Quantifying accuracy and heterogeneity in single-molecule super-resolution microscopy

Mazidi, Hesam; Ding, Tianben; Nehorai, Arye; Lew, Matthew D.

doi:10.1038/s41467-020-20056-9

Cited by 11 publications

(10 citation statements)

References 47 publications

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“…S5, S6) to optimize their concentrations for the analysis of MNPs @ SiO 2 (RITC) with d STORM imaging. Because NPs are not reagents to which specific antibodies are bound or stained for specific organelles in cells, the optimization of the concentration of NPs was determined through intuitive characterization rather than localization density [ 48 ]. At a higher concentration (0.1 μg/mL), RAW 264.7 cells demonstrated the uptake of numerous NPs, making the task of distinguishing individual NPs difficult in practice.…”

Section: Resultsmentioning

confidence: 99%

Quantifying intracellular trafficking of silica-coated magnetic nanoparticles in live single cells by site-specific direct stochastic optical reconstruction microscopy

et al. 2021

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Background Nanoparticles have been used for biomedical applications, including drug delivery, diagnosis, and imaging based on their unique properties derived from small size and large surface-to-volume ratio. However, concerns regarding unexpected toxicity due to the localization of nanoparticles in the cells are growing. Herein, we quantified the number of cell-internalized nanoparticles and monitored their cellular localization, which are critical factors for biomedical applications of nanoparticles. Methods This study investigates the intracellular trafficking of silica-coated magnetic nanoparticles containing rhodamine B isothiocyanate dye [MNPs@SiO2(RITC)] in various live single cells, such as HEK293, NIH3T3, and RAW 264.7 cells, using site-specific direct stochastic optical reconstruction microscopy (dSTORM). The time-dependent subdiffraction-limit spatial resolution of the dSTORM method allowed intracellular site-specific quantification and tracking of MNPs@SiO2(RITC). Results The MNPs@SiO2(RITC) were observed to be highly internalized in RAW 264.7 cells, compared to the HEK293 and NIH3T3 cells undergoing single-particle analysis. In addition, MNPs@SiO2(RITC) were internalized within the nuclei of RAW 264.7 and HEK293 cells but were not detected in the nuclei of NIH3T3 cells. Moreover, because of the treatment of the MNPs@SiO2(RITC), more micronuclei were detected in RAW 264.7 cells than in other cells. Conclusion The sensitive and quantitative evaluations of MNPs@SiO2(RITC) at specific sites in three different cells using a combination of dSTORM, transcriptomics, and molecular biology were performed. These findings highlight the quantitative differences in the uptake efficiency of MNPs@SiO2(RITC) and ultra-sensitivity, varying according to the cell types as ascertained by subdiffraction-limit super-resolution microscopy. Graphical Abstract

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Section: Resultsmentioning

confidence: 99%

Quantifying intracellular trafficking of silica-coated magnetic nanoparticles in live single cells by site-specific direct stochastic optical reconstruction microscopy

et al. 2021

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“…Indeed, previous correlative AFM and fluorescence imaging of amyloids has revealed strong heterogeneity in labelling, [25,36] which can be topography‐dependent and likely due to high polymorphism in amyloids [25] . Furthermore, the interaction and binding orientations to the amyloid fibrils has recently been shown to affect the quality of SMLM reconstructed images [37] …”

Section: Resultsmentioning

confidence: 99%

“…[25] Furthermore, the interaction and binding orientations to the amyloid fibrils has recently been shown to affect the quality of SMLM reconstructed images. [37]…”

Section: Resultsmentioning

confidence: 99%

Versatile Near‐Infrared Super‐Resolution Imaging of Amyloid Fibrils with the Fluorogenic Probe CRANAD‐2

Torra

Viela

Megı́as

et al. 2022

Chemistry A European J

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CRANAD-2 is a fluorogenic curcumin derivative used for near-infrared detection and imaging in vivo of amyloid aggregates, which are involved in neurodegenerative diseases. We explore the performance of CRANAD-2 in two super-resolution imaging techniques, namely stimulated emission depletion (STED) and single-molecule localization microscopy (SMLM), with markedly different fluorophore requirements. By conveniently adapting the concentration of CRANAD-2, which transiently binds to amyloid fibrils, we show that it performs well in both techniques, achieving a resolution in the range of 45-55 nm. Correlation of SMLM with atomic force microscopy (AFM) validates the resolution of fine features in the reconstructed super-resolved image. The good performance and versatility of CRANAD-2 provides a powerful tool for near-infrared nanoscopic imaging of amyloids in vitro and in vivo.

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“…Fourth, to compensate for optical aberrations, we used images of bright fluorescent beads and a maximum-likelihood estimator to retrieve the pupil phase function, as reported in 23,78 . In short, optical aberrations were modeled as a phase mask at the pupil consisting of the sum of Zernike modes.…”

Section: Methodsmentioning

confidence: 99%

Resolving the nanoscale structure of β-sheet assemblies using single-molecule orientation-localization microscopy

Zhou,

O’Neill,

Ding

et al. 2023

Preprint

Self Cite

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Synthetic peptides that self-assemble into cross-β fibrils have remarkable utility as engineered biomaterials due to their modularity and biocompatibility, but their structural and morphological similarity to amyloid species has been a long-standing concern for their translation. Further, their polymorphs are difficult to characterize using spectroscopic and imaging techniques that rely on ensemble averaging to achieve high resolution. Here, we utilize single-molecule orientation-localization microscopy (SMOLM) to characterize fibrils formed by the designed amphipathic enantiomers, KFE8Land KFE8D, and the pathological amyloid-beta peptide Aβ42. SMOLM reveals that the orientations of Nile red, as it transiently binds to both KFE8 and Aβ42, are consistent with a helical (bilayer) ribbon structure and convey the precise tilt of the fibrils' inner and outer backbones. SMOLM also finds polymorphic branched and curved morphologies of KFE8 whose backbones exhibit much more heterogeneity than those of more typical straight fibrils. Thus, SMOLM is a powerful tool to interrogate the structural differences and polymorphism between engineered and pathological cross β-rich fibrils.

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Quantifying accuracy and heterogeneity in single-molecule super-resolution microscopy

Cited by 11 publications

References 47 publications

Quantifying intracellular trafficking of silica-coated magnetic nanoparticles in live single cells by site-specific direct stochastic optical reconstruction microscopy

Quantifying intracellular trafficking of silica-coated magnetic nanoparticles in live single cells by site-specific direct stochastic optical reconstruction microscopy

Versatile Near‐Infrared Super‐Resolution Imaging of Amyloid Fibrils with the Fluorogenic Probe CRANAD‐2

Resolving the nanoscale structure of β-sheet assemblies using single-molecule orientation-localization microscopy

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