Confusion about the equation for numerical perture

Posted by John Oreopoulos on
URL: http://confocal-microscopy-list.275.s1.nabble.com/How-to-measure-objective-transmission-curves-tp590172p590182.html

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