Re: FW: LSM 510 META computer upgrade

[Claire Brown]

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We had very similar issues on our 510. It has never run as well as it did
with the old AIM software. The way I understand it is that ZEN if programed
to get certain response times from the equipment. The older equipment takes
longer than the newer equipment to respond. The software times out while
waiting for a response and you can an error or a crash. The other problem is
we got stuck at Zen2009 because some of our hardware was not compatible with
the newer versions. So we had bugs but no one was working on fixing them.
Which I totally understand from a company perspective but...

We also found that there are some odd fixes like you can change the order of
the tracks and everything works (collect blue green red instead of red green
blue) you might try this for the PMT that is not working. I would be very
surprised if something suddenly happened to the microscope.

My lesson from all this is I do not upgrade older equipment to new software
if I don't have to. Do you still have the old computer? Maybe you could just
put some more memory or a larger HD in that computer instead?

Luckily we got some funding for new equipment so we'll be retiring the 510 soon.

Sorry to not have better news.

Claire

[Rosemary.White]

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Has anyone written a plugin for ImageJ/Fiji that will calculate the
perpendicular distance between an inner and outer line at various
positions?

We need to measure the wall thickness on isolated plant cells which, some
of which look like cross-sections of a cylinder. Now, if they were all
nearly circular cylinders with even wall thickness, that'd be easy. But
these cells are far from perfectly cylindrical in shape and the wall
thickness is uneven.

In the past, we just made 4 measurements of the wall at fixed N-S-E-W
positions and calculated the average thickness. But with the imaging tools
available today, it should be possible to write a plugin that will do
something like this - run a ball along the wall which shrinks and swells
according to the wall thickness and record the ball diameter at every
position, for example. That would not only give us the average thickness
but some useful stats about variability (per cell and per sample) as well.

If anyone knows of something that will do this, you will have our eternal
gratitude!

thanks,
Rosemary

Dr Rosemary White
CSIRO Black Mountain
GPO Box 1600
Canberra, ACT 2601
Australia

T 61 2 6246 5475
E [hidden email]

[Hashmath Inayath Hussain]

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Hi,

Try Orthogonal z-projections using the reslice tool in ImageJ. Extended depth of field images using an ImageJ plugin (refer to the following paper " Complex Wavelets for Extended Depth-of-Field: A New Method for the Fusion of Multichannel Microscopy Images")

As an alternate to imagej you may wish to try http://www.plant-image-analysis.org/software/cellset or http://www.plant-image-analysis.org/software/celer


Hope this helps.

Regards,
Hashmath

-----Original Message-----
From: Confocal Microscopy List [mailto:[hidden email]] On Behalf Of [hidden email]
Sent: Wednesday, 12 August 2015 3:10 PM
To: [hidden email]
Subject: measuring average wall thickness

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Has anyone written a plugin for ImageJ/Fiji that will calculate the perpendicular distance between an inner and outer line at various positions?

We need to measure the wall thickness on isolated plant cells which, some of which look like cross-sections of a cylinder. Now, if they were all nearly circular cylinders with even wall thickness, that'd be easy. But these cells are far from perfectly cylindrical in shape and the wall thickness is uneven.

In the past, we just made 4 measurements of the wall at fixed N-S-E-W positions and calculated the average thickness. But with the imaging tools available today, it should be possible to write a plugin that will do something like this - run a ball along the wall which shrinks and swells according to the wall thickness and record the ball diameter at every position, for example. That would not only give us the average thickness but some useful stats about variability (per cell and per sample) as well.

If anyone knows of something that will do this, you will have our eternal gratitude!

thanks,
Rosemary

Dr Rosemary White
CSIRO Black Mountain
GPO Box 1600
Canberra, ACT 2601
Australia

T 61 2 6246 5475
E [hidden email]

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[Michael Model]

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We measure thickness of live animal cells of any shape in monolayer by dye
exclusion (Microscopy Today
<http://journals.cambridge.org/action/displayJournal?jid=MTO> / Volume 20 /
Issue 03 / May 2012, pp 32-37). It might work on plant cells too though we
have not tried that.

Mike Model

On Wed, Aug 12, 2015 at 1:10 AM, <[hidden email]> wrote:

> *****
> To join, leave or search the confocal microscopy listserv, go to:
> http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy
> Post images on http://www.imgur.com and include the link in your posting.
> *****
>
> Has anyone written a plugin for ImageJ/Fiji that will calculate the
> perpendicular distance between an inner and outer line at various
> positions?
>
> We need to measure the wall thickness on isolated plant cells which, some
> of which look like cross-sections of a cylinder. Now, if they were all
> nearly circular cylinders with even wall thickness, that'd be easy. But
> these cells are far from perfectly cylindrical in shape and the wall
> thickness is uneven.
>
> In the past, we just made 4 measurements of the wall at fixed N-S-E-W
> positions and calculated the average thickness. But with the imaging tools
> available today, it should be possible to write a plugin that will do
> something like this - run a ball along the wall which shrinks and swells
> according to the wall thickness and record the ball diameter at every
> position, for example. That would not only give us the average thickness
> but some useful stats about variability (per cell and per sample) as well.
>
> If anyone knows of something that will do this, you will have our eternal
> gratitude!
>
> thanks,
> Rosemary
>
> Dr Rosemary White
> CSIRO Black Mountain
> GPO Box 1600
> Canberra, ACT 2601
> Australia
>
> T 61 2 6246 5475
> E [hidden email]
>

[olivier.burri]

> > Has anyone written a plugin for ImageJ/Fiji that will calculate the
> > perpendicular distance between an inner and outer line at various
> > positions?

