Localization of a fluorescent source without numerical fitting

We present an algebraic solution to the problem of localizing a single fluorescent particle with sub-diffraction-limit accuracy. The algorithm is derived and its performance studied experimentally. Isolated 20 nm fluorescent beads were imaged using a wide-field microscope at two different positions separated by 100 nm and at a range of signal-to-noise ratios (SNR). The data were analyzed using both the new algorithm and the standard approach of fitting the data to a Gaussian profile. Results indicate that the proposed approach is nearly as accurate as Gaussian fitting across a wide range of SNR while executing over 200 times faster. In addition, the new algorithm is able to localize at lower SNR than the fitting method. (c) 2008 Optical Society of America