Hello from Austria

What is your microscopy history? What are your interests? What equipment do you use?
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Microscopy_is_fun
Posts: 11
Joined: Sun Nov 21, 2021 6:11 pm
Location: Austria

Hello from Austria

#1 Post by Microscopy_is_fun » Sun Nov 21, 2021 9:28 pm

Hi everyone,

I am a hobby microscopist from Austria and just happened to find this forum. It seems to be a very friendly community, and that's why I signed up. My focus is mostly on botany, meaning that I collect all kinds of plants which I find in my garden or during hiking. I am also interested in the technical aspects of microscopy, as well as procedures for fixing, slicing and staining of my samples. Below are some recent pictures: a bright field image of a stained section of a mistletoe and fluorescence micrograph of a spruce needle.
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2021-11-06 21-33-12 _stack_fluo.jpg
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Dubious
Posts: 248
Joined: Sun May 09, 2021 7:55 pm

Re: Hello from Austria

#2 Post by Dubious » Sun Nov 21, 2021 9:54 pm

Welcome! Beautiful images. The cross section of mistletoe resembles a mosaic work of art.

Microscopy_is_fun
Posts: 11
Joined: Sun Nov 21, 2021 6:11 pm
Location: Austria

Re: Hello from Austria

#3 Post by Microscopy_is_fun » Sun Nov 21, 2021 10:03 pm

Hi Dubious,

thanks for your feedback!

charlie g
Posts: 1543
Joined: Mon Oct 20, 2014 7:54 pm

Re: Hello from Austria

#4 Post by charlie g » Sun Nov 21, 2021 10:21 pm

Welcome to a terrific community/online forum ( thank you, Oliver Kim). Did you process the two botanical stained sections you share with us in this post? Wonderful sections. Please share with us the workup process and stain schedule for these two plant sections.

Please use this forums search feature to enjoy the botanical microtechnique of member: "Mr.Sonchas" .

I have purchased wonderful stained plant slides from Mr.Sonchas..please yourself 'take a peek' at his low cost/high quality botanical slides.

welcome, welcome...err: 'bruderschaft microscopy'!

Microscopy_is_fun
Posts: 11
Joined: Sun Nov 21, 2021 6:11 pm
Location: Austria

Re: Hello from Austria

#5 Post by Microscopy_is_fun » Sun Nov 21, 2021 10:50 pm

Hi Charlie,

glad to read that you like the images, and thanks for the hint to Mr. Sonchas!

I made booth slides by myself, using a self-made sliding microtome (see picture). The mistletoe is a 30µm section which I stained using Acridine red (10min), then Acriflavine (15sec) and Astra blue (1min). After washing with aqua dest. the sections were dehydrated in isopropanol and then mounted in Euparal.

The fluorescence image is from a freshly cut needle (35-40µm thick) which was mounted in water and imaged with blue excitation (Leica filter I3).
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charlie g
Posts: 1543
Joined: Mon Oct 20, 2014 7:54 pm

Re: Hello from Austria

#6 Post by charlie g » Sun Nov 21, 2021 11:45 pm

Wow, both your sections seem a lot thinner than: '30-40 microns' the 'bare spots' in the mistletoe section..clear of 30-40 microns column of tissue..yet with crisp defined cell walls...and the crisp nuclei in the pine needle section seem too clear of 'above/ beneath tissues' to be 30-40 micron sections'..great achievement, thanks for sharing your process.

welcome, charlie g

Hobbyst46
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Joined: Mon Aug 21, 2017 9:02 pm

Re: Hello from Austria

#7 Post by Hobbyst46 » Mon Nov 22, 2021 9:05 am

Welcome, what nice micro images ! I like the auto fluorescence image of the needle !

And the microtome is impressive - most of it is made from standard industrial profiles I see. I believe many would find interest in the construction details.
Zeiss Standard GFL+Canon EOS-M10, Olympus VMZ stereo

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Javier
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Location: Buenos Aires, Argentina

Re: Hello from Austria

#8 Post by Javier » Mon Nov 22, 2021 12:49 pm

Welcome!

