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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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