blind unmixing

[Marc Thibault]

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HI,

We are experiencing some difficulties doing blind unmixing with the Olympus
FV100 on double-orange beads.

Have someone been successful with this technique ? If not, we could also
enter the spectral data of the 2 fluorophores to do normal unmixing, but
this too seems problematic. Any info or protocol would be enormously
appreciated.

Thank you very much in advance.

Marc

 

 

Marc Thibault, PhD

Chemical Engineering Department  

Ecole Polytechnique of Montreal

tel: [phone] ext.3968

 

 

[Zac Arrac Atelaz]

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Hello Marc: We have realized some blind unmixing, almost always we use this function, you have to select the number of expected channels in order for this to work better, sometimes a change in the width of the spectral capture also helps, so we used a combination of opening the confocal aperture with a shorter step size, 5nm or 2nm depending on the distance between peaks of the dyes involved, and a band width at 7nm or less, this usually compensates when we see a low emission on the sample.  I hope this helps Gabriel OH
 > Date: Wed, 12 Oct 2011 10:55:21 -0400

> From: [hidden email]
> Subject: blind unmixing
> To: [hidden email]
>
> *****
> To join, leave or search the confocal microscopy listserv, go to:
> http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy
> *****
>
> HI,
>
> We are experiencing some difficulties doing blind unmixing with the Olympus
> FV100 on double-orange beads.
>
> Have someone been successful with this technique ? If not, we could also
> enter the spectral data of the 2 fluorophores to do normal unmixing, but
> this too seems problematic. Any info or protocol would be enormously
> appreciated.
>
> Thank you very much in advance.
>
> Marc
>
>  
>
>  
>
> Marc Thibault, PhD
>
> Chemical Engineering Department  
>
> Ecole Polytechnique of Montreal
>
> tel: [phone] ext.3968
>
>  
>
>  

     

[mcammer]

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We have a question about using Indo1 with two-photon excitation.  We are using 740 nm to excite the dye and have collection spectral ranges set to 370 to 442 nm and 455 to 600 nm.

We expected that treating the cells with ionomycin would make the shorter wavelength range more intense and the longer wavelength range less intense.  However, what really happens is that the shorter wavelength drops a little and  the longer wavelength drops a lot.  So the raw data look wrong, but after ratioing the data appear correct (i.e. high Ca yields 1.0 and low Ca yields 0.5).  Also, washing out the ionomycin with EGTA does reverse the ratio most of the way.

When we run the experiments of cells interacting, the trend of the ratios match published results and previous results we got with Fluo4 by widefield.  However, we are worried because the lower wavelength range does not increase intensity.  This means that the changes are in the range of only twofold, i.e. 0.5 to 1.0.

Does anybody know what the problem is?

Thanks!

________________________________________________________
Michael Cammer, Assistant Research Scientist
Skirball Institute of Biomolecular Medicine
Lab: [phone]  Cell: [phone]

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[Mark Cannell]

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In our earlier work we used a much shorter wavelength for excitation (~ 700nm). Have you tried tuning shorter?
See: Jones, K.T., Soeller, C. & Cannell, M.B. (1998) The passage of Ca2+ and fluorescent markers between the sperm and egg after fusion in the mouse. Development 125, 4627-4635

Cheers Mark

On 13/10/2011, at 8:59 PM, Cammer, Michael wrote:

> *****
> To join, leave or search the confocal microscopy listserv, go to:
> http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy
> *****
>
> We have a question about using Indo1 with two-photon excitation.  We are using 740 nm to excite the dye and have collection spectral ranges set to 370 to 442 nm and 455 to 600 nm.
>
> We expected that treating the cells with ionomycin would make the shorter wavelength range more intense and the longer wavelength range less intense.  However, what really happens is that the shorter wavelength drops a little and  the longer wavelength drops a lot.  So the raw data look wrong, but after ratioing the data appear correct (i.e. high Ca yields 1.0 and low Ca yields 0.5).  Also, washing out the ionomycin with EGTA does reverse the ratio most of the way.
>
> When we run the experiments of cells interacting, the trend of the ratios match published results and previous results we got with Fluo4 by widefield.  However, we are worried because the lower wavelength range does not increase intensity.  This means that the changes are in the range of only twofold, i.e. 0.5 to 1.0.
>
> Does anybody know what the problem is?
>
> Thanks!
>
> ________________________________________________________
> Michael Cammer, Assistant Research Scientist
> Skirball Institute of Biomolecular Medicine
> Lab: [phone]  Cell: [phone]
>
> ------------------------------------------------------------
> This email message, including any attachments, is for the sole use of the intended recipient(s) and may contain information that is proprietary, confidential, and exempt from disclosure under applicable law. Any unauthorized review, use, disclosure, or distribution is prohibited. If you have received this email in error please notify the sender by return email and delete the original message. Please note, the recipient should check this email and any attachments for the presence of viruses. The organization accepts no liability for any damage caused by any virus transmitted by this email.
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