Re: Coverslips

Posted by simon walker (BI)-2 on
URL: http://confocal-microscopy-list.275.s1.nabble.com/Coverslips-tp6865241p6869167.html

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Thanks for everyone's input on this, it's really appreciated.  Julien - although it doesn't mention it specifically I think the Zeiss link makes the point about the requirement for precise cover glass thickness with SIM.
If you're to achieve the best resolution possible you have to use glass of a thickness that most closely matches that for which the objective is designed (usually 170 um).  Any deviation from this will introduce sperical aberration.  My guess is that because the resolution of a confocal system is lower, you won't notice much of a difference between an image acquired using 170 and a 180 um glass, whereas on a SIM system you will.
Simon



-----Original Message-----
From: Confocal Microscopy List [mailto:[hidden email]] On Behalf Of Julien Cau
Sent: 07 October 2011 08:26
To: [hidden email]
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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