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Hi Dmitry,

I am unaware of any publications on excitation-emission matrix 
spectroscopy ("3D landscape") of mCherry. Vitaly and others may want to 
comment on any odd behaviors of mCherry. It has cousins, rsCherry and 
rsCherryRev for example, that do interesting things. There is also the 
issue that measurements in cells and tissues can differ from pure protein.

Diagnosing peak shift using 561 vs 594 excitation ... spectral scans of:
1. test with solution of pure mCherry, rather than cells/tissue.
2. excite (pure solution) with 514 nm - should be the same as 561 nm 
excitation, whether using identical spectral range as with 561, or 
starting at shorter wavelength (i.e. at 525 nm).
3. They have an MP laser - in fact, MP-OPO, so Daniela could use 
multiple MP(OPO) wavelengths and spectral emission scanning.
4. Buy a PARISS (www.lightforminc.com) with excitation confocal slit and 
appropriate excitation options (including excitation slit), or go visit 
Keith Lidke to use his clone 
http://www.systemscenters.org/wordpress/wp-content/uploads/2011/01/nm-ncbs-aug-8-11-lidke.pdf 

and test on this imaging spectrometer.

Also: never rely on just the peak. The area under the curve of a 
fluorescence spectrum carries a lot more information.

Bob Zucker & Robert Lief published a couple of papers showing various 
artifacts with early spectral confocal microscopes. When they used the 
latter's MIDL calibrated lamp, most (all?) Leica SP1's had an 
interesting behavior where the spectra differed depending on start 
wavelength (i.e. 500 vs 501, vs 502 vs 503 vs 504 nm).

George
p.s. bonus: some confocal microscopes adjust the pinhole size based on 
(excitation) wavelength. For example, LSM710 if the user clicks "1 Airy" 
button after switching the laser line selected. The pinhole is part of 
the emission path, so this affects the spectral resolution (probably not 
enough for most LSM users to notice if going from 561 to 594 nm). More 
generally, any manuscript that uses a spectral confocal microscope and 
fails to state pinhole size is a waste of effort. Easy test anyone with 
a spectral confocal microscope can perform: compare spectra at pinhole 1 
vs wide open.

On 2/16/2013 10:48 PM, Dmitry Sokolov wrote:
> 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 ?????:
>> *****
>> To join, leave or search the confocal microscopy listserv, go to:
>> http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy
>> *****
>>
>> 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:
>>> *****
>>> To join, leave or search the confocal microscopy listserv, go to:
>>> http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy
>>> *****
>>>
>>> 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
>>>
>