http://confocal-microscopy-list.275.s1.nabble.com/Oil-vs-water-objectives-tp7579114p7579136.html
I am sure that Guy and John (and T.P. Burkhardt!)
objective in use. (i.e., On a given set-up, it
plane). Therefore, what we might call
surface. It will also add more out-of-focus light
having an RI that is higher than that specified.
how thick the layer of the "wrong" oil will be).
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>
>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).
>
>Here is a section in the discussion that I think fits your description, Guy:
>
>"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.
>
>Cheers,
>
>John Oreopoulos
>Research Assistant
>Spectral Applied Research
>Richmond Hill, Ontario
>Canada
>www.spectral.ca
>
>
>On 2012-10-04, at 8:40 PM, Guy Cox wrote:
>
>> *****
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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:
[hidden email]] On
>>Behalf Of Scot C Kuo
>> Sent: Friday, 5 October 2012 2:35 AM
>> To:
[hidden email]
>> Subject: Re: Oil vs water objectives
>>
>> *****
>> To join, leave or search the confocal microscopy listserv, go to:
>>
http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy>> *****
>>
>> 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:
[hidden email]...............
>> ...Director, Microscope Facility, JHU-SOM, www.hopkinsmedicine.org/micfac...
>> ..Assoc Professor, Biomedical Engineering & Cell Biology, www.jhu.edu/cmml..
>>
>>
>> ----- Original Message -----
>> From: Gabriel Lapointe <
[hidden email]>
>> Date: Thursday, October 4, 2012 9:16 am
>> Subject: [CONFOCALMICROSCOPY] Oil vs water objectives
>> To:
[hidden email]
>>
>>
>>> *****
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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
>>>