Precision analysis for standard deviation measurements of immobile single fluorescent molecule images.

Michael C Desantis,Shawn H Decenzo,Je-Luen Li,Y M Wang

doi:10.1364/oe.18.006563

Michael C Desantis, Shawn H Decenzo + Show 2 more

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https://doi.org/10.1364/oe.18.006563

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Journal: Optics Express	Publication Date: Mar 15, 2010
Citations: 21	License type: cc-by

Affiliation: University of Washington

Abstract

Standard deviation measurements of intensity profiles of stationary single fluorescent molecules are useful for studying axial localization, molecular orientation, and a fluorescence imaging system's spatial resolution. Here we report on the analysis of the precision of standard deviation measurements of intensity profiles of single fluorescent molecules imaged using an EMCCD camera.We have developed an analytical expression for the standard deviation measurement error of a single image which is a function of the total number of detected photons, the background photon noise, and the camera pixel size. The theoretical results agree well with the experimental, simulation, and numerical integration results. Using this expression, we show that single-molecule standard deviation measurements offer nanometer precision for a large range of experimental parameters.

Highlights

Single-molecule-fluorescence imaging has been a powerful tool in particle localization and tracking studies [1,2,3,4]
We report our study of 2D ∆sx,rms using four different methods: (1) experimental measurements, (2) simulations, (3) numerical integrations of Eq (10), and (4) analytical calculations using Eq (17)
To clearly illustrate the change in the standard deviation (SD) error, which is measured as the point spread function (PSF) SD minus sx, the 1D intensity profiles of the PSFs are plotted in Fig. 1B as opposed to their 2D intensity profiles for clarity

Summary

Introduction

Single-molecule-fluorescence imaging has been a powerful tool in particle localization and tracking studies [1,2,3,4]. In single-molecule imaging, the fluorescence intensity profile of a point light source is called a point spread function (PSF). While the PSF is described by an Airy function, it is, in practice, approximated by a Gaussian. A Gaussian fit to the PSF of a stationary single fluorophore has two fitting parameters: centroid and standard deviation (SD). While the centroid determines the lateral position of the particle in the imaging plane, the standard deviation determines its axial position [5,6,7] and orientation [8,9,10], as well as the spatial resolution of the fluorescence imaging system [11]

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