*****
To join, leave or search the confocal microscopy listserv, go to:
http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy
*****

Christof, 

I don't actually have an answer to your problem. However, my feeling is that going from let's say water to 20 mM propionate should have only a very minimal effect on refractive index (RI water = 1.333, 100% propionate: 1.386; 20 mM propionate maybe ~ 1.335; check tables/charts listing RI as a function of concentration for propionate or another similar substance). Adjusting the refractive index collar as suggested by Vladimir is one good idea, if you have one; those however are found most likely on high NA immersion objectives, not so much on dipping objectives. I guess what may be worth knowing is which specific objectives you are using, and also what the distortions look like. Is it possible that you are getting your cells dry and wet again (in which case maybe the cells themselves may be doing something funky); or they may not like to be in propionic acid; or is it possible you are trapping air bubbles under your objective as you replace medium?


Julio Vazquez, 
Fred Hutchinson Cancer Research Center

http://www.fhcrc.org

==

On Mar 18, 2013, at 9:24 AM, Vladimir Ghukasyan wrote:

> *****
> To join, leave or search the confocal microscopy listserv, go to:
> http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy
> *****
> 
> Hello Christof,
> 
> As you mentioned, one of the most prominent reasons for the
> distortions you see is change of the refractive index. Lenses are
> usually corrected for chromatic and spherical aberrations for some
> defined refractive index. The more you deviate from it, the more will
> be the extent of the aberrations affecting your image. One way of
> overcoming this is using lenses with adjustable refractive index
> correction collar.
> 
> With regards,
> Vladimir
> 
> On Mon, Mar 18, 2013 at 11:29 AM, Christof Schwiening <[log in to unmask]> wrote:
>> *****
>> To join, leave or search the confocal microscopy listserv, go to:
>> http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy
>> *****
>> 
>> 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