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Hi Christof ,
 
whatever is the reason for image distortion (Schlieren  or mechanical movement)
why don't you exclude the changing solution from the optical path, i.e. image your
cells from below while adding solution from the top. Any confocal on an inverted
stand would allow you to do this (you may need to switch to a dish with a coverslip
window at the center).
 
If switching to the inverted scope is not feasable/practical then try to improve your
injection/perfusion. I recall my cell perfusion experiments on an inverted scope
 (some with simultaneous patch clamp), and the perfusion practically never was 
a problem both for image or patch clamp under condition that perfusion is properly 
designed ( flow <1ml/min, opening diameter ~0.5 mm and located few mm apart
from the imaged/clamped cells. observing).
 
Moreover, some organic compounds do not dissolve well (eg. when dissolving BA
or BB in ethanol you can see Schlieren effect by a naked eye for several minutes).
Try to very well dissolve (vortex?)  your compound at elevated temperature, or try
more gradual (slower) addition and replacement of the compound. Regarding possible
formation of bubbles try cool the solution below room temperature ( at elevated temperatures
gas solubility in liquid decreases and bubbles are not produced. Maybe your compound
releases a lot of heat during dissolution, or changes the surfaces tension - if you introduce
it more gradually  all spatial inhomogeneties should be reduced. 
 
My first choice in your situation would be to try an inverted configuration. 
 
Best,
Arvydas


>>> Christof Schwiening <[log in to unmask]> 3/19/2013 10:02 AM >>>
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Dear Arvydas,
The graph is here: https://dl.dropbox.com/u/24087924/Convallaria%20slide.jpg
I have tried three long working distance objectives (20-63X). I don't have a 
good example of the distorted image since it shifts faster than my resonnant 
scanner can capture it. However, the ROI plot shows the magnitude of the 
shifts on the fluorescence signal. We do see the same effect on fixed samples 
(which is what the image above shows). I am sure that it is some kind of 
Schlieren type of effect (refractive index). I think that in areas of high 
contrast even small optical deviations cause quite marked intensity changes.....
Greetings,
Christof

On Mon, 18 Mar 2013 16:25:51 -0400, Arvydas Matiukas 
<[log in to unmask]> wrote:

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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