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Very good points!
I am sure that Guy and John (and T.P. Burkhardt!)
are describing a very important effect.
Perhaps it is also worth pointing out that this
effect will only occur if the epi-illumination
system really fills the full NA of the specific
objective in use. (i.e., On a given set-up, it
might work with 100x NA 1.4 but not so well with
a 40x 1.4, where the diameter of the entrance
pupil is 2.5x larger).
Also, deconvolution systems assume that the total
excitation illumination power does not vary with
z (although its power density in mw/µm*2 will, of
course, vary with the distance from the focus
plane). Therefore, what we might call
TIRF-enhancement will tend to exaggerate
quantitative results from features near the glass
surface. It will also add more out-of-focus light
to every plane than is justified by the
fluorescent markers present in the specimen and
it very seems likely to distort the z-axis of any
the PSF standard obtained from a sub-resolution
bead located at the glass surface.
I agree that a straight PSF obtained in this way
would not, in any case, have the proper SA
correction for planes inside the specimen,
however some workers have reduced this problem by
using an NA 1.4 objective with an immersion oil
having an RI that is higher than that specified.
This can work pretty well for a specific, narrow
range of distances into the cell (exactly where
these planes are depends on RI of the oil and the
working distance of the objective, which defines
how thick the layer of the "wrong" oil will be).
However, it only works well if one determines the
PSF using small objects located within this focus
range.
Cheers,
Jim Pawley
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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:
>
>> *****
>> To join, leave or search the confocal microscopy listserv, go to:
>> http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy
>> *****
>>
>> 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
>>
>> *****
>> 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: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
>>>
--
James and Christine Pawley, PO Box 2348, 5446
Burley Place (PO Box 2348), Sechelt, BC, Canada,
V0N3A0, 604-885-0840 NEW! Cell (when I remember
to turn it on!) 1-765-637-1917,
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