SNSMIL, a real-time single molecule identification and localization algorithm for super-resolution fluorescence microscopy.

Yunqing Tang,Junbai Li,Nadine Gruteser,Alexandros Katranidis,Annika Meisenberg,Xiaoming Zhang,Xiaoming Fan,Johnny Hendriks,Thomas Gensch,Arnd Baumann,Luru Dai

doi:10.1038/srep11073

Abstract

Single molecule localization based super-resolution fluorescence microscopy offers significantly higher spatial resolution than predicted by Abbe’s resolution limit for far field optical microscopy. Such super-resolution images are reconstructed from wide-field or total internal reflection single molecule fluorescence recordings. Discrimination between emission of single fluorescent molecules and background noise fluctuations remains a great challenge in current data analysis. Here we present a real-time, and robust single molecule identification and localization algorithm, SNSMIL (Shot Noise based Single Molecule Identification and Localization). This algorithm is based on the intrinsic nature of noise, i.e., its Poisson or shot noise characteristics and a new identification criterion, QSNSMIL, is defined. SNSMIL improves the identification accuracy of single fluorescent molecules in experimental or simulated datasets with high and inhomogeneous background. The implementation of SNSMIL relies on a graphics processing unit (GPU), making real-time analysis feasible as shown for real experimental and simulated datasets.

Highlights

Cellular structures as well as morphological features in nanostructured materials and devices have dimensions in the range from 5 to 500 nm
Due to experimental implementation and sample preparation, background is uneven in many measurements, local signal-to-noise ratio (SNR) (Signal-to-Noise Ratio) analysis is essential and being used in excellent single molecule localization microscopy (SMLM) software such as rapidSTORM, QuickPALM, Localizer, MaLiang, etc
SNSMIL defines a new metric for identification, QSNSMIL, that allows for an easy assessment of the quality of an identified single molecule

Summary

Introduction

Cellular structures (e.g., mitochondria, endoplasmic reticulum, Golgi apparatus) as well as morphological features in nanostructured materials and devices have dimensions in the range from 5 to 500 nm. All SMLM program packages require that the user needs to choose a set of parameters that influence the performance of the program and have a considerable impact on the super-resolution image obtained It is, rather difficult for experimentalists not involved in the development of the analysis programs or not trained in single molecule microscopy to perceive and predict the influence of a certain parameter on the final result (the super-resolution image) and it is complicated to make reasonable choices. Especially when the imaged biological structures extend in z-direction, a variable width in the Gaussian fitting model is needed to allow “out of focus” single molecules to be accepted For those cases a second parameter - localization precision filtration for single emitters – is introduced. The performance of SNSMIL is compared to several other SMLM program packages

Methods

Results

Conclusion