Histochemistry and Cell Biology

, Volume 144, Issue 2, pp 123–131 | Cite as

Artifacts in single-molecule localization microscopy

Original Paper


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.


Super-resolution microscopy Localization microscopy dSTORM Artifacts Photoswitching 

Supplementary material

418_2015_1340_MOESM1_ESM.avi (889 kb)
dSTORM movie_1Microtubules labeled U2OS cells shown in Fig. 1 excited at 641 nm with ~7 kW cm−2 at a frame rate of 50 Hz (AVI 889 kb)
418_2015_1340_MOESM2_ESM.avi (1.5 mb)
dSTORM movie_2Microtubules labeled U2OS cells shown in Fig. 1 excited at 641 nm with ~0.3 kW cm−2 at a frame rate of 50 Hz. (AVI 1574 kb)
418_2015_1340_MOESM3_ESM.avi (1.5 mb)
dSTORM movie_3Microtubules labeled U2OS cells shown in Fig. 1 excited at 641 nm with ~0.1 kW cm−2 at a frame rate of 50 Hz. (AVI 1585 kb)
418_2015_1340_MOESM4_ESM.avi (1.8 mb)
dSTORM movie_4Alexa Fluor 647-WGA-labeled glycans in the basal plasma membrane of U2OS cells shown in Fig. 1 excited at 641 nm with ~7 kW cm−2 at a frame rate of 50 Hz. (AVI 1879 kb)
418_2015_1340_MOESM5_ESM.avi (2.3 mb)
dSTORM movie_5Alexa Fluor 647-WGA-labeled glycans in the basal plasma membrane of U2OS cells shown in Fig. 1 excited at 641 nm with ~0.3 kW cm−2 at a frame rate of 50 Hz. (AVI 2394 kb)
418_2015_1340_MOESM6_ESM.avi (2.4 mb)
dSTORM movie_6Alexa Fluor 647-WGA-labeled glycans in the basal plasma membrane of U2OS cells shown in Fig. 1 excited at 641 nm with ~0.1 kW cm−2 at a frame rate of 50 Hz. (AVI 2456 kb)


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Copyright information

© Springer-Verlag Berlin Heidelberg 2015

Authors and Affiliations

  1. 1.Department of Biotechnology and BiophysicsUniversity WürzburgWürzburgGermany

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