The fidelity of stochastic single-molecule super-resolution reconstructions critically depends upon robust background estimation.
The quality of super resolution images obtained by stochastic single-molecule microscopy critically depends on image analysis algorithms. We find that the choice of background estimator is often the most important determinant of reconstruction quality. A variety of techniques have found use, but many have a very narrow range of applicability depending upon the characteristics of the raw data. Importantly, we observe that when using otherwise accurate algorithms, unaccounted background components can give rise to biases on scales defeating the purpose of super-resolution microscopy. We find that a temporal median filter in particular provides a simple yet effective solution to the problem of background estimation, which we demonstrate over a range of imaging modalities and different reconstruction methods.
1] Section of Molecular Cytology and Van Leeuwenhoek Centre of Advanced Microscopy, Swammerdam Institute for Life Sciences, University of Amsterdam Science Park 904, NL-1098 XH Amsterdam The Netherlands [2].
Eelco
E
Hoogendoorn
1] Section of Molecular Cytology and Van Leeuwenhoek Centre of Advanced Microscopy, Swammerdam Institute for Life Sciences, University of Amsterdam Science Park 904, NL-1098 XH Amsterdam The Netherlands [2] [3].
Kevin C
KC
Crosby
1] Division of Cell Biology and Van Leeuwenhoek Centre of Advanced Microscopy, The Netherlands Cancer Institute, Amsterdam, The Netherlands [2].
Daniela
D
Leyton-Puig
Section of Molecular Cytology and Van Leeuwenhoek Centre of Advanced Microscopy, Swammerdam Institute for Life Sciences, University of Amsterdam Science Park 904, NL-1098 XH Amsterdam The Netherlands.
Ronald M P
RM
Breedijk
Division of Cell Biology and Van Leeuwenhoek Centre of Advanced Microscopy, The Netherlands Cancer Institute, Amsterdam, The Netherlands.
Kees
K
Jalink
Section of Molecular Cytology and Van Leeuwenhoek Centre of Advanced Microscopy, Swammerdam Institute for Life Sciences, University of Amsterdam Science Park 904, NL-1098 XH Amsterdam The Netherlands.
Theodorus W J
TW
Gadella
Section of Molecular Cytology and Van Leeuwenhoek Centre of Advanced Microscopy, Swammerdam Institute for Life Sciences, University of Amsterdam Science Park 904, NL-1098 XH Amsterdam The Netherlands.
Marten
M
Postma
D000199:Actins / Q000648:ultrastructure; D000465:Algorithms; D002232:Carbocyanines; D045744:Cell Line, Tumor; D005456:Fluorescent Dyes; D006367:HeLa Cells; D006801:Humans; D007089:Image Enhancement / Q000379:methods*; D007090:Image Interpretation, Computer-Assisted / Q000379:methods*; D024507:Nonmuscle Myosin Type IIA / Q000648:ultrastructure; D062345:Nuclear Microscopy / Q000379:methods*; D016596:Vinculin / Q000648:ultrastructure
D000199:Actins; C569686:Alexa Fluor 647; D002232:Carbocyanines; D005456:Fluorescent Dyes; D016596:Vinculin; D024507:Nonmuscle Myosin Type IIA