http://confocal-microscopy-list.275.s1.nabble.com/Oil-vs-water-objectives-tp7579114p7579129.html
take the time to adjust the collar properly. "
collar - easy to reach and use. Of course the customer buying an
Another option with respect to S.A. is to move the correction outside
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>
> 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:
[hidden email]
>> 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:
[hidden email]] On Behalf Of Steffen Dietzel
>> Sent: Thursday, October 04, 2012 7:30 AM
>> To:
[hidden email]
>> Subject: Re: Oil vs water objectives
>>
>> *****
>> To join, leave or search the confocal microscopy listserv, go to:
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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
>>>
[hidden email]
>>> 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
>
>