spectral unmixing with Zeiss 510 meta

[Martin Wessendorf-2]

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Dear List--

I am trying to learn how to use the spectral unmixing function on the
Zeiss 510.  I've loaded individual spectra of the three fluorophores
that I'm using.  I then took an image of a region in which there's a lot
of triple labeling and loaded the stored spectra to allow linear
unmixing.  The program displays a resulting image.  However, the
program appears not to display structures in which there are coexisting
labels--only the few regions in which there is single-labeling.

Am I missing something?  Or is this how the program is designed to work?

Any suggestions welcome!

Thanks--

Martin Wessendorf
--
Martin Wessendorf, Ph.D.                   office: [phone]
Assoc Prof, Dept Neuroscience                 lab: [phone]
University of Minnesota             Preferred FAX: [phone]
6-145 Jackson Hall, 321 Church St. SE    Dept Fax: [phone]
Minneapolis, MN  55455                    e-mail: [hidden email]

[Craig Brideau]

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It really depends on how the algorithm handles assignment.  For each pixel,
the algorithm has to decide whether to false-color it (for example) red,
blue, or green, depending on whether the algorithm decides that that pixel
is mostly the flurophore assigned to red, or blue, or green.  Some
algorithms can't make a proportional call, for instance they can't give you
information that a pixel is 40% the blue-assigned flurophore and 60% the
red-assigned fluorphore.  In this case it would show the pixel only as red,
because the red-assigned fluorphore dominates that pixel.  I'm not familiar
with the Zeiss spectral unmix algorithm, but unless there's just a setting
somewhere I suspect this is what it is doing to you.  Our in-house software
will actually 'dither' pixels by proportion of fluorophore present in each
pixel to deal with this problem.  The Zeiss software may also be able to do
this, but I'm not sure.

Craig


On Wed, May 23, 2012 at 9:32 AM, Martin Wessendorf <[hidden email]> wrote:

> *****
> To join, leave or search the confocal microscopy listserv, go to:
> http://lists.umn.edu/cgi-bin/**wa?A0=confocalmicroscopy<http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy>
> *****
>
> Dear List--
>
> I am trying to learn how to use the spectral unmixing function on the
> Zeiss 510.  I've loaded individual spectra of the three fluorophores that
> I'm using.  I then took an image of a region in which there's a lot of
> triple labeling and loaded the stored spectra to allow linear unmixing.
>  The program displays a resulting image.  However, the program appears not
> to display structures in which there are coexisting labels--only the few
> regions in which there is single-labeling.
>
> Am I missing something?  Or is this how the program is designed to work?
>
> Any suggestions welcome!
>
> Thanks--
>
> Martin Wessendorf
> --
> Martin Wessendorf, Ph.D.                   office: [phone]
> Assoc Prof, Dept Neuroscience                 lab: [phone]
> University of Minnesota             Preferred FAX: [phone]
> 6-145 Jackson Hall, 321 Church St. SE    Dept Fax: [phone]
> Minneapolis, MN  55455                    e-mail: [hidden email]
>

[G. Esteban Fernandez]

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In co-labeled regions ZEN software will proportionately assign the
signal to both labels, so you should see the structures in both
regions (I remember LSM/AIM doing the same but I could be wrong).  How
did you measure the spectra that you put into the unmixing algorithm?
The best way may be to measure the spectra within the same image if
you can, by marking areas that you know are single-labeled.  Is there
autofluorescence?

-Esteban


On Wed, May 23, 2012 at 11:21 AM, Craig Brideau <[hidden email]> wrote:

> *****
> To join, leave or search the confocal microscopy listserv, go to:
> http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy
> *****
>
> It really depends on how the algorithm handles assignment.  For each pixel,
> the algorithm has to decide whether to false-color it (for example) red,
> blue, or green, depending on whether the algorithm decides that that pixel
> is mostly the flurophore assigned to red, or blue, or green.  Some
> algorithms can't make a proportional call, for instance they can't give you
> information that a pixel is 40% the blue-assigned flurophore and 60% the
> red-assigned fluorphore.  In this case it would show the pixel only as red,
> because the red-assigned fluorphore dominates that pixel.  I'm not familiar
> with the Zeiss spectral unmix algorithm, but unless there's just a setting
> somewhere I suspect this is what it is doing to you.  Our in-house software
> will actually 'dither' pixels by proportion of fluorophore present in each
> pixel to deal with this problem.  The Zeiss software may also be able to do
> this, but I'm not sure.
>
> Craig
>
>
> On Wed, May 23, 2012 at 9:32 AM, Martin Wessendorf <[hidden email]> wrote:
>
>> *****
>> To join, leave or search the confocal microscopy listserv, go to:
>> http://lists.umn.edu/cgi-bin/**wa?A0=confocalmicroscopy<http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy>
>> *****
>>
>> Dear List--
>>
>> I am trying to learn how to use the spectral unmixing function on the
>> Zeiss 510.  I've loaded individual spectra of the three fluorophores that
>> I'm using.  I then took an image of a region in which there's a lot of
>> triple labeling and loaded the stored spectra to allow linear unmixing.
>>  The program displays a resulting image.  However, the program appears not
>> to display structures in which there are coexisting labels--only the few
>> regions in which there is single-labeling.
>>
>> Am I missing something?  Or is this how the program is designed to work?
>>
>> Any suggestions welcome!
>>
>> Thanks--
>>
>> Martin Wessendorf
>> --
>> Martin Wessendorf, Ph.D.                   office: [phone]
>> Assoc Prof, Dept Neuroscience                 lab: [phone]
>> University of Minnesota             Preferred FAX: [phone]
>> 6-145 Jackson Hall, 321 Church St. SE    Dept Fax: [phone]
>> Minneapolis, MN  55455                    e-mail: [hidden email]
>>

