Jessica,

I agree with Ramin.

> It seems to me that your sampling is infrequent for the target.  That is
to
>

Your sampling in z seems too coarse. To reproduce the 'field of view' on a
widefield microscope you should at least sample at around:

dz ~ 0.5 n w_em / NA^2

n... RI of immersion
w_em ... emission wavelength

This represents a quarter of the Rayleigh distance in z under aberration
free conditions and within the paraxial approximation (NA<=0.5). Half of the
Rayleigh distance is the commonly used resolution estimate. It is between
about 80% of the maximum of the peak. By going with that definition, this
distance should be sampled twice to comply with the Nyquist criterion (the
0.5 factor accounts for that). So, in your case for the Alexa488 you should
space your samples at around 0.257 microns or round it up to at least 0.3
microns. For the Cy5 you should be fine, since that becomes oversampled then
by using the same distance. Are you applying any deconvolution post
processing to your image stacks? Then it is even more important to sample as
fine as possible and to include enough above and below out of focus image
slices.

> 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?
>

I think you rather dealing here with spherical aberrations. They are as
wavelength dependent as a perhaps appearent RI mismatch would be. How close
is the RI of your embedding media to the 1.33 of water? Then, being far away
from the coverslip amplifies SA of any RI mismatch. Despite using a water
immersion lens and closely matching the RI's, you should always try to mount
the sample as close as possible to the coverslip. If your PlanApo has a
collar for the coverslip thickness, carefully set it to its measured
thickness. Chromatic aberrations are omni-present as well but they usually
won't contribute and manifest itself that much that you will see a reversed
interference pattern.


Cheers
Lutz

______________________________________
Lutz Schaefer
Advanced Imaging Methodology Consultation
16-715 Doon Village Rd.
Kitchener, Ontario
N2P 2A2, Canada
Email: [log in to unmask]
Phone, FAX: (519)-894-8870
______________________________________



----- Original Message -----
From: 'Ramin' 'Rahbari' <[log in to unmask]>
To: <[log in to unmask]>
Sent: Thursday, September 13, 2001 12:16 PM
Subject: Re: colocalization with 488 & 647 fluorochromes


> Jessica-
> It seems to me that your sampling is infrequent for the target.  That is
to
> say you may be getting one good image through the target, and then imaging
> above and below the target.  Since the PSF is axial in the direction of
the
> scan, z, it may not be unreasonable for the two fluors to be spatially
> separated in the z. Your target may be blocking, casting a shadow,
blocking
> transmission of light for the respective halves of the PSF you are not
> detecting.
>
> -----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