Re: Confusion about the equation for numerical perture

Posted by Peng Xi-2 on
URL: http://confocal-microscopy-list.275.s1.nabble.com/How-to-measure-objective-transmission-curves-tp590172p590195.html

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Hi John,
    The perfect imaging plane for a typical objective is right after the
coverglass, and the coverglass is usually compensated through the
objective collar. So the refractive index n is referring to the
immersion fluid, which helps to "bend" the light for a higher NA.
 

--
Peng
Dantus Research Group
Department of Chemistry
Michigan State University
East Lansing, MI 48824
Tel: [phone] x319
Email: [hidden email]
http://www.msu.edu/~xipeng/


John Oreopoulos wrote:

> Search the CONFOCAL archive at
> http://listserv.acsu.buffalo.edu/cgi-bin/wa?S1=confocal Hi, I'm a
> little confused about the traditional equation for a microscope's
> numerical aperture:
>
> NA = n x sin(theta)
>
> where theta is the maximum half angle subtended by the lens's light
> collection cone. It's the "n", the refractive index I'm confused
> about. I've now read several sources and books that say n is the
> refractive index of the medium that the sample is embedded in (above
> the coverslip), and in other places, I read that n is the refractive
> index of the immersion fluid, say oil (below the coverslip). I
> understand that in the ideal cases, both of these refractive indexes
> should be matched for the best possible imaging, but in reality they
> never really are, right? So which medium does the "n" refer too? And
> what about in the case for TIRF microscopy, where it is required that
> you have a oil immersion objective to image a sample in water for
> example? Which n should I use to calculate my objective NA?
>
>
> John Oreopoulos, BSc,
>
> PhD Candidate
>
> University of Toronto
>
> Institute For Biomaterials and Biomedical Engineering
>
> Centre For Studies in Molecular Imaging
>
>
> Tel: W:416-946-5022
>
>
>