If you can create a closed surface out of your internal and external line you can use the Local Thickness plugin that comes with Fiji. It does just what you want
Best
http://fiji.sc/Local_Thickness

Oli

[Rosemary.White]

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We tried that, but it gives spurious results.
thanks,
Rosemary

On 12/08/15 11:07 PM, "Burri Olivier" <[hidden email]> wrote:

>> > Has anyone written a plugin for ImageJ/Fiji that will calculate the
>> > perpendicular distance between an inner and outer line at various
>> > positions?
>
>If you can create a closed surface out of your internal and external line
>you can use the Local Thickness plugin that comes with Fiji. It does just
>what you want
>Best
>http://fiji.sc/Local_Thickness
>
>Oli

[Lloyd Donaldson]

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Hi Rosemary

Difficult to visualise exactly what your cells are like. However an approach we used years ago was to binarize the cell wall and make two images, a Euclidian distance map, and a watershed. Then you use the watershed as a mask to read out the local maxima of the Euclidian distance map. I did this interactively in imageJ - sorry no plugin. This yields a continuous plot of wall thickness around the cell. If your cells are isolated rather than in a tissue then the values will be half wall thickness.
If that won't work maybe you can send me a test image and I will think about it.

Cheers - Lloyd

Dr Lloyd Donaldson
Microscopy & Wood Identification
Senior Scientist - Plant Cell Walls & Biomaterials
Scion - Forests, Products, Innovation
49 Sala Street, Rotorua 3010
New Zealand
Ph 07 343 5581
www.scionresearch.com



-----Original Message-----
From: Confocal Microscopy List [mailto:[hidden email]] On Behalf Of [hidden email]
Sent: Thursday, August 13, 2015 10:23 AM
To: [hidden email]
Subject: Re: measuring average wall thickness

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We tried that, but it gives spurious results.
thanks,
Rosemary

On 12/08/15 11:07 PM, "Burri Olivier" <[hidden email]> wrote:

>> > Has anyone written a plugin for ImageJ/Fiji that will calculate the
>> > perpendicular distance between an inner and outer line at various
>> > positions?
>
>If you can create a closed surface out of your internal and external line
>you can use the Local Thickness plugin that comes with Fiji. It does just
>what you want
>Best
>http://fiji.sc/Local_Thickness
>
>Oli

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[Guy Cox-2]

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Rosemary,

               Perhaps the problem is that the question is not well defined.  Here's one suggestion that will give an answer which seems to meet your requirements but ...

Segment out the wall (from exterior of the cell to the plasma membrane).  Measure the area of the cell wall profile.  Now skeletonize your segmented area to a line.  This should be your effective wall midline.  Measure the length of that, and divide your wall area by the length of the midline.  This should be, on one criterion at least, your 'average' wall thickness.  

                                                      Guy

Guy Cox, Honorary Associate Professor
School of Medical Sciences

Australian Centre for Microscopy and Microanalysis,
Madsen, F09, University of Sydney, NSW 2006

-----Original Message-----
From: Confocal Microscopy List [mailto:[hidden email]] On Behalf Of [hidden email]
Sent: Thursday, 13 August 2015 8:23 AM
To: [hidden email]
Subject: Re: measuring average wall thickness

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We tried that, but it gives spurious results.
thanks,
Rosemary

On 12/08/15 11:07 PM, "Burri Olivier" <[hidden email]> wrote:

>> > Has anyone written a plugin for ImageJ/Fiji that will calculate the
>> > perpendicular distance between an inner and outer line at various
>> > positions?
>
>If you can create a closed surface out of your internal and external
>line you can use the Local Thickness plugin that comes with Fiji. It
>does just what you want Best http://fiji.sc/Local_Thickness
>
>Oli

[Sathya]

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Hi Rosemary,
Out of curiosity was searching the web and found this article which is similar to your question:
"Measuring Cell wall dimensions using the distance transform" in Annals of Botany 75: 545-552, 1995
 
In this article they have used SCIL-Image from TDP-TNO, Delft, Netherlands. I tried to find something about it, but it takes me to a place where this software is available as trial version. No details if it is still in market or its availability.  Maybe someone who is good with ImageJ/ FIJI plugins can go through the steps described in the article and create a Macros or a instant plugin. Or if the authors of this paper can be found, they can be contacted directly. The article is freely available online. If not, contact me offline and I can send you the PDF I have downloaded. Good luck.
 
