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Andreas,

 		Always look for the best refractive index match.  Be aware that some 'glycerol' mounting media are NOT pure glycerol but more like 50%.  I have demonstrated experimentally that in this case you can do better with a water immersion lens setting the correction collar as if for a very thick coverslip, than you can with an oil objective.  IF all oil immersion objectives were equipped with correction collars the situation could be different.   Let's be clear that they should be - oil varies hugely in refractive index with temperature.  If you are looking at living samples in aqueous media you should always use a water (coverslip) lens - at least they always have correction collars.

		The corollary is that you do need to learn how to use a correction collar - it's a skill, and not a difficult one, but it does need to be mastered.  Simply setting it to what 'should' be the correct value never works.  

                                         Guy

Optical Imaging Techniques in Cell Biology
by Guy Cox.  2nd edition 2012    CRC Press 
     http://www.guycox.com/optical.htm
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Guy Cox, MA, DPhil(Oxon), Honorary Associate, 
Australian Centre for Microscopy & Microanalysis, 
Madsen Building F09, University of Sydney, NSW 2006 

Phone +61 2 9351 3176     Fax +61 2 9351 7682
             Mobile 0413 281 861
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      http://www.guycox.net
 

-----Original Message-----
From: Confocal Microscopy List [mailto:[log in to unmask]] On Behalf Of Vonderheit, Andreas
Sent: Tuesday, 31 July 2012 7:19 PM
To: [log in to unmask]
Subject: Immersion Objectives

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Dear list,

I learned and I thought that the different types of immersion media are there to get the best refractive index match to the media or cells behind the cover glass, in order to minimize spherical aberration.
Now some microscope companies say that this discussion is tedious since the error of the lenses/objectives are higher than the effect of the refractive index match, is that true?

Totally understandable: when I use a mounting medium, which would be close to the refractive index of the used oil (refractive index : 1.52) and the cover glass (refractive index: 1.52), this would be a perfect match.

But what happens, when I am using a mounting medium based on glycerol (refractive index: 1.47), still the cover glass has 1.52. Should I use a glycerol objective and glycerol as an immersion medium, or an oil objective with oil as immersion medium?

I thought when I do live microscopy in an aqueous solution (refractive index of water: 1.33) the best choice would be a water-immersion objective.

Now one company also offers silicone-oil-immersion objectives, the silicone oil has a refractive index of 1.4, which would be closer to the refractive index of the sample which they claim to be at 1.38.

I have the feeling that I opened the box of Pandora and all that I wanted was the best solution to a setup, where one of my users wants to look at C.elegans in aqueous solution, which objective type should be used?

Thanks, best regards
Andreas