MicrobeHunter.com Microscopy Forum

Nothing to brag about, but as a newbie I spent hours of hand sectioning, fixing, staining, mounting, stacking and stitching:


36 set of 3 focus planes (total of 108 pictures) stitched and stacked. Plan achro 10x, 0.25/ EOS 50D / stitched in PS, stacked in Helicon Focus.

No DIC or any special type of illumination. Feel free to bombard me with critiques and comments about my mistakes (in fact I'd be glad).

I think it looks phenomenal, you put a lot of work into it and it shows

I unfortunately can't give you any critiques but maybe some of the more experienced guys will have some tips for you

That looks awesome! You did a great job.

I agree with the members above me. This is a really good result!

Would be interesting to get some details on your sectioning technique as well as fixing, staining and mounting.

I'm with Wes. Let's hear the details. Very nice result.
Tom W.

Hi Darousha,

great image, thank you for sharing! The result is well worth the effort. It also is an interesting specimen with a somewhat special structure. Can you name the species of the pine?
What stain was this? The combination looks very nice and differentiates well.
These bigger plant sections actually look better stitched than in of single shot with a lower power objective, though the full resolution can't be shown here in the forum. I don't know why, in theory one single shot should do for the 1024 pixel images here.
These botanic sections sometimes warp in the mountant and are less flat than one would expect. Here a couple of more shots could have improved the image further. But at already 36 frames...

Bob

Wow, I'm surprised. Never thought you would like it. I was gonna delete all of pictures and felt disappointed with myself (I didn't like it due to the lack of sharpness over the whole final image). until the wife insisted on posting it somewhere to hear more comments.

I'll write a complete procedure of what I have done later at the night from the man cave.

Bob, the picture is stack of 3 focus planes not 36. I stitched 36 photos 3 times and stacked the 3 large images into one final image .
That's why some areas are not tack sharp...

Thank you guys for encouraging me.

I can smell it!
Great job, looks damn fine.

I always find it very important not to value things by their worst corner but by the better ones (ok, a car with a puncture on one wheel is an exception ). I can guaratee you: When you get everything tack sharp there will appear an air bubble out of nowhere! Also diatom slides with bubbles or not quite enough Pleurax: No reason to worry.

Really nice photo !

Guys, I'm speechless, thank you.

I promised earlier today to write a few short set of steps and procedures, but wondered it'd be more responsible to the community to write a rather lengthy and comprehensive protocol.

Basically I fix the tissue in the FPA (immediately after taking sample) , softening the tissue in Ethylenediamine or Hydrofluoric Acid (nasty chemicals, forget the HF, it bites/burns you in the a**), embed it in to a paraffin block, hand sectioning with razor sandwiches, remove the paraffin, re-hydrate the tissue again, clear the cut sections in the Sodium Hypochlorite, wash the sample in water, stain it safranain/alcian blue + ammonia-fuchsin/carmine, remove the extra stains, fix the stains and then dehydrate it again with series of alcohol solution and then into 2 series of Xylene to completely remove the alcohol. And finally mounting it with Entellan. It's a week or even more (considering that I have to go to work and spend time with family, etc...) for me prepare a sample like that and I think it's very very inefficient. Let's not talk about the cost of chemicals

I'll will come up with all explanations and exact protocol soon. I just need a free day in the man cave.