http://confocal-microscopy-list.275.s1.nabble.com/Coverslips-tp6865241p6870238.html
collar. The black one is said to be for room temp
>
>John Oreopoulos
>Research Assistant
>Spectral Applied Research
>Richmond Hill, Ontario
>Canada
>www.spectral.ca
>
>
>On 2011-10-07, at 12:02 PM, Unruh, Jay wrote:
>
>> *****
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>>
http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy>> *****
>>
>> A couple of things I have noticed about
>>coverslips. Firstly, if you ever do measure
>>commercial coverslips, you will notice that
>>within a batch, most are within a 5 um
>>tolerance with a few outliers. From batch to
>>batch you will notice far more differences (I
>>had one batch of #1.5's that was all almost 190
>>um). Secondly, the comment about oil vs. water
>>objectives is a very important one. Oil
>>objectives are designed to image just above the
>>coverslip. Since the oil, coverslip, and front
>>lens are all of the same refractive index, it
>>makes no difference how thick the coverslip
>>is--that ends up being compensated by the
>>increased oil thickness necessary to achieve
>>the same focal depth into the sample. For
>>water, I have never noticed a 2 um difference
>>by eye, but fluorescence correlation
>>spectroscopy is uniquely sensitive to these
>>things and I don't see a significant difference
>>as long as I stay within a 5 um tolerance. I
>>used to think that the solution was to
>>carefully adjust the correction collar on water
>>objectives and soon discovered that the lag in
>>the correction collar is as much as 100 um! As
>>a result, the numbers on the correction collar
>>are essentially meaningless unless you always
>>adjust from the same direction (note that I
>>have only tested this for Zeiss objectives).
>>The best practice is to adjust the collar with
>>a sensitive sample and then leave it and use
>>measured or high precision coverslips for the
>>remainder of the experiment.
> >
>> Jay
>>
>> -----Original Message-----
>> From: Confocal Microscopy List
>> [mailto:
[hidden email]] On Behalf Of James Pawley
>> Sent: Friday, October 07, 2011 10:32 AM
>> To:
[hidden email]
>> Subject: Re: Coverslips
>>
>> *****
>> To join, leave or search the confocal microscopy listserv, go to:
>>
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>>
>>> *****
>>> To join, leave or search the confocal microscopy listserv, go to:
>>>
http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy>>> *****
>>>
>>> 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
>>>
>>> *****
>>> To join, leave or search the confocal microscopy listserv, go to:
>>>
http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy>>> *****
>>>
>>> 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/C66A2521>>> E5 24C891852575A200721A7C for a direct comparison of imaging with
>>> #1.5 vs
>>> #2 coverglass.
>>>
>>> Le 07/10/2011 03:26, Martin Wessendorf a écrit :
>>>> *****
>>>> To join, leave or search the confocal microscopy listserv, go to:
>>>>
http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy>>>> *****
>>>>
>>>> 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
>>>
>>> --
>>>
>>> ____________________________________________
>>>
>>> */Julien Cau, PhD./*
>>>
>>> /Montpellier RIO Imaging Facility manager/Responsable technique MRI/
>>>
>>> Montpellier RIO Imaging
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> >>
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>>> ____________________________________________
>>
>>
>> --
>> James and Christine Pawley, 21 N. Prospect Ave.
>> Madison, WI, 53726 Phone: 608-238-3953
James and Christine Pawley, 21 N. Prospect Ave.