http://confocal-microscopy-list.275.s1.nabble.com/5-color-imaging-6-color-would-be-even-better-tp7579662p7579733.html
> 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
>>
>> *Advanced Knowledge Management*
>> for *MICROSCOPY *and *Image Analysis *
>> ------------------------------------------------------------------------
>> *Dmitry Sokolov*, Ph.D.
>> Mob: *+64 21 063 5382***
>>
[hidden email] <mailto:
[hidden email]>
>>
>>
>>
>> 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
>>>>
>>
>