Re: 5 color imaging (6 color would be even better!)

Posted by Dmitry Sokolov on
URL: http://confocal-microscopy-list.275.s1.nabble.com/5-color-imaging-6-color-would-be-even-better-tp7579662p7579728.html

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Hi George,

could you please comment more on:
> "reference spectra for each FP (Figure 1A). Unexpectedly, we observed
> that the mCherry spectrum was dependent on the excitation wavelength,
> and that 594 nm excitation resulted in ~12-nm-peak red-shift compared
> with 561-nm excitation."
>
> which is more likely to be instrument than protein related.
What experiment would diagnose the reason for the peak shift?
Would you suggest the reference on the continuous ("3D") fluorescence
landscape of mCherry please?

Thank you,
Dmitry

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17.02.2013 7:01, George McNamara ?????:

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>
> Hi Daniela,
>
> First paragraph of your discussion states, " ... mCherry (based on our
> novel observation of mCherry photoconversion to a red-shifted species)"
>
> where is data on this?
>
> Is this referring to your observation,
>
> "reference spectra for each FP (Figure 1A). Unexpectedly, we observed
> that the mCherry spectrum was dependent on the excitation wavelength,
> and that 594 nm excitation resulted in ~12-nm-peak red-shift compared
> with 561-nm excitation."
>
> which is more likely to be instrument than protein related.
>
> Are you going to add a blue FP?
> Are you adding fusion proteins, ex. cyan-yellow (re: CY11.5,
> CyPet-YPet) with moderate to high FRET, enabling more colors (if using
> sequential scanning ... may also better take advantage of MP/NDDs)?
> How about using plasma membrane targeting to outline the cell
> surfaces? This would leave a cleaner signal for taking advantage of my
> Tattletales concept to multiplex fluorescent reporters and live cells
> (http://works.bepress.com/gmcnamara/26/ - best so start with the Feb 5
> pdf and ppt).
>
> Sincerely,
>
> George
>
>
> On 2/15/2013 1:22 PM, Daniela Malide wrote:
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>> http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy
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>>
>> We have a recent publication in Blood -imaging 5 fluorescent proteins
>> on a
>> commercial Leica SP5 confocal system:
>>
>> Dynamic clonal analysis of murine hematopoietic stem and progenitor
>> cells
>> marked by 5 fluorescent proteins using confocal and multiphoton
>> microscopy
>>
>> Daniela Malide, Jean-Yves Métais and Cynthia E. Dunbar
>> Blood December 20, 2012 vol. 120 no. 26 e105-e116
>>
>> http://bloodjournal.hematologylibrary.org/content/120/26/e105.long
>>
>> I can email a pdf of the manuscript if interested -as require
>> subscription to
>> Blood.
>>
>> Daniela Malide
>> Staff Scientist
>> NHLBI Light microscopy core facility
>>