We are trying to determine whether we can colocalize two antibodies,
directly labelled with Alexa 488 and Cy5, to a target that is
approximately 0.5 - 1 um (in diameter).  The sample is mounted in
Mowiol mounting media (polyvinyl alcohol in a tris/glycerol buffer),
approximately 5 um from the coverslip.  Using a 60x water immersion
planapo objective (NA 1.2), a series of X/Y images are collected at
0.7 um steps and then 3-D reconstructed to produce a flattened top
view.

We believe we are seeing a spatial shift between the green and far
red fluorescent images due to chromatic aberration but can't
eliminate the possibility that we are correctly localizing two
closely spaced objects.  Is it realistic to try to colocalize a 0.5
um object with fluorophores as widely spaced as the Alexa488 & Cy5?

A further refinement of this problem is seen when we view X/Z cross
sections of the stacks.  In some image stacks, the "green" object
(labeled by Alexa488) has a tear drop shape with the narrow peak of
the tear drop facing east while the "red" object has the opposite
symmetry with the tear drop shape facing west.  Is this simply
further evidence of chromatic aberration subtly changing the PSF in
opposite ways for these two different wavelengths?

Thanks in advance,
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Jessica Wagner
Senior Research Lab Technician
Molecular Cytology Core Facility
University of Missouri
2 Tucker Hall
Columbia, MO 65211
573-882-4895