Quantifying and optimizing single-molecule switching nanoscopy at high speeds.

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

Use of High-Resolution Geospatial and Genomic Data to Characterize Recent Tuberculosis Transmission, Botswana.

Combining genomic and geospatial data can be useful for understanding Mycobacterium tuberculosis transmission in high-burden tuberculosis (TB) settings. We performed whole-genome sequencing on M. tuberculosis DNA extracted from sputum cultures from a population-based TB study conducted in Gaborone, Botswana, during 2012-2016. We determined spatial distribution of cases on the basis of shared genotypes among isolates. We considered clusters of isolates with ≤5 single-nucleotide polymorphisms identified by whole-genome sequencing to indicate recent transmission and clusters of ≥10 persons to be outbreaks. We obtained both molecular and geospatial data for 946/1,449 (65%) participants with culture-confirmed TB; 62 persons belonged to 5 outbreaks of 10-19 persons each. We detected geospatial clustering in just 2 of those 5 outbreaks, suggesting heterogeneous spatial patterns. Our findings indicate that targeted interventions applied in smaller geographic areas of high-burden TB identified using integrated genomic and geospatial data might help interrupt TB transmission during outbreaks

Quantifying and Optimizing Single-Molecule Switching Nanoscopy at High Speeds

<div><p>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.</p></div

Effect of photoswitching dynamics on the density of active fluorophores.

<p>(<b>A, B</b>) Fraction of Alexa Fluor 647 molecules in the ON-state (the singlet state, S) and the OFF-states (the triplet state, T, the dark state, D and the long-lived dark state, LLD) over time upon irradiation with 31 kW/cm² (<b>A</b>) and 2 kW/cm² (<b>B</b>) of 642-nm light. The horizontal black lines mark the threshold for the optimal fraction of fluorophores in the ON-state, which corresponds to the optimal density of active fluorophores (1 active fluorophore per 700 nm length of microtubule) in the case of an artificial microtubule as illustrated in <b><a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0128135#pone.0128135.g008" target="_blank">Fig 8 A</a></b>. (<b>C</b>) The fraction of fluorophores in the ON-state at equilibrium at different excitation intensities.</p

Effect of laser intensity on Alexa Fluor 647 photoswitching kinetics.

<p>(<b>A</b>) Model of Alexa Fluor 647 photoswitching mechanism [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0128135#pone.0128135.ref020" target="_blank">20</a>, <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0128135#pone.0128135.ref021" target="_blank">21</a>]. Upon irradiation, the fluorophore can undergo intersystem crossing and switch from the fluorescence-emitting ON-state to the triplet state with rate <i>k</i>_<i>12</i>. The triplet state (T) can either recover to the singlet ground state or react with a thiolate to form the radical anion of the fluorophore (dark state, D). The dark state can be oxidized to recover to the singlet ground state or form a thiol adduct [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0128135#pone.0128135.ref022" target="_blank">22</a>] (long-lived dark state, LLD). (<b>B—G</b>) Photoswitching rates at different excitation laser intensities extracted from single-molecule experiments. At low intensities (1.0–16 kW/cm², blue dots), data were recorded at 200 fps to allow for high temporal resolution and high signal-to-noise ratio. At high intensities (31–97 kW/cm², red triangles), data were recorded at 800 fps because the ON-state lifetime is reduced to a few milliseconds and requires higher temporal resolution.</p

Effect of photobleaching on localization density at different laser intensities.

<p>(<b>A</b>) Localization density accumulated with three pairs of imaging parameters over long term imaging (160,000–400,000 frames per data set). (<b>B, C</b>) Total number of SMSN images obtained before the spatiotemporal resolution decreases due to photobleaching. (<b>B</b>) When imaging at 3.9 kW/cm² and 50 fps with 405-nm light, 46 SMSN images were generated with a localization density of ~800 localizations per 1 μm MT and temporal resolution of 100 s per image. (<b>C</b>) When imaging at 31 kW/cm² and 800 fps with 405-nm light, 37 SMSN images were generated with the same localization density and improved temporal resolution of 5 s per image. Scale bars: 1 μm.</p

The influence of single-molecule switching kinetics and camera frame rate on SMSN imaging.

<p>A schematic shows the accumulation of localized molecules over time for different scenarios. The fluorescence emission (red circles) of each molecule labeling a ring-like structure is fit to yield its position (black dots). Both fast switching kinetics and high camera speed are required for the most efficient localization of molecules (case III).</p

Image resolution comparison of high-speed and conventional SMSN.

<p>(<b>A</b>) FRC resolution values calculated from SMSN images reconstructed from 20,000 raw camera frames acquired at different camera speeds and laser intensities. (<b>B</b>, <b>C</b>) Example SMSN images of Alexa Fluor 647-immunolabeled microtubules from conventional (purple arrow, <b>B</b>) and high-speed SMSN (red arrow, <b>C</b>) demonstrate comparable image quality with ~40 nm FRC resolution. Insets show further magnification of the white box areas respectively. The images are reconstructed from data acquired in 160 s at 50 fps and 3.9 kW/cm² with a density of 1,572 localizations per 1 μm MT (<b>B</b>), and data acquired in 10 s at 800 fps and 62 kW/cm² with a density of 1,091 localizations per 1 μm MT (<b>C</b>). Scale bars: 1 μm; inset 100 nm.</p

Simulation reproduces experimentally-observed dependence of localization density on laser intensity and frame rate.

<p>Localization density obtained in the first 1 s of data acquisition from both experiment (solid lines; extracted from the same raw data as <b><a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0128135#pone.0128135.g003" target="_blank">Fig 3A</a></b>) and simulation (dash-dot lines) data. Alexa Fluor 647 photoswitching model and switching rates used in the simulation were reported in <b><a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0128135#pone.0128135.g006" target="_blank">Fig 6</a></b>. The structure of artificial microtubules used in our simulations is illustrated in <b><a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0128135#pone.0128135.g008" target="_blank">Fig 8A</a></b>.</p