maybe / might be very rare - at least as compared to other "popular" objects of current research.... but you for sure might find some image(s) on scrutinizing original scientific literature, even via keywording in Google Scholar.

3) In strict consideration of the circumstances (i. e. image / structural resolution of the optic nerve bundles / axons) - and if you have the possibilities of histological specimen preparation - I would suggest you to process at least one or two O.N.-heads [CAVE: CUT already into maximally 1.0 – 1.5 mm thick slices **) ] by histological methods (similarly as you might have processed ? your µCT-specs) but not only fix them with ONE fixative (e. g. buffered FA, or FA-GA-solution, or any other suited fixative) and –after Osmication - dehydrate…. BUT wash your specs in suited buffer, then osmicate (at least 1% OsO4 in A. bidest, better in a suited buffer) at least for 1-1.5 hrs at RT, wash once or twice for at least 5 mins each, start dehydration [ step I: 5 min] with 50% EtOH, to the (Step II: 70%EtOH but add 1% w/v para-phenylendiamine (PPD) and incubate for at least 30 min at RT (specimen rotator would be of benefit). Then wash 2-3 times with pure 70% EtOH to get rid of the brown unbound PPD, then proceed with dehydration as usual and end up with…(embedding in ?? e.g. Paraffin).

Cut paraffin sections in a thickness comparable to the tickness (or a bit thicker than) you do for your µCT-analysis and examine unstained (for orientation you could stain a companion section with, e.g., H&E or any other histological dye used for staining nerve tissue).

I do hope that you don’t think I would feel the need to lecture you on the matter…. but I felt your really fascinating and somewhat 'fancy' research task needs to be supported anyhow and anyway (:--)).

Hope you meanwhile have had access to the literature references I provided and you already found some promising images....

Nevertheless: If you feel embarrassed by my former reply, feel free to flame to me…

Best wishes and regards,

Wolfgang

**) because the Penetration Rate of Osmiumtetroxide solution is limited to about 0.6 - 0.7 mm / hr at RT (cf.: HAGSTRÖM&BAHR_1960: ‚PENETRATION RATES OF OSMIUM TETROXIDE WITH DIFFERENT FIXATION VEHICLES‘, @ https://link.springer.com/article/10.1007/BF00736486 or better:

NB: delete the underscore characters in between http _ and ://link springer first before clicking the URL in your browser: https__://link.springer.com/article/10.1007/BF00736486 ).

Dear Laura Paletto , thank you for your efforts to include an image of your proposed axon preparation as imaged by µCT.

As I am no specialist in µCT-Image interpretation, I cannot give you a SERIOUS "YES, these are axons" ...

I still found your reply and request for "certification" of your opinion.....it is late now and I cannot afford the time for searching or reasoning about the technique....so I frankly ask if you know what "light" or transparent areas in achieved images are of nature: either e-dense or e-transparent matter or is it the opposite: dark areas in image represent loose / less dense material/biologic matter...

For another reason I have tried to fool my and perhaps others' eyes and inverted the contrast in your images (cf. the enclosed image).....just to get an impression how this look like... resulting in: it could be that roundish structure represent myelinated axons (myelin sheaths would be interacting with OsO4, resulting in heavy precipitation of Os to OsO2 = Osmium black due to processing = binding of OsO4 to lipids / lipoproteins of myelin sheath, continous and consecutive precipitation **(oxidation)**(see correction below as of 2019-03-25) by / during dehydration (Ethanol, especially in the higher concentrations).

Do you see a 'similarity' with the images as enclosed in Kevin M. Gribbins or my previous answer?

What lacks for me would be an information about the magnification bars in your images: would you mind to inform us about the distance/lentgh the bars represent in the images? Thank you, have a beautiful working day before a hopefully splendid weekend...

my best wishes and regards,

cordially yours, Wolfgang

**oxidation** (see above)

NB: Correction actually needed for the following (done 2019-03-25 16:00 local time):

When I came back to this thread today I found that 10 days ago I've erroneously indicated an "oxidation" leading to a precipitation of OsO4 in tissue (sections or specimens) instead of the correct term: "reduction" of OsO4 to OsO2

Dear Wolfgang H. Muss ,

thank you for your comments, helpful for sure.

yes the scale bars are roughly 100 um. I think I agree with your suggestions, I will try to think more about it.

