Fluorescent doughnut-shaped mitochondria from Arabidopsis roots

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Wes
Posts: 270
Joined: Sat Mar 09, 2019 12:58 pm

Fluorescent doughnut-shaped mitochondria from Arabidopsis roots

#1 Post by Wes » Sat Oct 12, 2019 2:51 pm

I got the opportunity to play with a Zeiss LSM880 confocal laser scanning microscope. For this purpose I had to prepare a specimen that was the root of Arabidopsis thaliana seedling. This particular strain of Arabidopsis expresses a green fluorescent protein which contains a mitochondrial targeting sequence. The signal you are seeing is the GFP fluorescence (I've replaced the 256 gray scale with a fire lookup table) coming from mitochondria which are shaped like doughnuts (I think the proper term is toroids). I made a Z-stack and from this a 3D rotating projection.

I thought you guys might find it interesting.

Image

Hobbyst46
Posts: 2133
Joined: Mon Aug 21, 2017 9:02 pm

Re: Fluorescent doughnut-shaped mitochondria from Arabidopsis roots

#2 Post by Hobbyst46 » Sat Oct 12, 2019 3:29 pm

There are specific mitochondrial stains, yet they are very expensive....
Zeiss Standard GFL+Canon EOS-M10, Olympus VMZ stereo

Wes
Posts: 270
Joined: Sat Mar 09, 2019 12:58 pm

Re: Fluorescent doughnut-shaped mitochondria from Arabidopsis roots

#3 Post by Wes » Sat Oct 12, 2019 3:33 pm

Hobbyst46 wrote:
Sat Oct 12, 2019 3:29 pm
There are specific mitochondrial stains, yet they are very expensive....
Yes, there was mitotracker in the lab but we had the GFP seedlings so it was one step less in the preparation

mintakax
Posts: 139
Joined: Mon Jul 29, 2019 12:06 am
Location: Boulder CO, USA

Re: Fluorescent doughnut-shaped mitochondria from Arabidopsis roots

#4 Post by mintakax » Sat Oct 12, 2019 3:42 pm

What a great experience, I'd be interested to hear more about it !

MicroBob
Posts: 1422
Joined: Sun Dec 25, 2016 9:11 am

Re: Fluorescent doughnut-shaped mitochondria from Arabidopsis roots

#5 Post by MicroBob » Sat Oct 12, 2019 3:45 pm

Hi Wes,
that rotation presentation is very nice! Which software did you use to generate it?
Can you say how big the picture object was altogether?

Bob

Wes
Posts: 270
Joined: Sat Mar 09, 2019 12:58 pm

Re: Fluorescent doughnut-shaped mitochondria from Arabidopsis roots

#6 Post by Wes » Sat Oct 12, 2019 3:53 pm

mintakax wrote:
Sat Oct 12, 2019 3:42 pm
What a great experience, I'd be interested to hear more about it !
Thanks mintakax, I had to learn to use the confocal microscope in order to capture and quantify single RNA molecules from in situ hybridization and as far as the microscopy part goes I used readily available fluorescent seedlings as a test subject to get a hang of the controls etc. Its a nice microscope with multiple lasers and top of the line planapo objectives. Maybe one day decades from now they will start popping up on ebay and come into the possession of the highest bidder.
MicroBob wrote:
Sat Oct 12, 2019 3:45 pm
Hi Wes,
that rotation presentation is very nice! Which software did you use to generate it?
Can you say how big the picture object was altogether?

Bob
Hi Bob,
The bigger mitochondria in the image measure around 1.5 µm, I used a 100/1.46 planapo for this image. I used ImageJ, also goes by the name of FIJI which means "FIJI Is Just ImageJ", basically the same thing. Its completely free and packed with functionality. I use it mostly for taking measurements of microbes.

Here is a root hair from the same Arabidopsis strain, notice the round mitochondria at the periphery of the cell.

Image
Last edited by Wes on Sat Oct 12, 2019 5:02 pm, edited 1 time in total.

