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Hi Christof,
 
More quantitative description of your imaging geometry/optics
would be useful. I have been observing similar effects when  imaging
a thick sample  on 
inverted scope using 40x water dipping lens in immersion mode.  
When water drop substantially shrinks after 2-3 hrs
focus starts shifting until disappears completely (from the set range).
This corresponds to ultimate refractive index change from 1.333 to 1.0.
The possible solution is to set big enough thickness of z-stack so that
your specimen still fits into imaged  focal range (taking into account the
z-range shift due to refractive index change). 
 
I would like to see your graph and the distorted image. I wonder
if your chemicals are not dissolving well or recrystalizing thus creating
nonhomogenous layers distorting  your image. Or maybe some vibrations
create inhomogeneities in the immersion/dipping medium. Do you observe this
type of distortion with fixed specimens as well?
 
Best regards,
Arvydas
--------------------------------


 
 
Arvydas Matiukas, Ph.D.
Director of Confocal&Two-Photon Core
Department of Neurosci& Physiology
SUNY Upstate Medical University
766 Irving Ave., WH 3167
Syracuse, NY 13210
tel.: 315-464-7997
fax: 315-464-8014
email: [log in to unmask]
>>> Christof Schwiening <[log in to unmask]> 3/18/2013 10:29 AM >>>
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Dear All,
I wonder if someone could throw some light on a problem we have been having 
for the past few years! We use a Leica SP5 confocal on an upright microscope 
with dipping lenses to image pH and calcium-sensitive fluorescent dyes. During 
solution changes - most notably the addition and removal of organic 
compounds such as propionate (20 mM)- we get severe image distortion and 
changes in focal depth. I attempted to attach a graph of a ROI from a fixed 
sealed specimen which we have imaged during solution changes with 3 
different objectives - however, the ListServer rejected every image format I 
tried...I can send it by email if anyone is interested. It is impossible that the 
propionate is actually getting to the specimen itself - I suspect it must be as a 
result of refractive index changes.

My questions are: How do others image small structures (i.e. dendritic spines 
or NMJs) during changes of solution with differing refractive indicies? Is there 
some well know trick? Or, am I using the wrong objectives? Does wide field 
microscopy have the same problems?
Many thanks,
Christof