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Nobody seems to have mentioned so far that the NA of an oil objective will NOT be 1.4 if it is imaging a sample in water. The maximum it can be is 1.33 - the refractive index of water. Anything over this will be beyond the critical angle and rays will not reach the specimen (in excitation) or the objective (in emission). So the oil objective has little or no advantage in NA and as Scot pointed out, the spherical aberration becomes horrendous very rapidly. So Gabriel's user is quite right. Actually I'd be surprised if you could see anything 100µm (0.1mm) into water with the oil lens.
So why do some people say they do better with an oil lens when imaging very close to the coverslip? The suggestion has been made in this list that they are seeing evanescent wave enhancement of fluorescence, and it seems highly believable to me. Those rays between NA 1.33 and 1.4 cannot reach the sample in the far field, but their evanescent wave can give a TIRF image, and we see this superimposed on the regular far-field image.
There is one further caveat, which Mark hinted at. If this is a Yokogawa spinning disk system it is designed for a 100x objective, and if used with 60x both pinhole size and pupil filling will not be optimal. But 100x water immersion objectives are rare beasts. (Apparently there are design constraints which prevent a Yokogawa head being optimised for a 60x objective).
Guy
-----Original Message-----
From: Confocal Microscopy List [mailto:[log in to unmask]] On Behalf Of Scot C Kuo
Sent: Friday, 5 October 2012 2:35 AM
To: [log in to unmask]
Subject: Re: Oil vs water objectives
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I've measured it quantitatively and the performance difference flips surprisingly close to the coverslip (see supplemental info, Fisher & Kuo 2009 PNAS 106, 133-138). For an Olympus 60x U-PlanApoS lenses, comparing 1.2 and 1.4 NA, the flip happens ~8 microns into an aqueous sample. For fluorescence closer than ~8um, oil is brighter, whereas for objects further, water immersion is brighter. If lenses aren't matched, then the cross-over can happen elsewhere, but the relative shapes of the curves are the same. Oil lenses (1.4NA) will have half the brightness by ~50um.
For the information you've provided (higher NA on water lens), I'd expect the cross-over to be closer to the coverslip surface.
-- Scot
============================================================================
...............Scot C. Kuo (410) 955-4536; email:skuo@jhu.edu...............
...Director, Microscope Facility, JHU-SOM, www.hopkinsmedicine.org/micfac...
..Assoc Professor, Biomedical Engineering & Cell Biology, www.jhu.edu/cmml..
----- Original Message -----
From: Gabriel Lapointe <[log in to unmask]>
Date: Thursday, October 4, 2012 9:16 am
Subject: [CONFOCALMICROSCOPY] Oil vs water objectives
To: [log in to unmask]
> *****
> To join, leave or search the confocal microscopy listserv, go to:
>
> *****
>
> 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
>
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