On Fri, 15 Sep 1995, Matthew Hannah wrote:
 
> I do not understand why there is any shift between the red and green
when doing
> a simultaneous scan? When we have spoken to Leica about such things they have
> said that as the red and green channel light paths are identical then there can
> be no shift. Is this a bit of an over-simplification? It sounded reasonable to
> me and I cannot say that I have ever noticed a real shift (ie red one side
> yellow in the middle and green the other side of all double labelled structures)
> when scanning simultaneously. At high zoom it sometimes appears that the red
> signal extends beyond the green, but I assumed this was due to the the reduced z
> resolution of the longer wavelength light or increased bleaching of the FITC
> relative to the TRITC!
 
The shift is due to lights of different wave lengths focus on different
focal plane (chromatic abberation).  This is particularly serious if one
uses two different excitation wave lengths on the red and green channels.
(eg 488 for FITC and 568 for texas red as mentioned in the original query).
Theoretically, this shift is in the z-axis only. Poor optical alignement,
a typical problem in beam scan systems, may translate the shift to the x-y
plane.
A compromise is to use a single excitation for both the red and the green.
eg use 514 for both FITC and Texas. With some sacrifice on fluorescence
intensity, the shift is minimized.
 
 
---------------------------------------------------------------------
Harry Leung,
Technical Officer, Electron Microscopy & Confocal Microscopy,
Zoology Dept., University of Western Ontario, London, Ont., Canada. N6A 5B7
Tel: 519-679-2111 x6733  fax: 519-661-2014
E-mail: <[log in to unmask]>
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