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Maybe the users are partly to blame by demanding inverted microscopes. I am convinced that at least 50% of the time an upright would do the job better, and be much easier to use. But how is it that people will happily spend hours each week learning to correct their golf swing, yet they will not spend minutes learning how to adjust a microscope properly?
Guy
-----Original Message-----
From: Confocal Microscopy List [mailto:[log in to unmask]] On Behalf Of George McNamara
Sent: Friday, 5 October 2012 8:56 PM
To: [log in to unmask]
Subject: Re: Oil vs water objectives
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Hi Jim and listserv,
Jim wrote, "I am convinced that it was because they just didn't want to take the time to adjust the collar properly. "
An additional explanation is that the microscope vendors have done a poor job making critical microscope features - such as the correction collar - easy to reach and use. Of course the customer buying an anti-ergonomic microscope are also contributing to the problem.
One solution of course is to "automate everything" - which simply changes the issue from being physically out of reach physically to one of budget.
The vendors should be able to design microscopes - and nanoscopes - that have excellent ergonomics and optics, and customers should take the time to buy such microscopes.
Another option with respect to S.A. is to move the correction outside the microscope, as with the mSAC
https://www.intelligent-imaging.com/msac.php
though this makes me wonder what happens if the mSAC is used with an objective lens whose "correction" collar is (badly) misadjusted.
George
On 10/4/2012 7:26 PM, James Pawley wrote:
> *****
> To join, leave or search the confocal microscopy listserv, go to:
> http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy
> *****
>
> Hi all,
>
> Looks like you have received a lot of good advice, but I would like to
> add one thing.
>
> Yes, do a test BUT don't forget to be very careful in adjusting the SA
> correction collar on the water lens. )Find a point object: focus up
> and down and adjust for symmetry (i.e., for the same image a little
> above focus as a little below focus. DON'T just try to "make it
> sharper!" The collar changes the focus plane.)
>
> The collars usually have markings in terms of the coverslip thickness
> that they are designed to correct for. One can easily see the
> difference created by mis-adjusting a 1.2 NA objective by 2
> µm-of-glass-replaced-by-water (my favorite unit for "measuring" SA).
>
> When modern water objectives first emerged in the early 1990s, many
> folk bought them and then went back to their oil objectives and I am
> convinced that it was because they just didn't want to take the time
> to adjust the collar properly.
>
> Because, if you do adjust it properly, you will get better (brighter?
> higher resolution?) images of aqeous specimens beyond about 3 µm
>
> Best,
>
> Jim Pawley,
>
> Near Harbin, China.
>
>
>
>> Make sure the coverslip is orthogonal to the image axis with your
>> water objective.
>>
>> A common aberration with water-immersion objective lenses.
>> J Microsc. 2004 Oct;216(Pt 1):49-51.
>> http://www.ncbi.nlm.nih.gov/pubmed/15369482
>>
>> Doug
>>
>> ^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^
>> Douglas W. Cromey, M.S. - Associate Scientific Investigator Dept. of
>> Cellular & Molecular Medicine, University of Arizona
>> 1501 N. Campbell Ave, Tucson, AZ 85724-5044 USA
>>
>> office: AHSC 4212 email: [log in to unmask]
>> voice: 520-626-2824 fax: 520-626-2097
>>
>> http://swehsc.pharmacy.arizona.edu/exppath/
>> Home of: "Microscopy and Imaging Resources on the WWW"
>>
>>
>> -----Original Message-----
>> From: Confocal Microscopy List
>> [mailto:[log in to unmask]] On Behalf Of Steffen
>> Dietzel
>> Sent: Thursday, October 04, 2012 7:30 AM
>> To: [log in to unmask]
>> Subject: Re: Oil vs water objectives
>>
>> *****
>> To join, leave or search the confocal microscopy listserv, go to:
>> http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy
>> *****
>>
>> I second Michael's comment.
>>
>> Apart from that, when you have an aqueous(!) preparation, spherical
>> aberration decreases image quality faster for the oil objective. So,
>> directly behind the coverslip the oil objective (1.4) will give you
>> the better image due to the higher NA, at some depth both objectives
>> will give you similar quality and beyond that, the water immersion
>> will be better.
>>
>> I seem to remember that for oil 1.4 and water 1.2 the crossing point
>> is
>> 10-20 µm into the sample.
>>
>> If you have the Handbook laying around, check the chapter on Lens
>> aberrations. Chapter 20 by Hell and Stelzer in the second edition,
>> chapter 20 by Egner and Hell in the 3rd edition.
>>
>> Needles to say that all above considerations assume that both
>> objectives have the same transparency for excitation and emission
>> wavelengths - which they probably don't. So we are back at Michael's
>> comment: You've got to test it. Beads attached to the coverslip and
>> the slide (various preparations with various distances between the
>> two) give good test objects.
>>
>> Steffen
>>
>> On 04.10.2012 15:15, Gabriel Lapointe wrote:
>>> *****
>>> To join, leave or search the confocal microscopy listserv, go to:
>>> http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy
>>> *****
>>>
>>> Hi,
>>>
>>> I have a user who insist that using a 1,27NA water immersion
>>> objective is brighter and would give better images than using a
>>> 1,4NA oil immersion. I understand that deeper into the media that would be true.
>>> But, in that particular case, we are talking about imaging GFP at
>>> less than 100 micron away with a spinning disk.
>>>
>>> So, I was wondering at which distance from the coverslips do we
>>> start seeing benefits of using a water immersion objective over an
>>> oil objective in aqueous media.
>>>
>>> Thanks for your help.
>>>
>>> Sincerely
>>> *Gabriel Lapointe, M.Sc.*
>> > Lab Manager / Microscopy Specialist
>>> Concordia University, Biology Department
>>> 7141 Sherbrooke St. West SP 534
>>> Montréal QC H4B 1R6 Canada
>>> [log in to unmask]
>>> cmac.concordia.ca
>>> http://gabriellapointe.ca
>>>
>>
>>
>> --
>> ------------------------------------------------------------
>> Steffen Dietzel, PD Dr. rer. nat
>> Ludwig-Maximilians-Universität München Walter-Brendel-Zentrum für
>> experimentelle Medizin (WBex) Head of light microscopy
>>
>> Mail room:
>> Marchioninistr. 15, D-81377 München
>>
>> Building location:
>> Marchioninistr. 27, München-Großhadern
>
>
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