Re: TIRF

Posted by Paul Maddox on
URL: http://confocal-microscopy-list.275.s1.nabble.com/reducing-size-of-illumination-in-epi-fluorescence-tp593586p593598.html

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I second Kurt's assessment.  At the coverslip surface, the higher NA = higher res and more light gather.  As you go into the sample, the RI of the sample begins to dominate.  This can be corrected with a collar, or if there is no collar, using oils of a different RI can also correct (you have to test empirically for your system and the room temp etc.).  

Paul

Paul S. Maddox, PhD
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-----Original Message-----
From: Confocal Microscopy List [mailto:[hidden email]] On Behalf Of Kurt Thorn
Sent: Thursday, July 24, 2008 2:46 PM
To: [hidden email]
Subject: Re: TIRF

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We have had very good luck using a 100x / 1.49 NA TIRF lens for imaging
in aqueous samples - typically yeast or mammalian cells grown on
coverslips - on our spinning disk confocal.  I'm not sure how much the
larger NA helps, but having the correction collar is really nice for
correcting the spherical aberration that comes from using an oil
immersion lens to image into an aqueous sample.  If you put the time
into setting the correction collar, the Z-profiles obtained with the
TIRF lens are much more symmetric and show less intensity fall off into
the sample than do images taken with a 100x/1.4 lens.  We've not,
however, compared this to using a water immersion lens.  The lens we're
using are Nikon ones.

So from my limited experience, there can be some benefit to using a TIRF
lens as compared to an equivalent non-TIRF lens.

Kurt

Mancini, Michael A wrote:

>
> Speaking of TIRF issues, perhaps list members would like to comment on
> some differing opinions I've been hearing about the usefulness of a
> TIRF lens for standard fluorescence. I've heard two extremes on the
> appropriateness/usefulness of a 1.49 NA TIRF lens for high mag/high
> res/high sensitivity imaging away from the substratum (for example,
> intranuclear structures). Although I'm a bit physics-challenged, it
> seems to make sense to me that the NA would not be realized in a
> glycerol mounting medium due to RI mismatch, and could be counter
> productive, in fact. Seems the vendors that sell TIRF lens-systems say
> they are great for every use, pumping up the high NA as a cure for
> everything, and vendors that don't sell them say they're only good for
> a true TIRF application.
>
> Comments are welcome both on/off the list.
>
> Cheers,
>
> Mike
> _______________________
> Michael A. Mancini, Ph.D
> Department of Molecular and Cellular Biology
> Baylor College of Medicine
> Houston, TX 77030
> [hidden email]
> 713.408.0179 cell
>

--
Kurt Thorn, PhD
Director, Nikon Imaging Center
University of California San Francisco

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