[Martin Wessendorf-2]

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On 5/24/2012 3:30 PM, G. Esteban Fernandez wrote:

> In co-labeled regions ZEN software will proportionately assign the
> signal to both labels, so you should see the structures in both
> regions (I remember LSM/AIM doing the same but I could be wrong).  How
> did you measure the spectra that you put into the unmixing algorithm?
> The best way may be to measure the spectra within the same image if
> you can, by marking areas that you know are single-labeled.  Is there
> autofluorescence?
>
> -Esteban

That is exactly the behavior that I did not see.

We have what I think are good quality spectra that were obtained from
regions with single labeling.  There's no significant autofluorescence.

I expect there's some box in the Zen system that I haven't checked, but
I think I've tried all of them that seemed obvious to try (i.e. all the
boxes on the Linear Unmixing window).

I must be missing something but am baffled by what it might be!

Martin

> On Wed, May 23, 2012 at 11:21 AM, Craig Brideau<[hidden email]>  wrote:
>> *****
>> To join, leave or search the confocal microscopy listserv, go to:
>> http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy
>> *****
>>
>> It really depends on how the algorithm handles assignment.  For each pixel,
>> the algorithm has to decide whether to false-color it (for example) red,
>> blue, or green, depending on whether the algorithm decides that that pixel
>> is mostly the flurophore assigned to red, or blue, or green.  Some
>> algorithms can't make a proportional call, for instance they can't give you
>> information that a pixel is 40% the blue-assigned flurophore and 60% the
>> red-assigned fluorphore.  In this case it would show the pixel only as red,
>> because the red-assigned fluorphore dominates that pixel.  I'm not familiar
>> with the Zeiss spectral unmix algorithm, but unless there's just a setting
>> somewhere I suspect this is what it is doing to you.  Our in-house software
>> will actually 'dither' pixels by proportion of fluorophore present in each
>> pixel to deal with this problem.  The Zeiss software may also be able to do
>> this, but I'm not sure.
>>
>> Craig
>>
>>
>> On Wed, May 23, 2012 at 9:32 AM, Martin Wessendorf<[hidden email]>  wrote:
>>
>>> *****
>>> To join, leave or search the confocal microscopy listserv, go to:
>>> http://lists.umn.edu/cgi-bin/**wa?A0=confocalmicroscopy<http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy>
>>> *****
>>>
>>> Dear List--
>>>
>>> I am trying to learn how to use the spectral unmixing function on the
>>> Zeiss 510.  I've loaded individual spectra of the three fluorophores that
>>> I'm using.  I then took an image of a region in which there's a lot of
>>> triple labeling and loaded the stored spectra to allow linear unmixing.
>>>   The program displays a resulting image.  However, the program appears not
>>> to display structures in which there are coexisting labels--only the few
>>> regions in which there is single-labeling.
>>>
>>> Am I missing something?  Or is this how the program is designed to work?
>>>
>>> Any suggestions welcome!
>>>
>>> Thanks--
>>>
>>> Martin Wessendorf
>>> --
>>> Martin Wessendorf, Ph.D.                   office: [phone]
>>> Assoc Prof, Dept Neuroscience                 lab: [phone]
>>> University of Minnesota             Preferred FAX: [phone]
>>> 6-145 Jackson Hall, 321 Church St. SE    Dept Fax: [phone]
>>> Minneapolis, MN  55455                    e-mail: [hidden email]
>>>

--
Martin Wessendorf, Ph.D.                   office: [phone]
Assoc Prof, Dept Neuroscience                 lab: [phone]
University of Minnesota             Preferred FAX: [phone]
6-145 Jackson Hall, 321 Church St. SE    Dept Fax: [phone]
Minneapolis, MN  55455                    e-mail: [hidden email]