ArticlePloS one2015
Quantifying and optimizing single-molecule switching nanoscopy at high speeds.
Article in PloS one, 2015. The graph could read no effect estimate from its abstract, so it casts no vote on the map. Cited by 36 papers.
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Who cites it
36 citing papers in PubMed.
- Single molecule super resolution microscopy reveals formation of Dengue NS2B3 cluster on mitochondrial network and its effect on fragmentation.Npj viruses · 2026Article
- Objective clustering protocol for single-molecule data: A lifetime vs. intensity study.Biophysical reports · 2026Article
- Unveiling Exciton-Plasmon Polariton Coupling Regions via Polarization-Enhanced Optical Nanoscopy.Advanced science (Weinheim, Baden-Wurttemberg, Germany) · 2025Article
- Deep Learning and Single-Molecule Localization Microscopy Reveal Nanoscopic Dynamics of DNA Entanglement Loci.ACS nano · 2025Article
- StayGold variants for molecular fusion and membrane-targeting applications.Nature methods · 2024Article
- An integrated platform for high-throughput nanoscopy.Nature biotechnology · 2023Article
- openFrame: A modular, sustainable, open microscopy platform with single-shot, dual-axis optical autofocus module providing high precision and long range of operation.Journal of microscopy · 2023Article
- Ultrafast single-molecule imaging reveals focal adhesion nano-architecture and molecular dynamics.The Journal of cell biology · 2023Article
- Development of ultrafast camera-based single fluorescent-molecule imaging for cell biology.The Journal of cell biology · 2023Article
- Temporally resolved SMLM (with large PAR shift) enabled visualization of dynamic HA cluster formation and migration in a live cell.Scientific reports · 2023Article
- Fluorescence fluctuation-based super-resolution microscopy: Basic concepts for an easy start.Journal of microscopy · 2022Review
- Estimating the dynamic range of quantitative single-molecule localization microscopy.Biophysical journal · 2021Article
- Advancing biological super-resolution microscopy through deep learning: a brief review.Biophysics reports · 2021Article
- Fast widefield scan provides tunable and uniform illumination optimizing super-resolution microscopy on large fields.Nature communications · 2021Article
- Extraction of rapid kinetics from smFRET measurements using integrative detectors.Cell reports. Physical science · 2021Article
- Generalizing HMMs to Continuous Time for Fast Kinetics: Hidden Markov Jump Processes.Biophysical journal · 2021Article
- Accelerating 3D single-molecule localization microscopy using blind sparse inpainting.Journal of biomedical optics · 2021Article
- Sample Preparation and Imaging Conditions Affect mEos3.2 Photophysics in Fission Yeast Cells.Biophysical journal · 2021Article
- Single-molecule localization microscopy.Nature reviews. Methods primers · 2021Article
- Three-Dimensional Single-Molecule Localization Microscopy in Whole-Cell and Tissue Specimens.Annual review of biomedical engineering · 2020Review
Corrections and comments
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Authors and funding
15 authors.
Funding
Abstract
Single-molecule switching nanoscopy overcomes the diffraction limit of light by stochastically switching single fluorescent molecules on and off, and then localizing their positions individually. Recent advances in this technique have greatly accelerated the data acquisition speed and improved the temporal resolution of super-resolution imaging. However, it has not been quantified whether this speed increase comes at the cost of compromised image quality. The spatial and temporal resolution depends on many factors, among which laser intensity and camera speed are the two most critical parameters. Here we quantitatively compare the image quality achieved when imaging Alexa Fluor 647-immunolabeled microtubules over an extended range of laser intensities and camera speeds using three criteria - localization precision, density of localized molecules, and resolution of reconstructed images based on Fourier Ring Correlation. We found that, with optimized parameters, single-molecule switching nanoscopy at high speeds can achieve the same image quality as imaging at conventional speeds in a 5-25 times shorter time period. Furthermore, we measured the photoswitching kinetics of Alexa Fluor 647 from single-molecule experiments, and, based on this kinetic data, we developed algorithms to simulate single-molecule switching nanoscopy images. We used this software tool to demonstrate how laser intensity and camera speed affect the density of active fluorophores and influence the achievable resolution. Our study provides guidelines for choosing appropriate laser intensities for imaging Alexa Fluor 647 at different speeds and a quantification protocol for future evaluations of other probes and imaging parameters.
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Registered trials
Read under generation 80e0d062 · epoch 390. Bibliography from PubMed, PubMed Central and OpenAlex; grants from NIH RePORTER; trial links from ClinicalTrials.gov; estimates, votes and beliefs from the OpenQuestion graph.