Question about 3D images from rapidSTORM

[pth...@gmail.com]

Hello all!

I'm currently working on implementing 3d by astigmatism in my dSTORM setup.
First results so far are promising, even though there's still a lot of fine-tuning to do.
I have, however, encountered an issue with the reconstruction of my data via rapidSTORM.
I basically followed the workflow from the manual to create a calibration curve for my measurements.
When I choose to reconstruct a color-coded 3d image (that is, vary color with z-position of localization), the resulting image is fine.
However, when I tick the "Make 3D image" option (I changed the image format to .tif), the resulting .tif file seems to contain only a single z-layer - it is also rather small (~100-400kB) and is showing only very few localizations.
No additional files are created either.
Is there something I'm missing?

Best regards,
Patrick

[Steve Wolter]

Hi Patrick,

sounds like a problem with the z range when creating the TIF file. Could you try adding an expression filter and setting maxposz and minposz by hand?

Best, Steve

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[pth...@gmail.com]

Hey Steve,

so I tried that, but it doesn't really seem to change anything.
I now have the adjusted values for min and max z showing up in the scalebar of the image which is being calculated, but that's it.
The resulting .tif file is still not containing anything but the lowest z layer (at least that's what I assume it contains).

Best,
Patrick

[Steve Wolter]

Fair enough. I'm afraid that I'm a bit out of ideas at this point.

Has someone on the list used 3D stacks recently?

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[Sven Proppert]

Hi Patrick, hi Steve,

I'm back at work and can spend a little time on your problem.

I really don't know, what's going on with your rapidSTORM as I never had these problems. What especially puzzles me is that your 2D (with z colorcoded) representation seems to look fine.

By now, I can only think of not too many more things. Here is one: Did you tick the "Y zero at bottom" checkbox? If yes, please uncheck it, because this one often causes trouble.

Could you please give me/us some more information about your system and your rapidSTORM release?
- Is it rapidSTORM 3.3.1 or any prior release?
- Windows or Linux?
- 32 bit or 64 bit?
- How many cores? (It seems unlikely but I once had a problem with rapidSTORM when I only gave it one single CPU.)
- In my opinion unlikely because rapidSTORM should raise an "out of memory" error, but: How much RAM do you have?

Some other thoughts:
Which voxelsize, i.e. resolution in x,y,z, did you set? Too small pixels cause big stacks that might cause an error.
Did you check, whether it really is the bottom layer you get after ticking the "Make 3D image" box? You could do that by restricting your 2D-image to the bottom-most layer with such a filter: posz < (minposz + resolution in z)   (e.g.: posz < (-500 nm +60 nm)

Most importantly: Could you please give me a use case? It would help, if you could provide me with a measurement, that shows the behavior you describe, and the output files you get (filename.tif, filename.png, filename.txt, AND filename-settings.txt). It does not need to be a full measurement as long as it shows the "(un-)desired" behavior. any ftp-server or wetransfer/wesendit/etc. would be just fine.
I want to compare your settings file with one I would create and I want to know, if I can reproduce your error on my computer.

So far, this is my two cents. I hope it helps and I look forward to some usecase.

Cheers,
Sven

Am Dienstag, 29. August 2017 19:26:06 UTC+2 schrieb Steve Wolter:

Fair enough. I'm afraid that I'm a bit out of ideas at this point.

Has someone on the list used 3D stacks recently?

On Fri, Aug 25, 2017 at 9:13 AM <pth...@gmail.com> wrote:

Hey Steve,

so I tried that, but it doesn't really seem to change anything.
I now have the adjusted values for min and max z showing up in the scalebar of the image which is being calculated, but that's it.
The resulting .tif file is still not containing anything but the lowest z layer (at least that's what I assume it contains).

Best,
Patrick

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[Sven Proppert]

Hi Patrick,

the answer is as simple as can be: Your settings-file was not suited for an evaluation. I created one that should work just fine for you.

Cheers,
Sven

[Patrick Then]

Thank you very much, but unfortunately it still behaves the same when I use your file...

I guess I could try an older version of rapidSTORM or install it on a Linux System to see if there's any difference.

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[pth...@gmail.com]

Well, never mind!
I hadn't read your other mail before I posted here. The problem was that I indeed had to uncheck the "Display dSTORM result image" option!