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Regardless of the number of channels to be displayed, my point was  
that selecting labels or assigning LUTs can have a significant impact  
on visibility with less  'fiddling with the histogram' to achieve the  
same result. Assign the bluer/bluish, tones to those items that are  
more readily resolved or that do not require the same level of  
prominence, as is often the case for counterstains meant to indicate  
the nucleus, cytoplasmic extent or membrane. Once you get to more  
than 3 blocks of wavelengths, then color assignment does become  
somewhat arbitrary, and requires similar treatment of controls.

Glen

On Mar 10, 2008, at 6:05 AM, Michael Weber wrote:

> Search the CONFOCAL archive at
> http://listserv.acsu.buffalo.edu/cgi-bin/wa?S1=confocal
>
> Glen,
>
> but what do you do if you have 4 or 5 stainings in your sample -  
> you have to involve blue. Or you select a green look-up-table for  
> Alexa 405. Or for quantitative approaches, one can use spectrum or  
> fire lut. Fiddling with the histogram is not always a good idea, at  
> least when it has to be scientific.
>
> Michael
>
>
> Glen MacDonald wrote:
>> Search the CONFOCAL archive at
>> http://listserv.acsu.buffalo.edu/cgi-bin/wa?S1=confocal
>> All of the Alexa fluor conjugates have a tendency to stick non- 
>> specifically via charge interactions of the anionic dye moiety.   
>> the Image-iT fx does helps greatly, especially in thick samples.
>> In regard to using the blue fluorophores for the more prominent  
>> targets in the tissue, the human visual system is only weakly  
>> responsive to blue wavelengths and lacks blue-sensitive cones in  
>> the fovea, the region of greatest visual acuity.  In general, if  
>> the smallest or rarest bits get the green, red or gray channel,  
>> they are noticeable with less histogram manipulation than if the  
>> smallest bits are viewed in the blue channel.
>>    Regards,
>> Glen
>> Glen MacDonald
>> Core for Communication Research
>> Virginia Merrill Bloedel Hearing Research Center
>> Box 357923
>> University of Washington
>> Seattle, WA 98195-7923  USA
>> (206) 616-4156
>> [log in to unmask]
>> ********************************************************************* 
>> ********* The box said "Requires WindowsXP or better", so I bought  
>> a Macintosh.
>> ********************************************************************* 
>> ********* On Mar 6, 2008, at 7:09 AM, Junya HIROI wrote:
>>> Search the CONFOCAL archive at
>>> http://listserv.acsu.buffalo.edu/cgi-bin/wa?S1=confocal
>>>
>>> Christophe,
>>>
>>> I was also looking for a fluorescent dye for 405 nm excitation,  
>>> and I compared Alexa 405
>>> with Pacific Blue.  Both dyes bleached to some extent with  
>>> ProlongGold, but Pacific Blue was
>>> absolutely brighter than Alexa 405, when compared at the same  
>>> dilution.  So, I recommend
>>> Pacific Blue, although Molecular Probes sells Pacific Blue for  
>>> flow cytometry.
>>> I found another problem on Alexa 405: it nonspecifically binds to  
>>> nuclei !  I asked Molecular
>>> Probes/Invitrogen about this problem, and they recommended me to  
>>> use their blocking
>>> solution, Image-iT FX Signal Enhancer.  It actually worked well,  
>>> but Pacific Blue does not have
>>> such a problem, so that is another reason I like Pacific Blue.
>>>
>>> Junya
>>>
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