Re: TIRF

Posted by John Oreopoulos on
URL: http://confocal-microscopy-list.275.s1.nabble.com/reducing-size-of-illumination-in-epi-fluorescence-tp593586p593600.html

Search the CONFOCAL archive at http://listserv.acsu.buffalo.edu/cgi-bin/wa?S1=confocal I'm going to say that in general, the TIRF objectives can be used for intranuclear structures both with confocal and regular epifluorescence, but I think most people on the listserver are going to say their preference would always be for a water objective with a live-cell specimen. Reducing aberrations caused by refractive index mismatch is probably more important to maintain BOTH resolution and high signal count. Jim Pawley has discussed a lot of these issues in his book, and at his live-cell microscopy course the detrimental effects of refractive mis-match were emphasized several times. High NA oil objectives perform the best when you're concerned with what's happening at or very close to the substrate. That's why TIRF is great for single-molecule in vitro experiments as well that require the sample to be diluted and immobilized on the surface.

John

On 24-Jul-08, at 2:32 PM, 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
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