>Acknowledging in advance that I am hardly a deconvolution expert,
>our Nikon 60x 1.49 provides a sharper PSF than our 40x plan fluor
>(hardly an apples-to-apples comparison, I know). We measured the
>PSF because our blind deconvolution software (Huygens) complained
>about the NA/medium mismatch. It still appeared to work fairly
>well.
>
>cheers,
>
>
>Tim
>
>Timothy Feinstein, PhD
>Postdoctoral Associate
>University of Pittsburgh Dept. of Pharmacology
>Pittsburgh, PA
Hi Tim,
You don't mention the NA of the 40x (or if we are talking widefield
or laser confocal, where underfilling might be less of a factor at
60x than at 40x) but I think that your comment about the useful
suggestion that the Huygens software made about mismatch might be
the nub of the argument.
I am assuming that Hans van der Voort's clever program makes this
warning when it detects that the PSF is asymmetrical in z. Such
asymmetry implies spherical aberration and this is usually caused by
a mismatch between the refractive index (RI) that the lens is
designed for and the actual RI of the specimen or using a coverslip
of the incorrect thickness.
None of the high NA 40x Fluor lenses I can think of include an SA
corection collar, while the Nikon NA 1.49 does do so (did you use
it?). So, if your specimen RI is not the same as that of oil or your
coverslip is the wrong thickness, you can correct for this on the
1.49 but not on the other 40x lens.
SA correction being equal, and assuming that your 40x is NA 1.3, I
would expect that the on-axis x-y resolution of the NA 1.49 would be
about 14% better (smaller spacing) than the 40x.
Cheers,
Jim P.
*********************************************************************************
Prof. James B. Pawley, Ph. 608-263-3147
Room 223, Zoology Research Building,
FAX 608-265-5315
1117 Johnson Ave., Madison, WI, 53706
[log in to unmask]
3D Microscopy of Living Cells Course, June 12-24, 2010, UBC, Vancouver Canada
Info: http://www.3dcourse.ubc.ca/ Applications still
being accepted
"If it ain't diffraction, it must be statistics." Anon.
>
>On Mar 24, 2010, at 7:33 PM, Cameron, Lisa wrote:
>
>> Yes, I concur with Kurt -
>>
>> Nikon 60x and 100x 1.49 NA TIRF objectives work well with the standard 1.515
>> Nikon oil.
>>
>>
>> ---------------------------------------
>> Lisa Cameron, Ph.D.
>> Director of Confocal and Light Microscopy
>> Dana Farber Cancer Institute
>>
>> -----Original Message-----
>> From: Confocal Microscopy List [mailto:[log in to unmask]] On
>> Behalf Of Kurt Thorn
>> Sent: Wednesday, March 24, 2010 7:28 PM
>> To: [log in to unmask]
>> Subject: Re: [CONFOCALMICROSCOPY] TIRF objective for routine imaging
>>
>> This isn't true - our Nikon 1.49 TIRF lens works with the standard
>> immersion oil.
>>
>> Barbara Foster wrote:
>>> Hi, all
>>>
>>> Just remember that the 1.49 NA only works with special, high
>>> refractive index liquids.
>>>
>>> *Barbara Foster, President and Sr. Consultant
>>>
>>> Microscopy/Microscopy Education
>>> *7101 Royal Glen Trail, Suite A
>>> McKinney TX 75070
>>> *P: *(972)924-5310 *Skype: *fostermme
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>>> www.MicroscopyEducation.com <http://www.microscopyeducation.com/>! And
>>> don't forget: MME is now scheduling customized, on-site courses for
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>>
>>
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*********************************************************************************
Prof. James B. Pawley, Ph. 608-263-3147
Room 223, Zoology Research Building,
FAX 608-265-5315
1117 Johnson Ave., Madison, WI, 53706
[log in to unmask]
3D Microscopy of Living Cells Course, June 12-24, 2010, UBC, Vancouver Canada
Info: http://www.3dcourse.ubc.ca/ Applications still
being accepted
"If it ain't diffraction, it must be statistics." Anon.
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