Posted by
Steffen Dietzel on
URL: http://confocal-microscopy-list.275.s1.nabble.com/Oil-vs-water-objectives-tp7579114p7579117.html
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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:
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>
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>
--
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Steffen Dietzel, PD Dr. rer. nat
Ludwig-Maximilians-Universität München
Walter-Brendel-Zentrum für experimentelle Medizin (WBex)
Head of light microscopy
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