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
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-----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
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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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> To join, leave or search the confocal microscopy listserv, go to:
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>
> 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
>
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