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Dear Sylvie,

I just follow up with a few comments, embedded below.  Thanks.

Sheng



> 1- Then I have a question to the list regarding this issue: Is unmixing an
> appropriate technique for experiments where one wants to extract
> quantitative intensity information from the image? Does anyone have any
> experience with this?


I think so.  When I worked for Peri, we published a few papers using
spectral FRET, including a 3-color FRET one.  I can send you the papers if
you are interested.  I also know quite a few other people use spectral
imaging and unmixing for steady-state and time-resolved (FLIM) FRET.





> In spectral unmixing, an algorithm is applied post acquisition to the
> image to extract the spectrum of each fluorophore from the complex spectrum
> collected by the detector. I think that the dimmer the signal, the lower
> the signal to noise ratio and so the higher the risk of errors by the
> algorithm. So I would be very cautious with calculating FRET efficiency (ie
> applying a second algorithm) from unmixed images.
>
> If your signal is very bright in both fluorophores (assuming you are not
> using biosensors), you can acquire reference spectra that are quite
> reliable. Then it is worth trying to unmix then calculate the FRET image
> but you will need to scrutinize your results for bias in the FRET
> efficiency in the dim vs bright areas.
>

You are right - the SNR is a fundamental issue to any quantitative
measurement.  The PFRET addresses this issue carefully and calculates the
bleedthrough percentages based on the intensity levels.



>
> 2- Concerning the autofluorescence, I assume you have checked that it is
> really autofluorescence (ie you can see it in the unstained tissue). In
> that case it might be worth trying to apply Sudan Black before imaging, if,
> of course it is fixed tissue you are imaging.
> If instead it is 'unwanted' fluorescence ('sticky' antibody), spectral
> unmixing will not help since the 'autofluorescence' will have the same
> spectrum as your signal. Increase the stringency of your labelling instead.
>
> All this applies only if you are not the unlucky one who wants to image
> non biosensor FRET is a live tissue giving strong autofluorescence.
> In that case I would turn to FLIM.
>
>
Thank you for mentioning FLIM.  I think it is a very good technique for
FRET.



> Best of luck :)
>
> Med vänlig hälsning / Best regards
>
> Sylvie
>
> @@@@@@@@@@@@@@@@@@@@@@@@
> Sylvie Le Guyader, PhD
> Live Cell Imaging Unit Manager
> Karolinska Institutet- Bionut Dpt
> Hälsovägen 7,
> Novum, G lift, floor 6
> 14157 Huddinge
> Sweden
> mobile: +46 (0) 73 733 5008
> office: +46 (0) 8 5248 1107
> LCI website
>
>
> -----Original Message-----
> From: Confocal Microscopy List [mailto:[log in to unmask]]
> On Behalf Of Feinstein, Timothy N
> Sent: den 20 november 2015 15:51
> To: [log in to unmask]
> Subject: Re: Autofluorescence in PFRET
>
> *****
> To join, leave or search the confocal microscopy listserv, go to:
> http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy
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> *****
>
> Spectral mode (confocal?) is a great idea in principle, but you have to
> keep in mind it is a highly inefficient mode of acquisition.  Many probes
> and experiments that are routine on a widefield will become quite hard to
> detect.  If have a bright enough signal that should be no problem.  If the
> signal is dim then make sure to use large pixels and open the pinhole much
> wider than usual.
>
> Best,
>
>
> Tim
>
> Timothy Feinstein, Ph.D.
> Research Scientist
> University of Pittsburgh Department of Developmental Biology
>
>
>
>
>
> On 11/19/15, 11:20 PM, "Confocal Microscopy List on behalf of Periasamy,
> Ammasi (ap3t)" <[log in to unmask] on behalf of
> [log in to unmask]> wrote:
>
> >Hello Neil
> >As Sheng mentioned you can use the spectral imaging approach. Or you
> >can use the unlabeled cells to collect the signal to use for background
> >subtraction. That will take care. The background subtraction should
> >remove any autofluorescence or detector noise involved in fluorescence
> >imaging.
> >Hope this helps.
> >
> >Dr. Ammasi Periasamy
> >Professor & Center Director
> >http://www.kcci.virginia.edu/people/profile/ap3t
> >Phone: (434) 243-7602 or 982-4869
> >Fax: (434) 982-5210
> >E-mail: [log in to unmask]
> >Office Location
> >W.M. Keck Center for Cellular Imaging
> >Physical and Life Sciences Building, (B 005) At the intersection of
> >Geldard dr and White head Rd., Mailing or Shipping Address:
> >W.M. Keck Center for Cellular Imaging (PLSB 005) University of Virginia
> >Biology, Gilmer Hall, 409 McCormick Rd.
> >Charlottesville, VA 22904, USA
> >FRET/FLIM Workshop-March 7-11, 2016:
> >http://www.kcci.virginia.edu/workshop
> >
> >
> >
> >-----Original Message-----
> >From: Confocal Microscopy List
> >[mailto:[log in to unmask]]
> >On Behalf Of yuansheng.sun
> >Sent: Thursday, November 19, 2015 9:27 PM
> >To: [log in to unmask]
> >Subject: Re: Autofluorescence in PFRET
> >
> >Then, it would be better to use spectral FRET.  Measure the reference
> >spectrum of the autofluorescence from unlabeled sample.  Use linear
> >unmixing to remove autofluorescence.
> >
> >Sheng
> >
> >
> >Sent from my T-Mobile 4G LTE Device
> >
> >
> >-------- Original message --------
> >From: "Anthony, Neil" <[log in to unmask]>
> >Date:11/19/2015  3:06 PM  (GMT-06:00)
> >To: [log in to unmask]
> >Cc:
> >Subject: Autofluorescence in PFRET
> >
> >*****
> >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.
> >*****
> >
> >Hi all, I hope the science treats you well.
> >
> >I was wondering if anybody's had any experience with autofluorescence
> >when using PFRET, or PeriFRET as I like to call it.  I understand that
> >autofluorescence is not accounted for in the algorithm?
> >
> >Peri, I hope all is well with you.  Can I ask for some pointers on how
> >think about the system if autofluorescence is the mix?
> >
> >Thanks in advance for your time.
> >
> >Neil
> >
> >________________________________
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