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Hi Martin,

Sequential acquisition of fluorophores that can be excited at the same 
wavelength is silly. On a multi-detector microscope like most point 
scanning confocal microscopes, such as Leica SP5/SP8 (preferably with 
HyD's) or Zeiss LSM780, the user has 5 (SP#) or up to 34 (LSM710 or 780, 
2 standard PMTs, 32 channel spectral detector ... on 780 with GaAsP 
spectral detector one would probably just use that). To take a simple 
scenario, with the following three FPs,

mTFP1 ("Teal")
CY11.5 (Cyan-Venus high FRET; see S.S. Vogel papers for a series of CY 
fusions with different FRET efficiencies)
mBeRFP (446ex, 615em), published in Yang et al 2013 PLoS One 
http://www.plosone.org/article/info%3Adoi%2F10.1371%2Fjournal.pone.0064849

one could sequentially excite at 458 nm (Argon ion laser), or 440 or 436 
nm (other light sources), with single channel acquisition - as I quoted 
previously. However, all three fluorophores (or even more with Vogel's 
C-Y series) are going to emit and/or photobleach. So, if you have enough 
detectors, best to acquire simultaneously.

***

One should pay attention to details when imaging, whether point scanning 
confocal or widefield. For example, Krylova et al 2013 PLoS One 
http://www.plosone.org/article/info%3Adoi%2F10.1371%2Fjournal.pone.0063286
imaged four FP-FP dimers (in different cells) with single excitation:
mCherry-nls-mCherry
mRaspberry-nls-mRaspberry
mKate2-nls-mKate2
mPlum-nls-mPlum     (note: mPlum's are dim, they would have been better 
off with mPlum-mPlum-mPlum(etc), Steve Vogel has gone up to Venus 6 [V6]).
My kudos to them for approximately doubling the brightness of each 
protein by using tandem dimers (though tdTomato might have been a better 
choice).

Krylova et al used a widefield microscope, 575/15 ex filter (593 
dichroic) with 655/40 [635-675nm]  and 628/40 [608-648 nm] exciters. 
Their figure 4 shows the two emission filters overlap in the range of 
635-648 nm (bandpass interference filters are not absolutely vertical 
cut on/off, but close enough). They would have been better with 
non-overlapping emission bands (and two cameras would have been nice for 
simultaneous acquisition). An optimal emission filter would be about 
$150, a lot less than the publication charges for PLoS One or authors 
time to work on this project. Ms. Vinita Popat, a summer student working 
in our lab, calculated for me that 600-630 nm for the short wavelenghth 
emission filter, and 630LP would be the optimal pair (under the 
simplifying assumption that each FP is equally bright ... as noted 
above, mPlum could have improved performance with a higher order multimer).

***

I recently (re)read a very nice paper from Richard Neher et al, showing 
advantages to using two or more EXCITATION wavelengths - sequentially - 
with the same emission bands, to obtain additional information (2x more 
images, "multiple excitations greatly facilitate the decomposition").

    Neher RA, Mitkovski M, Kirchhoff F, Neher E, Theis FJ, Zeug A 2009
    Blind source separation techniques for the decomposition of multiply
    labeled fluorescence images. </pubmed/19413985> Biophys J. 96:
    3791-800. doi: 10.1016/j.bpj.2008.10.068.

    PMID: 19413985    
    http://www.cell.com/biophysj/retrieve/pii/S0006349509000927

    Methods of blind source separation are used in many contexts to
    separate composite data sets according to their sources. Multiply
    labeled fluorescence microscopy images represent such sets, in which
    the sources are the individual labels. Their distributions are the
    quantities of interest and have to be extracted from the images.
    This is often challenging, since the recorded emission spectra of
    fluorescent dyes are environment- and instrument-specific. We have
    developed a nonnegative matrix factorization (NMF) algorithm to
    detect and separate spectrally distinct components of multiply
    labeled fluorescence images. It operates on spectrally resolved
    images and delivers both the emission spectra of the identified
    components and images of their abundance. We tested the proposed
    method using biological samples labeled with up to four spectrally
    overlapping fluorescent labels. In most cases, NMF accurately
    decomposed the images into contributions of individual dyes.
    However, the solutions are not unique when spectra overlap strongly
    or when images are diffuse in their structure. To arrive at
    satisfactory results in such cases, we extended NMF to incorporate
    preexisting qualitative knowledge about spectra and label
    distributions. We show how data acquired through excitations at two
    or three different wavelengths can be integrated and that multiple
    excitations greatly facilitate the decomposition. By allowing
    reliable decomposition in cases where the spectra of the individual
    labels are not known or are known only inaccurately, the proposed
    algorithms greatly extend the range of questions that can be
    addressed with quantitative microscopy.

ImageJ plugins at       
http://www.mh-hannover.de/cellneurophys/poissonNMF/NMF/

I hope vendors start including this capability. I do wish Prof. Neher 
had chosen a less mathematically smelly word than decomposition - one 
reason I prefer to call this spectral unmixing.

Enjoy,

George




On 6/23/2013 2:23 PM, Martin Wessendorf wrote:
> Hey, George--
>
> On 6/23/2013 9:56 AM, George McNamara wrote:
>
>> I disagree with a couple of the authors imaging advice on page 544 (most
>> of the advice is very good):
>>
>> Authors: "Fluorophores with overlap in the excitation or emission
>> spectra should be imaged sequentially rather than simultaneously to
>> minimize fluorescence cross-talk and thereby optimize color separation".
>>
>> On a typical confocal microscope (ex. Leica SP5, SP8 or Zeiss LSM710,
>> 780) there are several detectors (ex. 5 on SP5 or SP8) or detector array
>> (ex. LSM710 or 780). Therefore, fluorophores that excite well at a given
>> excitation wavelength should be imaged simultaneously. I also recommend
>> the latest detectors, ex., HyD for leica, GaAsP for Zeiss, photon
>> counting mode if available (and TCSPC lifetime if available).
>
> I'm confused about why you think this is bad advice.  If you have 
> coexpression of labels, it'll be much easier to determine whether or 
> not a particular cell is single-labeled or multiple-labeled if you use 
> a laser that doesn't excite all the candidates (or conversely, if you 
> use a barrier filter that doesn't pass all the candidates).  This is 
> particularly true when you have strong expression of one and weak 
> expression of the other.  Even with an detector array, there's a limit 
> to what you can ferret out (--unless you have infinite photons, of 
> course!)
>
> However, my confusion probably means I'm missing something so explain 
> away!
>
> Thanks--take care--
>
> Martin Wessendorf
>
>


-- 



George McNamara, Ph.D.
Single Cells Analyst
L.J.N. Cooper Lab
University of Texas M.D. Anderson Cancer Center
Houston, TX 77054