Precision analysis for standard deviation measurements of single fluorescent molecule images

TL;DR

This paper develops an analytical model to quantify the precision of standard deviation measurements of single fluorescent molecules, demonstrating nanometer accuracy under various experimental conditions.

Contribution

The authors introduce a new analytical expression for the measurement error of standard deviation in single-molecule fluorescence imaging, validated through experiments and simulations.

Findings

Measurement error depends on photon count, background noise, and pixel size.

The model predicts nanometer-level precision for standard deviation measurements.

Experimental results agree with theoretical and simulation predictions.

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.