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I second Mark's statement.   
Glen MacDonald
	Core for Communication Research
Virginia Merrill Bloedel Hearing Research Center
	Cellular Morphology Core
Center on Human Development and Disability
Box 357923
University of Washington
Seattle, WA 98195-7923  USA
(206) 616-4156
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On Mar 19, 2013, at 8:08 AM, Mark Cannell <[log in to unmask]> wrote:

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> 
> It looks like a movement problem to me…
> 
> Cheers
> 
> On 19/03/2013, at 3:02 PM, Christof Schwiening <[log in to unmask]> wrote:
> 
>> *****
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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
> 
> Mark  B. Cannell Ph.D. FRSNZ
> Professor of Cardiac Cell Biology
> School of Physiology&  Pharmacology
> Medical Sciences Building
> University of Bristol
> Bristol
> BS8 1TD UK
> 
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