Those images are quite impressive. I'm looking forward to seeing more of your work.

Microscopy_is_fun
Posts: 11
Joined: Sun Nov 21, 2021 6:11 pm
Location: Austria

Re: Hello from Austria

#9 Post by Microscopy_is_fun » Mon Nov 22, 2021 7:19 pm

Hi Javier and Hobyst46,

thanks for the welcome and heads up! When I have some time, I will collect te details about the DIY-microtome. Its main benefits are the low weight (<3kg) compared to professional sliding microtomes and the use of mostly off-the-shelf components. It works very well for soft and medium hard specimen, here is another example: a section from the root of a water lilly. These water plants are not easy to cut, since their tissues are very weak and soft. Embedding in PEG 1500 and slicing with the microtome worked well. In my view it is quite amazing that the internal crosslinks, made up by chains of single cells, survived the procedure so well (40µm thickness, staining with Etzold FCA).
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Wes
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Re: Hello from Austria

#10 Post by Wes » Mon Nov 22, 2021 7:28 pm

Welcome! Your photos are really good and that microtome looks pretty amazing too. Looking forward to more of your posts :)
Zeiss Photomicroscope III BF/DF/Pol/Ph/DIC/FL/Jamin-Lebedeff
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mete
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Location: Switzerland

Re: Hello from Austria

#11 Post by mete » Mon Nov 22, 2021 11:04 pm

Great images. May I ask the procedure for PEG 1500 embedding ? It is preferred over higher molecular weights because it is a weak tissue or for other reasons ?

Microscopy_is_fun
Posts: 11
Joined: Sun Nov 21, 2021 6:11 pm
Location: Austria

Re: Hello from Austria

#12 Post by Microscopy_is_fun » Tue Nov 23, 2021 9:24 pm

@Wes: thanks for welcoming me!

@mete: I took the original recipe for PEG from the internet, unfortunately I forgot the source. I slightly modified it, and use the following procedure now:

(1) fixing the samples in AFE
(2) rinsing in aqua dest
(3) around 6h in 20wt%PEG 1500/80wt% aqua dest
(4) around 4h in 50%PEG/50%aqua dest.
(5) around 6h in PEG 1500 @55°C
(6) casting a PEG block with the embedded sample for slicing
(7) after slicing dissolution of PEG in 20wt% PEG / 80wt% aqua dest
(8) staining and mounting as desired

It is important to replace the water in the sample gently with PEG: water diffuses faster than PEG, and if the exchange process happens to fast (e.g. by skipping the 50/50-stage or putting the slices directly into aqua dest.), the sample gets easily destroyed.

Overall a fairly easy process, involving no problematic solvents or other nasty chemicals.

mete
Posts: 82
Joined: Thu Aug 26, 2021 8:31 am
Location: Switzerland

Re: Hello from Austria

#13 Post by mete » Wed Nov 24, 2021 7:22 am

Microscopy_is_fun wrote:
Tue Nov 23, 2021 9:24 pm
@mete: I took the original recipe for PEG from the internet, unfortunately I forgot the source. I slightly modified it, and use the following procedure now:

(1) fixing the samples in AFE
(2) rinsing in aqua dest
(3) around 6h in 20wt%PEG 1500/80wt% aqua dest
(4) around 4h in 50%PEG/50%aqua dest.
(5) around 6h in PEG 1500 @55°C
(6) casting a PEG block with the embedded sample for slicing
(7) after slicing dissolution of PEG in 20wt% PEG / 80wt% aqua dest
(8) staining and mounting as desired

It is important to replace the water in the sample gently with PEG: water diffuses faster than PEG, and if the exchange process happens to fast (e.g. by skipping the 50/50-stage or putting the slices directly into aqua dest.), the sample gets easily destroyed.

Overall a fairly easy process, involving no problematic solvents or other nasty chemicals.
Thanks, I think I will try PEG, I have a few questions but I will create a new post.

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