Artifacts in single-molecule localization microscopy

被引：65

作者：

Burgert, Anne ^{[1
]}

Letschert, Sebastian ^{[1
]}

Doose, Soeren ^{[1
]}

Sauer, Markus ^{[1
]}

机构：

[1] Univ Wurzburg, Dept Biotechnol & Biophys, D-97074 Wurzburg, Germany

来源：

HISTOCHEMISTRY AND CELL BIOLOGY | 2015年 / 144卷 / 02期

关键词：

Super-resolution microscopy; Localization microscopy; dSTORM; Artifacts; Photoswitching; NUCLEAR-PORE COMPLEX; SUPERRESOLUTION MICROSCOPY; PLASMA-MEMBRANE; REVEALS; GLYCOSYLATION; FLUORESCENCE; ORGANIZATION; PERFORMANCE;

D O I：

10.1007/s00418-015-1340-4

中图分类号：

Q2 [细胞生物学];

学科分类号：

071009 ; 090102 ;

摘要：

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.

引用

页码：123 / 131

页数：9

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