MicroBob
Posts: 1422
Joined: Sun Dec 25, 2016 9:11 am

Re: Fluorescent doughnut-shaped mitochondria from Arabidopsis roots

#7 Post by MicroBob » Sat Oct 12, 2019 4:13 pm

Again a great rotating image, Wes!
To make the shape of things understandable these animations are very interesting.
Would your new toy be useful to genetare some stunning mitosis pictures? ;)

Wes
Posts: 270
Joined: Sat Mar 09, 2019 12:58 pm

Re: Fluorescent doughnut-shaped mitochondria from Arabidopsis roots

#8 Post by Wes » Sat Oct 12, 2019 5:04 pm

MicroBob wrote:
Sat Oct 12, 2019 4:13 pm
Again a great rotating image, Wes!
To make the shape of things understandable these animations are very interesting.
Would your new toy be useful to genetare some stunning mitosis pictures? ;)
Thanks Bob, I believe it would be the perfect instrument to catch high resolution mitotic scenery. Below is an example of some of the images you can get with this scope (but most confocals would delivery comparable quality). The added bonus to the LSM880 is the gallium-arsenide AiryScan detector which improves resolution beyond the diffraction limit.

Image

Hobbyst46
Posts: 2133
Joined: Mon Aug 21, 2017 9:02 pm

Re: Fluorescent doughnut-shaped mitochondria from Arabidopsis roots

#9 Post by Hobbyst46 » Sat Oct 12, 2019 7:20 pm

Wes wrote:
Sat Oct 12, 2019 3:53 pm
...Its a nice microscope with multiple lasers and top of the line planapo objectives. Maybe one day decades from now they will start popping up on ebay and come into the possession of the highest bidder.
Those multiple lasers, among other features, make present day confocal an unlikely candidate for average hobby home use, even with eBay prices decades from now...although I am thinking of earlier LSM models (or Leica confocal models)
Zeiss Standard GFL+Canon EOS-M10, Olympus VMZ stereo

MicroBob
Posts: 1422
Joined: Sun Dec 25, 2016 9:11 am

Re: Fluorescent doughnut-shaped mitochondria from Arabidopsis roots

#10 Post by MicroBob » Sat Oct 12, 2019 8:16 pm

I have used Fiji before, but never used it much and so it dropped of my radar. I remember it for having very powerful but also quite hidden functions.
It seems as if I really should have a look at it again.

According the future hobby use of todays high-tech-Equipment: I think in gerneral this is becoming more and more difficult. While an open source 3D printer can easily be renovated and updated, a proprietary closed system will be a problem a while after manufacturer support is no longer available or affordable. Some electonics also are not long term dependable and difficult to repair when they break.
Our microscopy group has set up an old Leitz SEM, I think from 1969: http://www.mikrohamburg.de/HomeRem_1.html
It has been updated to digital imaging and is running well.It was already a difficult task to get this instrument that had been modified for special tasks at DESY to run in normal use again and this was with competent professional service in the background. I doubt that this could be done in 34 years with an instrument of today - we will see!

Bob

Wes
Posts: 270
Joined: Sat Mar 09, 2019 12:58 pm

Re: Fluorescent doughnut-shaped mitochondria from Arabidopsis roots

#11 Post by Wes » Sat Oct 12, 2019 8:30 pm

Hobbyst46 wrote:
Sat Oct 12, 2019 7:20 pm
Wes wrote:
Sat Oct 12, 2019 3:53 pm
...Its a nice microscope with multiple lasers and top of the line planapo objectives. Maybe one day decades from now they will start popping up on ebay and come into the possession of the highest bidder.
Those multiple lasers, among other features, make present day confocal an unlikely candidate for average hobby home use, even with eBay prices decades from now...although I am thinking of earlier LSM models (or Leica confocal models)
I think I made a flawed parallel to mid 20th century research grade scopes to their contemporary counterparts. While a Zeiss Universal research microscope is a beautifully engineered optical machine its nowhere near as complicated as an LSM. Perhaps a few specialist hobbyists well-versed in electronics and coding their own drivers for high-end cameras and laser devices might make use of it and even then it seems questionable. Even then there are a ton of components that can break and they normally do which is why these scopes require annual (if not more frequent) maintenance.
MicroBob wrote:
Sat Oct 12, 2019 8:16 pm
I have used Fiji before, but never used it much and so it dropped of my radar. I remember it for having very powerful but also quite hidden functions.
It seems as if I really should have a look at it again.
If you have a stage micrometer you can take an image of it and then measure the distance from one graduation to the next (say 10 or 100 µm) and tell the program that this many pixels correspond to the given distance. You set this as a global scale and you can measure straight lines, curved lines, circumference, random paths, area and more. All you have to do is photograph your sample under the same conditions you photographed the stage micrometer. You will never have to use a reticle (as long as you take an image of your specimen).

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