Single-image axial localization precision analysis for individual fluorophores

Bio-mechanism investigations demand single particle tracking with high spatial and temporal resolutions which require single fluorophore 3D localization measurements with matching precision and speed. Although the precision for lateral-localization measurements is well described by an analytical expression, for the axial direction, it is often obtained by repeating location measurements or by estimating a lower bound. Here, we report a precision expression for an axial-localization method that analyzes the standard deviations of single fluorophores’ intensity profiles. Like the lateral-localization precision, this expression includes all relevant experimental effects measurable from a Gaussian intensity profile of the fluorophore. This expression completes the precision analysis for single-image 3D localization of individual fluorophores and lifts the temporal resolution to the typical exposure timescales of milliseconds.