Are you saying though that when osmium stains, structures appear dark? if that' the case and I will not change the LUT of my original image, then potentially it has not worked, as they look grey ish and not dark (as the surrounding fibre).

it's difficult to understand

Thank you

wish you great week end too

Hey Laura

as Wolfgang has said OsO4 stains lipid so is taken up by cell membranes and myelin, so these will be dark in the image.

here is a rat ON section taken 2mm posterior to the globe, where axons become myelinated. In the 63x image you can see the myelin bundles, you need much higher magnification to see cell structures.

your staining will be most affected by the tissue prep. Wax processing will wash out a lot of the lipids through the solvents used, you can get away with it so long as you don't use xylene as the final clearer.

how did you acquire the images above?

no problem, your images look really interesting, and doing some regions at higher magnification would help. Of course, the images I shared are cross sections and are from the myelinated region of the ON so not directly comparable but at least give you something to go on.

Good luck, looking forward to seeing the results when you publish :)

Hope all is good in Cardiff!

Since I deleted my intended answer some second ago (due to the circumastance that the written text was deleted automatically after I tried to change the "ResearchGate system's linking to the RG-data=article as deposited in ResearchGate) I try it a second time and hope to be short as possible.

Dear James Tribble , thank you really for adding /uploading the cross sections of the osmicated rat ON in a low as well as a higher power magnification. If I were working actively in my former profession I would - for sure - have managed to process optic nerve tissue only with an osmication and to image that for upload into this thread.

But neither that would have been able to reply with a serious answer to Laura's initial request.

Knowing that OsO4 (even by perfusion fixation) of brain or other tissues has been used for long (trying) to demonstrate nerve structures and neuronal morphologies with all their connectivities (cf. Palay, in the 1960ies), but unfortunately did not show all structures and therefore did not permit precise mapping of connectional orgaanization.

Now that we graciously were informed about the imaging method (no sections, but 'whole mount tissue "block ONLY stained by Osmium") as well as the technique wich was used to acquire images I would have said that there is a great difference in using Osmium (e.g. as hydrous OsO4-solution ) and the anticipated BROPA -method (brain-wide reduced-osmium staining with pyrogallol-mediated amplification), as published by MIKULA and DENK 2015 (see Laura's reply 12 above):

For convenience of any further reader or interested colleague I copy and paste the bibliographic information here:

High-resolution whole-brain staining for electron microscopic circuit reconstruction.

MIKULA S, DENK W

Nat Methods. 2015 Jun; 12(6):541-6.

Article High-resolution whole-brain staining for electron microscopi...

Quote:

"The BROPA method (Brain-wide formamide-Reduced-Osmium staining with Pyrogallol-mediated osmium Amplification) results in the preservation and staining of the cellular ultrastructure throughout the brain at a resolution sufficient for reliably tracing neurites and identifying synapses, which are both necessary and sufficient for the reconstruction of brain-wide neuronal circuits (Mikula and Denk, 2015). Further improvements of the BROPA method and its application to larger species (e.g., rat, marmoset and macaque) are currently underway. " End of quote

together with another really interesting article on the technique(s) published later on by

MIKULA Shawn 2016: to be found at

Article Progress Towards Mammalian Whole-Brain Cellular Connectomics

Front Neuroanat. 2016; 10: 62. Published online 2016 Jun 30. doi: 10.3389/fnana.2016.00062 PMCID: PMC4927572 PMID: 27445704

as well as another, most recently published article by GENAUD et al, 2018,

"Fast homogenous en bloc staining of large tissue samples for Volume Electron Microscopy"

Quote: " Modifications of the original BROPA protocol include drastically reduced incubation timesand a lead aspartate incubation to increase sample conductivity. ..."

to be found at:

https://pdfs.semanticscholar.org/639f/bb63503def90c38fee08e48a948c77bdf1c3.pdf

Just guessing about the abbreviation [LUT] given by Laura in Re [010] which I could not find a meaningful explanation despite searching in 5-10 acronym finder databases (except for : "Look Up Table")....

Also I am glad to read about the original magnification of the Images in Re [008] which - according to Laura, have been 2.5 times only (or is 2.50x ment to be 25.0x?)

So, dear James Tribble - you made my day in confirming my opinion that OsO4 would stain / impregnate brain tissue -predominantly lipid(s) or lipoid material and some components of brain (neurons/myelin sheats) BLACK .... to the contrary: > axons < (at least in specimen biological processing for TEM) usually don't take up much OsO4 and therefore usually generate only faint or pale, even NO blackening of structures - IN ULTRATHIN SECTIONS.

MY best wishes and regards and good luck for further experiments,

W.H.M.

Thank you Jimmy

James Tribble

I thought I cannot compare directly but it gives me an idea, which I did not have before.

Everything looks fine here, hope you're enjoying Sweden!

I am about to submit (probably in a month or so) so this experiment specifically has to wait, I needed something to use for discussion

Thank you and wish you good luck too!

Spectacular and identical photos