Artifacts in single-molecule localization microscopy

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• 作者：Anne Burgert ; Sebastian Letschert ; S?ren Doose…
• 关键词：Super ; resolution microscopy ; Localization microscopy ; dSTORM ; Artifacts ; Photoswitching
• 刊名：Histochemistry and Cell Biology
• 出版年：2015
• 出版时间：August 2015
• 年：2015
• 卷：144
• 期：2
• 页码：123-131
• 全文大小：2,852 KB
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• 作者单位：Anne Burgert (1)
  Sebastian Letschert (1)
  S?ren Doose (1)
  Markus Sauer (1)
  
  1. Department of Biotechnology and Biophysics, University Würzburg, Am Hubland, 97074, Würzburg, Germany
  
• 刊物类别：Medicine
• 刊物主题：Medicine & Public Health
  Anatomy
  Medicine/Public Health, general
  
• 出版者：Springer Berlin / Heidelberg
• ISSN：1432-119X

文摘

Single-molecule localization microscopy provides subdiffraction resolution images with virtually molecular resolution. Through the availability of commercial instruments and open-source reconstruction software, achieving super resolution is now public domain. However, despite its conceptual simplicity, localization microscopy remains prone to user errors. Using direct stochastic optical reconstruction microscopy, we investigate the impact of irradiation intensity, label density and photoswitching behavior on the distribution of membrane proteins in reconstructed super-resolution images. We demonstrate that high emitter densities in combination with inappropriate photoswitching rates give rise to the appearance of artificial membrane clusters. Especially, two-dimensional imaging of intrinsically three-dimensional membrane structures like microvilli, filopodia, overlapping membranes and vesicles with high local emitter densities is prone to generate artifacts. To judge the quality and reliability of super-resolution images, the single-molecule movies recorded to reconstruct the images have to be carefully investigated especially when investigating membrane organization and cluster analysis.