Hi all,

We are trying to set up a four colour combination for tissues sections  
on our SP5 and are looking for recommendations.

The only colour that is currently "fixed" is AlexaFluor 488 (there is  
a difficult reagent that is working well and the researchers don't  
want to change unless they absolutely have to).

We have 405, multi-line Argon, 561 and 633 lasers on a Leica SP5.

We are considering the following and would appreciate comments:-

* Pacific Blue or Marina Blue (probably Marina based on Mike Ignatius'  
comments on and off this list)

* AlexaFluor 488

* AlexaFluor 546 or 555 or 594
	- not sure which of these will be best from the point of view of (i)  
brightness, (ii) photostability and (iii) overlap with the other dyes  
(eg from the spectral viewer it looks like the 546 will be slightly  
excited by the 488 laser for AF488, while the red dyes (below) will be  
slightly excited with 561)
	- interestingly Molecular Probes include 555 in the some of their  
triple label kits - I'm wondering why they have chosen that as 546  
looks at least comparable on the spectral viewer (obviously more going  
on -  as I hope Mike will reveal ;)

* AlexaFluor 647 or 700
	- my experience with AF700 with 633nm excitation in flow has not be  
fantastic but it can be a useful dye there and it certainly has less  
overlap with 546 or 594 - no idea how it performs for confocal.

	OR
  Qdot 705 or Qdot 800
	- I believe the SP5 will detect out to 800 so a lot of Qdot800 will  
be lost but there is still a good part available according to the  
spectral viewer.


Currently the researchers are leaning towards Marina Blue, AF488,  
AF546, AF647.... anyone have a better idea?

Regards,

Adrian