Single-molecule localization uncertainties induced by molecular labeling

Wei-Hong Yeo; Yang Zhang; Junghun Kweon; Cheng Sun; Hao  F. Zhang

doi:10.1117/12.3001243

13 March 2024 Single-molecule localization uncertainties induced by molecular labeling

Wei-Hong Yeo, Yang Zhang, Junghun Kweon, Cheng Sun, Hao F. Zhang

Proceedings Volume PC12849, Single Molecule Spectroscopy and Superresolution Imaging XVII; PC128490K (2024) https://doi.org/10.1117/12.3001243
Event: SPIE BiOS, 2024, San Francisco, California, United States

Abstract

This study explores uncertainties in fluorescence labeling, a complication in Single-Molecule Localization Microscopy (SMLM) image interpretation. We examine variability caused by antibody and fluorophore attachment, orientation, and photobleaching, focusing on protein tagging and indirect immunofluorescence, techniques known for their specificity but prone to introducing variable label densities. We use a Monte Carlo (MC) model to simulate SMLM images, providing a 'ground truth' for comparison. This model also investigates the balance between labeling size and density, considering the possibility of single fluorophore attachment in protein tagging and multiple fluorophores in indirect immunofluorescence. We propose methods to quantify the effects of labeling strategies on image quality and accuracy, considering parameters such as labeling linker length and fluorophore photoswitching. Our work enhances the accuracy of SMLM image interpretation and guides the selection of labeling strategies, advancing super-resolution microscopy.

Conference Presentation

Citation Download Citation

Wei-Hong Yeo, Yang Zhang, Junghun Kweon, Cheng Sun, and Hao F. Zhang "Single-molecule localization uncertainties induced by molecular labeling", Proc. SPIE PC12849, Single Molecule Spectroscopy and Superresolution Imaging XVII, PC128490K (13 March 2024); https://doi.org/10.1117/12.3001243

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KEYWORDS

Fluorophores

Proteins

Monte Carlo methods

Image quality

Image visualization

Microscopy

Nanostructures

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