http://confocal-microscopy-list.275.s1.nabble.com/Oil-vs-water-objectives-tp7579114p7579125.html
Just to add to Guy's last point, I recently read an interesting paper by Thomas Burghardt which thoroughly investigated the effect he just described (about evanescent wave enhancement near the coverslip):
Burghardt, T.P., Evanescent field shapes excitation profile under axial epi-illumination. Journal of Biomedical Optics, 2012. 17(6).
"The highest NA objectives available are TIRF objectives for through-the-objective total internal reflection because they achieve excitation incidence angles beyond critical angle for the glass/ aqueous interface. Generally, TIRF or epi-illumination excitations pertain to evanescent or propagating field microscopies that are appropriate for different applications. I show here that the TIRF objective under common axial epi-illumination conditions produces an evanescent field that favorably remodels the excitation volume for samples near the coverslip.
Point source fluorescent spheres were imaged from a region where the excitation evanescent field contributes to excitation and from a region where the evanescent field is necessarily absent. To do so, I constructed a microfluidic PDMS spacer that separates two glass coverslips (Fig. 1). The lower coverslip optically contacts the oil immersion objective whereas the upper coverslip has an intervening 20um-thick slab of water. The 100um objective working distance ensures that either object can be brought into focus by vertical movement of the objective. Objects at the lower coverslip are subjected to both evanescent and propagating exciting fields whereas objects at the upper coverslip feel only the propagating field. Figure 7 shows a one-beam intensity profile measured by axial translation of the objective over 1500 nm, indicating the narrowing effect of the evanescent field. Profile computation agrees with observation. Figure 5 indicates the expected half-width remodeling of the axial dependence for exciting light as a function of probe position relative to the lower coverslip interface. I also observed a 2- to 4-fold intensity enhancement for the fluorescent sphere at the lower coverslip that is attributable to the discontinuous enhancement of the exciting normal electric field on the aqueous side at the lower coverslip, the selective collection of near-field emission from a sphere at the lower coverslip, and the effect of light scattering in the intervening water layer on both exciting and emission light for the sphere at the upper coverslip. Other effects may be significant, including the presence of the aqueous/glass interface at the upper coverslip...
... evanescent excitation contributes to observed fluorescence whenever a TIRF objective is used and suggests that the sample material nearest the coverslip disproportionally contributes to the observed fluorescence signal."
To this point I would add that even a 1.4 NA oil immersion objective is technically a TIRF objective since even this NA subtends (just barely) an angle greater than the glass-water interface critical angle.
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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
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> -----Original Message-----
> From: Confocal Microscopy List [mailto:
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> Sent: Friday, 5 October 2012 2:35 AM
> To:
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> 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:
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>
> ----- Original Message -----
> From: Gabriel Lapointe <
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> Date: Thursday, October 4, 2012 9:16 am
> Subject: [CONFOCALMICROSCOPY] Oil vs water objectives
> To:
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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
>>
[hidden email]
>> cmac.concordia.ca
>>