Sathya Srinivasan
Manager
The Regeneration Unit in Neurobiology (RUN) Advanced Optical Microscopy Facility
(www.ucalgary.ca/runcore)
University of Calgary
Calgary, AB T2N4N1
Canada
 

> Date: Wed, 12 Aug 2015 05:10:22 +0000
> From: [hidden email]
> Subject: measuring average wall thickness
> To: [hidden email]
>
> *****
> To join, leave or search the confocal microscopy listserv, go to:
> http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy
> Post images on http://www.imgur.com and include the link in your posting.
> *****
>
> Has anyone written a plugin for ImageJ/Fiji that will calculate the
> perpendicular distance between an inner and outer line at various
> positions?
>
> We need to measure the wall thickness on isolated plant cells which, some
> of which look like cross-sections of a cylinder. Now, if they were all
> nearly circular cylinders with even wall thickness, that'd be easy. But
> these cells are far from perfectly cylindrical in shape and the wall
> thickness is uneven.
>
> In the past, we just made 4 measurements of the wall at fixed N-S-E-W
> positions and calculated the average thickness. But with the imaging tools
> available today, it should be possible to write a plugin that will do
> something like this - run a ball along the wall which shrinks and swells
> according to the wall thickness and record the ball diameter at every
> position, for example. That would not only give us the average thickness
> but some useful stats about variability (per cell and per sample) as well.
>
> If anyone knows of something that will do this, you will have our eternal
> gratitude!
>
> thanks,
> Rosemary
>
> Dr Rosemary White
> CSIRO Black Mountain
> GPO Box 1600
> Canberra, ACT 2601
> Australia
>
> T 61 2 6246 5475
> E [hidden email]

     

[Deanne Veronica Catmull]

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Thanks for that Claire, can I ask what version of the LSM 510 META you have? I am learning that versions 2.5 and less have experienced more problems with this upgrade than the newer versions? We have version 3.5 and we purchased in 2005.

Kind regards,
Deanne.

Deanne Catmull | Research Assistant and HSR
Melbourne Dental School | Faculty of Medicine, Dentistry and Health Sciences
Level 6, 720 Swanston St
The University of Melbourne, Victoria 3010 Australia
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-----Original Message-----
From: Confocal Microscopy List [mailto:[hidden email]] On Behalf Of Claire Brown
Sent: Wednesday, 12 August 2015 4:51 AM
To: [hidden email]
Subject: Re: FW: LSM 510 META computer upgrade

*****
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*****

We had very similar issues on our 510. It has never run as well as it did with the old AIM software. The way I understand it is that ZEN if programed to get certain response times from the equipment. The older equipment takes longer than the newer equipment to respond. The software times out while waiting for a response and you can an error or a crash. The other problem is we got stuck at Zen2009 because some of our hardware was not compatible with the newer versions. So we had bugs but no one was working on fixing them.
Which I totally understand from a company perspective but...

We also found that there are some odd fixes like you can change the order of the tracks and everything works (collect blue green red instead of red green
blue) you might try this for the PMT that is not working. I would be very surprised if something suddenly happened to the microscope.

My lesson from all this is I do not upgrade older equipment to new software if I don't have to. Do you still have the old computer? Maybe you could just put some more memory or a larger HD in that computer instead?

Luckily we got some funding for new equipment so we'll be retiring the 510 soon.

Sorry to not have better news.

Claire

[Francisco J H Blazquez]

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HI.
Maybe a simple method that I used to measure the spread of lucifer yelow
in a monolayer of cells in culture is to define a length (if you have a
straigth surface) and measure the area under this line. The length can
be the longer axis of the microphotography. In this case I could easily
threshold the area. Then you have one dimension (length) and the area,
now you can calculate the mean thickness if the other side of the length
is irregular.

Francisco Blazquez

Em 12/08/2015 02:10, [hidden email] escreveu:

> *****
> To join, leave or search the confocal microscopy listserv, go to:
> http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy
> Post images on http://www.imgur.com and include the link in your posting.
> *****
>
> Has anyone written a plugin for ImageJ/Fiji that will calculate the
> perpendicular distance between an inner and outer line at various
> positions?
>
> We need to measure the wall thickness on isolated plant cells which, some
> of which look like cross-sections of a cylinder. Now, if they were all
> nearly circular cylinders with even wall thickness, that'd be easy. But
> these cells are far from perfectly cylindrical in shape and the wall
> thickness is uneven.
>
> In the past, we just made 4 measurements of the wall at fixed N-S-E-W
> positions and calculated the average thickness. But with the imaging tools
> available today, it should be possible to write a plugin that will do
> something like this - run a ball along the wall which shrinks and swells
> according to the wall thickness and record the ball diameter at every
> position, for example. That would not only give us the average thickness
> but some useful stats about variability (per cell and per sample) as well.
>
> If anyone knows of something that will do this, you will have our eternal
> gratitude!
>
> thanks,
> Rosemary
>
> Dr Rosemary White
> CSIRO Black Mountain
> GPO Box 1600
> Canberra, ACT 2601
> Australia
>
> T 61 2 6246 5475
> E [hidden email]
>