title: Quantifying and optimizing single-molecule switching nanoscopy at high speeds.
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.
authors.affiliation: Department of Cell Biology, Yale University School of Medicine, New Haven, Connecticut, United States of America; Department of Biomedical Engineering, Yale University, New Haven, Connecticut, United States of America; Integrated Graduate Program in Physical and Engineering Biology, Yale University, New Haven, Connecticut, United States of America.
authors.forename: Yu
authors.initials: Y
authors.lastname: Lin
authors.affiliation: Yale College, Yale University, New Haven, Connecticut, United States of America.
authors.forename: Jane J
authors.initials: JJ
authors.lastname: Long
authors.affiliation: Department of Cell Biology, Yale University School of Medicine, New Haven, Connecticut, United States of America.
authors.forename: Fang
authors.initials: F
authors.lastname: Huang
authors.affiliation: Department of Cell Biology, Yale University School of Medicine, New Haven, Connecticut, United States of America.
authors.forename: Whitney C
authors.initials: WC
authors.lastname: Duim
authors.affiliation: Institute for Molecular Biophysics, The Jackson Laboratory, Bar Harbor, Maine, United States of America.
authors.forename: Stefanie
authors.initials: S
authors.lastname: Kirschbaum
authors.affiliation: Department of Cell Biology, Yale University School of Medicine, New Haven, Connecticut, United States of America.
authors.forename: Yongdeng
authors.initials: Y
authors.lastname: Zhang
authors.affiliation: Department of Cell Biology, Yale University School of Medicine, New Haven, Connecticut, United States of America.
authors.forename: Lena K
authors.initials: LK
authors.lastname: Schroeder
authors.affiliation: Integrated Graduate Program in Physical and Engineering Biology, Yale University, New Haven, Connecticut, United States of America; Department of Physics, Yale University, New Haven, Connecticut, United States of America.
authors.forename: Aleksander A
authors.initials: AA
authors.lastname: Rebane
authors.affiliation: Department of Cell Biology, Yale University School of Medicine, New Haven, Connecticut, United States of America; Department of Biomedical Engineering, Yale University, New Haven, Connecticut, United States of America; Integrated Graduate Program in Physical and Engineering Biology, Yale University, New Haven, Connecticut, United States of America.
authors.forename: Mary Grace M
authors.initials: MG
authors.lastname: Velasco
authors.affiliation: Integrated Graduate Program in Physical and Engineering Biology, Yale University, New Haven, Connecticut, United States of America; Department of Mechanical Engineering and Material Science, Yale University, New Haven, Connecticut, United States of America.
authors.forename: Alejandro
authors.initials: A
authors.lastname: Virrueta
authors.affiliation: Integrated Graduate Program in Physical and Engineering Biology, Yale University, New Haven, Connecticut, United States of America; Department of Molecular Biophysics and Biochemistry, Yale University, New Haven, Connecticut, United States of America.
authors.forename: Daniel W
authors.initials: DW
authors.lastname: Moonan
authors.affiliation: Department of Cell Biology, Yale University School of Medicine, New Haven, Connecticut, United States of America; Integrated Graduate Program in Physical and Engineering Biology, Yale University, New Haven, Connecticut, United States of America.
authors.forename: Junyi
authors.initials: J
authors.lastname: Jiao
authors.affiliation: Department of Biomedical Engineering, Yale University, New Haven, Connecticut, United States of America; Integrated Graduate Program in Physical and Engineering Biology, Yale University, New Haven, Connecticut, United States of America.
authors.forename: Sandy Y
authors.initials: SY
authors.lastname: Hernandez
authors.affiliation: Department of Cell Biology, Yale University School of Medicine, New Haven, Connecticut, United States of America; Integrated Graduate Program in Physical and Engineering Biology, Yale University, New Haven, Connecticut, United States of America.
authors.forename: Yongli
authors.initials: Y
authors.lastname: Zhang
authors.affiliation: Department of Cell Biology, Yale University School of Medicine, New Haven, Connecticut, United States of America; Department of Biomedical Engineering, Yale University, New Haven, Connecticut, United States of America; Integrated Graduate Program in Physical and Engineering Biology, Yale University, New Haven, Connecticut, United States of America.
authors.forename: Joerg
authors.initials: J
authors.lastname: Bewersdorf
mesh_terms: D000465:Algorithms; D000818:Animals; D019556:COS Cells; D002522:Chlorocebus aethiops; D007091:Image Processing, Computer-Assisted / Q000379:methods*; D008856:Microscopy, Fluorescence; D018715:Microscopy, Video; D008870:Microtubules / Q000648:ultrastructure; D036103:Nanotechnology / Q000379:methods*; D012984:Software