Localization Precision in Stepwise Photobleaching Experiments

Abstract

The precise determination of the position of fluorescent labels is essential for the quantitative study of biomolecular structures by various localization microscopy techniques. Localization by stepwise photobleaching is especially suited for measuring nanometer-scale distances between two labels; however, the precision of this method has remained elusive. Here, we show that shot noise from other emitters and error propagation compromise the localization precision in stepwise photobleaching. Incorporation of point spread function-shaped shot noise into the variance term in the Fisher matrix yielded fundamental Cràmer-Rao lower bounds (CRLBs) that were in general anisotropic and depended on emitter intensity and position. We performed simulations to benchmark the extent to which different analysis procedures reached these ideal CRLBs. The accumulation of noise from several images accounted for the worse localization precision in image subtraction. Propagation of fitting errors compromised the CRLBs in sequential fitting using fixed parameters. Global fitting of all images was also governed by error propagation, but made optimal use of the available information. The precision of individual distance measurements depended critically on the exact bleaching kinetics and was correctly quantified by the CRLBs. The methods presented here provide a consistent framework for quantitatively analyzing stepwise photobleaching experiments and shed light on the localization precision in some other bleaching- or blinking-assisted techniques.

Molecular architecture of native fibronectin fibrils

“…The localization precision in stepwise photobleaching experiments was determined according to the Cramèr-Rao lower bound as recently described 70 . For all localizations, the precision was better than 2 nm; the error of the distance between two localizations was accordingly smaller than ∼3 nm.…”

In vitro labeling strategies for in cellulo fluorescence microscopy of single ribonucleoprotein machines

“…Given the stochastic nature of biological processes in the cellular environment, single‐molecule assays are pivotal not only for probing biochemical mechanisms but also for understanding how biology is controlled spatiotemporally, ultimately deconvolving what separates disease from archetypical. Some examples of intracellular SMFM techniques used to measure these phenomena are fixed‐cell particle counting through single‐molecule fluorescence in situ hybridization (smFISH) to assess expression levels, single‐particle tracking for interaction kinetics and cellular localization, fixed‐cell stepwise photobleaching counting of subunit stoichiometry, and super‐resolution microscopy for 3D architecture, among others . Despite the value that SMFM approaches provide, there is still an insufficient number of tools available to label RNA components of RNP nanomachines by comparison to those that are protein based .…”