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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