Assessing resolution in super-resolution imaging

Demmerle, Justin; Wegel, Eva; Schermelleh, Lothar; Dobbie, Ian M.

doi:10.1016/j.ymeth.2015.07.001

Cited by 56 publications

(44 citation statements)

References 32 publications

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“…1p,q). The visual impression of increasing lateral resolution was confirmed in the Fourier spectrum 29 (Supplementary Figure 2a) and by Fourier Ring Correlation 30 (Supplementary Figure 2b).…”

Section: Resolution and Quality Controlmentioning

confidence: 73%

Nuclear pores as versatile reference standards for quantitative superresolution microscopy

Thevathasan¹,

Kahnwald²,

Cieslinski³

et al. 2019

Preprint

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Quantitative fluorescence and superresolution microscopy are often limited by insufficient data quality or artifacts. In this context, it is essential to have biologically relevant control samples to benchmark and optimize the quality of microscopes, labels and imaging conditions.Here we exploit the stereotypic arrangement of proteins in the nuclear pore complex as in situ reference structures to characterize the performance of a variety of microscopy modalities. We created four genome edited cell lines in which we endogenously labeled the nucleoporin Nup96 with mEGFP, SNAP-tag or HaloTag or the photoconvertible fluorescent protein mMaple. We demonstrate their use a) as 3D resolution standards for calibration and quality control, b) to quantify absolute labeling efficiencies and c) as precise reference standards for molecular counting.These cell lines will enable the broad community to assess the quality of their microscopes and labels, and to perform quantitative, absolute measurements.

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Section: Resolution and Quality Controlmentioning

confidence: 73%

Nuclear pores as versatile reference standards for quantitative superresolution microscopy

Thevathasan¹,

Kahnwald²,

Cieslinski³

et al. 2019

Preprint

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“…However, as stated by Demmerle et al 22,. ‘resolution in single-molecule imaging is especially challenging'.…”

Section: Resultsmentioning

confidence: 92%

Complementarity of PALM and SOFI for super-resolution live-cell imaging of focal adhesions

et al. 2016

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Live-cell imaging of focal adhesions requires a sufficiently high temporal resolution, which remains a challenge for super-resolution microscopy. Here we address this important issue by combining photoactivated localization microscopy (PALM) with super-resolution optical fluctuation imaging (SOFI). Using simulations and fixed-cell focal adhesion images, we investigate the complementarity between PALM and SOFI in terms of spatial and temporal resolution. This PALM-SOFI framework is used to image focal adhesions in living cells, while obtaining a temporal resolution below 10 s. We visualize the dynamics of focal adhesions, and reveal local mean velocities around 190 nm min−1. The complementarity of PALM and SOFI is assessed in detail with a methodology that integrates a resolution and signal-to-noise metric. This PALM and SOFI concept provides an enlarged quantitative imaging framework, allowing unprecedented functional exploration of focal adhesions through the estimation of molecular parameters such as fluorophore densities and photoactivation or photoswitching kinetics.

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“…Figures S1 and S7) offers a combination of super-resolution, high-speed, multi-color imaging and low photobleaching and phototoxicity that makes it well suited for studying intracellular dynamics. Compared to TIRF or TIRF-SIM, GI-SIM probes events $103 further past the sample substrate with $103 greater fluorescence intensity; compared to SDCM, it provides $23 better spatial resolution and $103 faster imaging speed; and compared to other super-resolution methods, it better balances the trade-offs between high spatial and temporal resolution and low photobleaching and phototoxicity in live-cell imaging over cellular-sized fields of view (Demmerle et al, 2015;Li et al, 2015;Tortarolo et al, 2018). Additional gains in resolution and speed beyond 97 nm at 266 frames/s can also be anticipated from continuing advancements in fluorophore photophysics (Wang et al, 2017).…”

Section: Plasticity Of Mouse Hippocampal Dendritic Spines and Dorsal mentioning

confidence: 99%

Visualizing Intracellular Organelle and Cytoskeletal Interactions at Nanoscale Resolution on Millisecond Timescales

Guo

Zhang

et al. 2018

Cell

498

488

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Assessing resolution in super-resolution imaging

Cited by 56 publications

References 32 publications

Nuclear pores as versatile reference standards for quantitative superresolution microscopy

Nuclear pores as versatile reference standards for quantitative superresolution microscopy

Complementarity of PALM and SOFI for super-resolution live-cell imaging of focal adhesions

Visualizing Intracellular Organelle and Cytoskeletal Interactions at Nanoscale Resolution on Millisecond Timescales

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