Coverslips

[Guy Cox-2]

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The only thing is, with water you do normally have a correction collar so you can get it right.  With oil (unless you use a TIRF lens) you can't.  In my one serious foray into super-resolution (STED) we used TDE mountant so that we knew we had one refractive index from the top of the coverslip all the way into and beyond the sample.  The difference compared to regular Citifluour mounts was obvious (even though the cells were mounted on the coverslip).  

Last time I looked at a Deltavision SI system they only offered an oil-immersion objective and when faced with samples in water or glycerol attempted to compensate by fiddling with different immersion oils.  While in principle this might work, I can't believe it would in practice.  

                                     Guy

Optical Imaging Techniques in Cell Biology
by Guy Cox    CRC Press / Taylor & Francis
     http://www.guycox.com/optical.htm
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Associate Professor Guy Cox, MA, DPhil(Oxon)
Australian Centre for Microscopy & Microanalysis,
Madsen Building F09, University of Sydney, NSW 2006

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-----Original Message-----
From: Confocal Microscopy List [mailto:[hidden email]] On Behalf Of James Pawley
Sent: Saturday, 8 October 2011 2:32 AM
To: [hidden email]
Subject: Re: Coverslips

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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
>

Just one more point: It makes a lot of difference
whether we are talking about oil or water lenses.

As immersion oil is almost the same RI as the
BK-7 glass that I think is used for coverslips,
changes in thickness from the optimum are much
less important than if you are using a water
lens. In the latter case, (RI = 1.515 vs Ri
1.334?) even a 2µm error in thickness is easily
noticeable by eye with an NA 1.2 objective. Other
factors that affect the PSF (such as the
temperature of the immersion oil!) are well
covered in Chapter 11 of the Handbook.

SIM is indeed curcially dependent of having a
known and unaberrated PSF and this is only
possible if you re free from SA. To check this,
always have some sub-resolution beads in your
preparation and ensure that they "go out of
focus" in a manner that looks the same whether
you focus up or focus down.

JP

>
>-----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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