Thanks to everyone who responded to my question.  It does sound  like
the shift we are seeing is most likely due to spherical aberration
(varying per wavelength of light), and not chromatic aberration.
We'll proceed with minimizing the spherical aberration and see how
things come out.

Cheers,
Jessica


>
>  >
>>  -----Original Message-----
>>  From: Jessica Wagner [mailto:[log in to unmask]]
>>  Sent: Thursday, September 13, 2001 11:18 AM
>>  To: [log in to unmask]
>>  Subject: colocalization with 488 & 647 fluorochromes
>>
>>
>>  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,
>>  --
>>  ---
>>  Jessica Wagner
>>  Senior Research Lab Technician
>>  Molecular Cytology Core Facility
>>  University of Missouri
>>  2 Tucker Hall
>>  Columbia, MO 65211
>>  573-882-4895