The authors cite a paper by Brandizzi et al (2002) in the same journal  
within their methods section at the end of the paper. Brandizzi's  
methods section details the filter settings and sequential laser  
acquisition that was used. Even with these settings there still may be  
a bit of bleedthrough, but it will be minimalized. To be absolutely  
sure, I agree with Joel that a set of controls with just GFP or just  
YFP should be imaged and observed in both channels (and presented  
somewhere in the publication).

John Oreopoulos

On 27-May-09, at 9:39 AM, Joel Sheffield <[log in to unmask]> wrote:

> The key is that the Leica allows you to set precise spectral settings
> for emission detection.  Sequential acquisition on the Leica SP2
> allows you to set these to limit bleed-through by looking at one
> channel at a time.  For each z slice, the microscope switches between
> the settings.  It's as if you were switching filter sets and
> superimposing the images.  On the other hand, it would have to be
> carefully controlled.  I would like to see that the "GFP" channel
> showed no signal from a YFP source, and vice-versa.
>
>
>
> Joel
>
>
> Date sent:          Wed, 27 May 2009 08:15:29 -0500
> Send reply to:      Confocal Microscopy List
> <[log in to unmask]>
> From:               Howard Berg <[log in to unmask]>
> Subject:            GFP/YFP colocalization?
> To:                 [log in to unmask]
>
>> Hi all:
>>
>> A paper for ''The Plant Cell'' (Raffaele et al.) just came online and
>> several figures are presented showing colocalization of GFP and YFP-
>> tagged proteins.  I am not surprised that they colocalized because I
>> expect there would be a severe bleedthrough for these two.  Am I
>> missing something here or have they presented an artifact?  Their
>> methods state that they used sequential acquisition (on a Leica TCS
>> SP2).
>>
>> Howard
>>
>>
>>
>> R. Howard Berg, Ph.D.
>> Director, Integrated Microscopy Facility
>> Danforth Plant Science Center
>> 975 N. Warson Rd.
>> St. Louis, MO 63132
>>
>> ph 314-587-1261    fx 314-587-1361   cell 314-378-2409
>> [log in to unmask]    www.danforthcenter.org
>> visit this educational resource: http://www.danforthcenter.org/Cells/
>
>
>
> Joel B. Sheffield, Ph.D
> Department of Biology
> Temple University
> Philadelphia, PA 19122
> Voice: 215 204 8839
> e-mail: [log in to unmask]
> URL:  http://astro.temple.edu/~jbs