Artifacts in single-molecule localization microscopy

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 simplici...

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Bibliographic Details
Published in:Histochemistry and cell biology 2015-08, Vol.144 (2), p.123-131
Main Authors: Burgert, Anne, Letschert, Sebastian, Doose, Sören, Sauer, Markus
Format: Article
Language:English
Subjects:
Biochemistry
Biomedical and Life Sciences
Biomedicine
Cell Biology
Cellular biology
Cluster Analysis
Developmental Biology
Humans
Membrane Proteins - chemistry
Microscopy
Microscopy, Fluorescence
Molecular biology
Original Paper
Proteins
Stochastic Processes
Tumor Cells, Cultured
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Summary: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.
ISSN:0948-6143
1432-119X
DOI:10.1007/s00418-015-1340-4