In message     Confocal Microscopy List writes:
>    We have recently been using the confocal microscope to examine
> colocalization of two proteins using MAb techniques. The signal level from
> one of the proteins is lower than the other by ca. a factor of 2-3.
> I am sure that many of you have observed that using such data it is possible
> by judicious selection of the intensities of the display colors (one for
> each protein) it is possible to make it appear that the proteins are
> colocalized or are not colocalized. The question is how does one establish
> 'reality'?
>     At first we assumed that if you could adjust the balance to show that
> they were colocalized then they were colocalized since for features that are
> not colocalized in our controls it is not possible to adjust the color scales
> to show colocalization. The problem with this assumption is that in more
> highly
> stained and thicker material, such as that with which we are working, there
> may be a sufficient level of out of focal plane signal contributing to our
> structures' signal. This structure might then be interpreted to be colocalized
> when it is not. Any ideas?
>
> Jim
 
I have 3 questions for you, first:
 
1)  what fluorophores are you using?
 
2)  Are you using an Ar-ion laser?
 
3)  If you're using a Kr/Ar-ion laser, are you trying to collect red and green
images simultaneously?
 
RSVP!
 
Best wishes,
 
Martin Wessendorf