http://confocal-microscopy-list.275.s1.nabble.com/Coverslips-tp6865241p6870051.html
Nikon's 1.45 and 1.49 NA's objectives have two separate sets of markings: One for room temp (the exact temp is printed and I do not remember) and a separate one for 37.C.
Most of the oil correction collars I have seen are to correct for glycerol immersion/mounting media. The temperature is an interesting point. I haven't seen this advertised by the microscope companies but that wouldn't be too surprising.
I can understand the argument for not needing exact coverslip thickness for oil objectives when imaging near the coverslip surface. Just out of curiosity then, why are there some oil objectives sold with correction collars as well? Are these collars there just for adjustment when imaging with an oil immersion objective at a temperature other than room temperature?
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> 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
>
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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/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 :
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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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