spectral unmixing with Zeiss 880

[Cammer, Michael]

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We are trying to do spectral unmixing with a series of Opal probes with the Zeiss 880 spectral detector.  We have collected the spectra of each one and saved them.


Question:  for each image that we want to use these references for, how can we automatically populate the unmixing settings with these.  We need to load six spectra and have the LUTs set in a standard manner.


We tried loading them and setting the LUTs, then saving the image we were using them with, and reusing the settings,  but the spectra profile do not get propagated.  We cannot set six spectra and their LUTs manually for every image that we need to unmix.


Help with this greatly appreciated.


Thank you.


Michael Cammer, Sr Research Scientist, DART Microscopy Laboratory

NYU Langone Health, 540 First Avenue, SK2 Microscopy Suite, New York, NY  10016

[hidden email]<mailto:[hidden email]>  http://nyulmc.org/micros  http://microscopynotes.com/

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[Sripad Ram-2]

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Hi Michael,
I am not surprised that the LUTs are not propagating when you reuse the
settings (typically the resuse feature on Zen propagates acquisition
parameters and a few other settings).

A few years back, I tried to do spectral unmixing for Opal dyes on Axioscan
images (Zen blue). I had the same problem that you encountered wherein I
had to manually load the LUTs for each image.

While the spectral unmixing step can be called in batch processing mode in
Zen blue, it does not allow you to do LUT based spectral unmixing as a
batch  (duh!!). So Zeiss wrote a set of marcos (clunky, but works) which
will perform spectral unmixing in batch mode using LUTs. I am guessing that
Zeiss may be able to give you the macro.

If you want to stay in Zen environment, then OAD module might be another
option but you would need to buy this module and code the batch script
yourself in python.

Hope this helps.

Sripad



On Wed, Aug 7, 2019 at 8:10 AM Cammer, Michael <
[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.
> *****
>
> We are trying to do spectral unmixing with a series of Opal probes with
> the Zeiss 880 spectral detector.  We have collected the spectra of each one
> and saved them.
>
>
> Question:  for each image that we want to use these references for, how
> can we automatically populate the unmixing settings with these.  We need to
> load six spectra and have the LUTs set in a standard manner.
>
>
> We tried loading them and setting the LUTs, then saving the image we were
> using them with, and reusing the settings,  but the spectra profile do not
> get propagated.  We cannot set six spectra and their LUTs manually for
> every image that we need to unmix.
>
>
> Help with this greatly appreciated.
>
>
> Thank you.
>
>
> Michael Cammer, Sr Research Scientist, DART Microscopy Laboratory
>
> NYU Langone Health, 540 First Avenue, SK2 Microscopy Suite, New York, NY
> 10016
>
> [hidden email]<mailto:[hidden email]>
> http://nyulmc.org/micros  http://microscopynotes.com/
>
> Voice direct only, no text or messages:  1-914-309-3270 and 1-646-501-0567
>

[G. Esteban Fernandez]

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What I do is open an unmixed image (i.e. resulting channel image not lambda
stack) and click Read Parameters From Image under Processing > Linear
Unmixing.

-Esteban

On Wed, Aug 7, 2019 at 8:10 AM Cammer, Michael <
[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.
> *****
>
> We are trying to do spectral unmixing with a series of Opal probes with
> the Zeiss 880 spectral detector.  We have collected the spectra of each one
> and saved them.
>
>
> Question:  for each image that we want to use these references for, how
> can we automatically populate the unmixing settings with these.  We need to
> load six spectra and have the LUTs set in a standard manner.
>
>
> We tried loading them and setting the LUTs, then saving the image we were
> using them with, and reusing the settings,  but the spectra profile do not
> get propagated.  We cannot set six spectra and their LUTs manually for
> every image that we need to unmix.
>
>
> Help with this greatly appreciated.
>
>
> Thank you.
>
>
> Michael Cammer, Sr Research Scientist, DART Microscopy Laboratory
>
> NYU Langone Health, 540 First Avenue
> <https://www.google.com/maps/search/540+First+Avenue?entry=gmail&source=g>,
> SK2 Microscopy Suite, New York, NY  10016
>
> [hidden email]<mailto:[hidden email]>
> http://nyulmc.org/micros  http://microscopynotes.com/
>
> Voice direct only, no text or messages:  1-914-309-3270 and 1-646-501-0567
>