Re: TIRF objective for routine imaging

Posted by James Pawley on
URL: http://confocal-microscopy-list.275.s1.nabble.com/TIRF-objective-for-routine-imaging-tp4778151p4798070.html

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  [phone]
1117 Johnson Ave., Madison, WI, 53706                                [hidden email]
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:[hidden email]] On
> Behalf Of Kurt Thorn
> Sent: Wednesday, March 24, 2010 7:28 PM
> To: [hidden email]
> 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
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>> McKinney TX 75070
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-- 

*********************************************************************************
Prof. James B. Pawley,                                     Ph.  608-263-3147 
Room 223, Zoology Research Building,                                  FAX  [phone]
1117 Johnson Ave., Madison, WI, 53706                                [hidden email]
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.