I agree that the cause of these focus shifts and halos is often chromatic
aberration but I don't believe that lateral aspect of the problem has
anything to do with misalignment.
 
Chromatic abberation refers to tendency of the focal length of any optical
system to vary with wavelength. (axial chromatic aberration: hence the
focus-plane shift problem).
 
However, this exact phenomenon is also the cause of "lateral chromatical
aberration" in beam-scanning systems.  This is because the distance that a
point in the image appears to be off the axis (read: x-y mirror position)
depends on the magnification of the optical system between the source (or
pinhole) and the focus plane in the specimen.  This, optical magnification,
in turn, depends inversely on the focal length of the objective.
 Therefore, if the focal length is shorter for the exciting wavelength than
for the emitted wavelength, the returning spot will be magnified less and
hence appear to be closer to the axis.  If a small pinhole is used, the
returning ray may miss the pinhole entirely and the image will appear dark.
If a larger pinhole is used, then the majority of the signal will be
recorded when, in fact an adjacent pixel is being illuminated. If this
effect is present, then the image will match will be worse as one moves
away from the axis.
 
In addition to these chromatic effects, it is possible, in systems (unlike
the new Leica) that have separate pinholes for each wavelength, for one or
both of the alignment mirrors of the two channels to be misadjusted.  This
could cause one (or both) of the mirrors to project the image of its
particular pinhole into a different point of the focus plane.  Again, this
will cause misalignment between images made at different wavelengths but it
should be about the same lateral amount and direction throughout the image.
 
Finally, if, in the region where both stains are present, one stain is much
brighter than the other,(or if the the gain of one channel is set much
higher than the other, this may artifactually enlarge the apparent size of
small features (or the edges of larger features) and this can create a halo
effect that occurs throughout the entire field but has no pronounced
direction.
 
Jim Pawley
>
>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]>
>        <[log in to unmask]>
 
                   *****************************************
Prof. James B Pawley,                                        Ph.  608-263-3147
Room 1235, Engineering Research Building,                    FAX  608-265-5315
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