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 t...
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| Main Authors: | , , , , , , , , , , , , , , |
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| Format: | Article |
| Language: | English |
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Public Library of Science (PLoS)
2015-01-01
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| Series: | PLoS ONE |
| Online Access: | https://doi.org/10.1371/journal.pone.0128135 |
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| author | Yu Lin Jane J Long Fang Huang Whitney C Duim Stefanie Kirschbaum Yongdeng Zhang Lena K Schroeder Aleksander A Rebane Mary Grace M Velasco Alejandro Virrueta Daniel W Moonan Junyi Jiao Sandy Y Hernandez Yongli Zhang Joerg Bewersdorf |
| author_facet | Yu Lin Jane J Long Fang Huang Whitney C Duim Stefanie Kirschbaum Yongdeng Zhang Lena K Schroeder Aleksander A Rebane Mary Grace M Velasco Alejandro Virrueta Daniel W Moonan Junyi Jiao Sandy Y Hernandez Yongli Zhang Joerg Bewersdorf |
| author_sort | Yu Lin |
| collection | DOAJ |
| description | 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. |
| format | Article |
| id | doaj-art-800b51504f3b4364919fe0c4049a6c05 |
| institution | OA Journals |
| issn | 1932-6203 |
| language | English |
| publishDate | 2015-01-01 |
| publisher | Public Library of Science (PLoS) |
| record_format | Article |
| series | PLoS ONE |
| spelling | doaj-art-800b51504f3b4364919fe0c4049a6c052025-08-20T02:34:10ZengPublic Library of Science (PLoS)PLoS ONE1932-62032015-01-01105e012813510.1371/journal.pone.0128135Quantifying and optimizing single-molecule switching nanoscopy at high speeds.Yu LinJane J LongFang HuangWhitney C DuimStefanie KirschbaumYongdeng ZhangLena K SchroederAleksander A RebaneMary Grace M VelascoAlejandro VirruetaDaniel W MoonanJunyi JiaoSandy Y HernandezYongli ZhangJoerg BewersdorfSingle-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.https://doi.org/10.1371/journal.pone.0128135 |
| spellingShingle | Yu Lin Jane J Long Fang Huang Whitney C Duim Stefanie Kirschbaum Yongdeng Zhang Lena K Schroeder Aleksander A Rebane Mary Grace M Velasco Alejandro Virrueta Daniel W Moonan Junyi Jiao Sandy Y Hernandez Yongli Zhang Joerg Bewersdorf Quantifying and optimizing single-molecule switching nanoscopy at high speeds. PLoS ONE |
| title | Quantifying and optimizing single-molecule switching nanoscopy at high speeds. |
| title_full | Quantifying and optimizing single-molecule switching nanoscopy at high speeds. |
| title_fullStr | Quantifying and optimizing single-molecule switching nanoscopy at high speeds. |
| title_full_unstemmed | Quantifying and optimizing single-molecule switching nanoscopy at high speeds. |
| title_short | Quantifying and optimizing single-molecule switching nanoscopy at high speeds. |
| title_sort | quantifying and optimizing single molecule switching nanoscopy at high speeds |
| url | https://doi.org/10.1371/journal.pone.0128135 |
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