Re: Coverslips

Posted by David Baddeley on
URL: http://confocal-microscopy-list.275.s1.nabble.com/Coverslips-tp6865241p6869169.html

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SIM is more sensitive to refractive index mismatch (including that produced by incorrect coverslip thickness) than more traditional microscopy techniques. This is partly because the improved resolution makes differences that are there for all microscopy types more visible, but also because refractive index (potentiall in the form of coverslip thickness) variations can distort the illumination pattern and lead to artefacts during the reconstruction process. Thus it probably is fair to say that SIM does require tighter tolerances than, e.g., confocal microscopy. Although moderately painful, testing each coverslip is not actually too bad once you get into the swing of it. When I was using a 4Pi microscope (which has a similar sensitivity to refractive index) I'd check each coverslip  in about 3 places using a digital micrometer and it only took around a minute/coverslip. Coverslip thickness is much more important if you are using water immersion.


cheers,
David


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From: Julien Cau <[hidden email]>
To: [hidden email]
Sent: Friday, 7 October 2011 8:26 PM
Subject: Re: Coverslips

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Hi Martin,

You are absolutely right and SIM does not need better coverslips than any other 0.17 objective-based microscopy technique.
Our users use these coverslips for any experiment (widefield, confocal, SIM) and they appreciated the difference.
The SIM point is more "don't use a fancy device that promiss you superesolution if you waste this potential with crap coverslips". If you buy a wonderfull coffee machine, will you put in it moldy coffee beans?
Wrong thickness coverslips induce decreased light collection, spherical aberrations and then reduced resolution.

It is worth using them for any type of experiment using a 0.17 lens. Alternatively, you can buy an expensive lens with a correction collar that can counteract the effects of the coverslip thickness error.

Best regards

PS : see for instance http://www.meditec.zeiss.com/4125681F004CA025/Contents-Frame/C66A2521E524C891852575A200721A7C for a direct comparison of imaging with #1.5 vs #2 coverglass.

Le 07/10/2011 03:26, Martin Wessendorf a écrit :

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>
> On 10/6/2011 3:43 AM, Simon Walker wrote:
>
>> Not strictly a confocal question, but I'm sure someone out there will be able to
>> help.  We are about to start using a SIM super res system and have been
>> advised that one of the critical factors in acquiring optimal images is the cover
>> glass.  Specifcally, the thickness of the glass needs to be consistant across
>> the whole coverslip (e.g. 170 um +/- 2 um), and reproducible between
>> coverslips.  One option is to measure each individual coverslip before use, but
>> this seems rather impractical to me.  Has anyone looked into this, and if so,
>> are there any manufacturers out there who can provide cover glass with this
>> high specification?
>
> My ignorance is showing here.  Why are higher-quality coverslips needed for SIM?  Or is this for live-cell imaging?
>
> Best wishes--
>
> Martin Wessendorf

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