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Challenges in quantitative single molecule localization microscopy
Author(s) -
Shivanandan A.,
Deschout H.,
Scarselli M.,
Radenovic A.
Publication year - 2014
Publication title -
febs letters
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.593
H-Index - 257
eISSN - 1873-3468
pISSN - 0014-5793
DOI - 10.1016/j.febslet.2014.06.014
Subject(s) - microscopy , fluorescence microscope , super resolution microscopy , quantitative biology , focus (optics) , photoactivated localization microscopy , superresolution , structural biology , resolution (logic) , nanotechnology , biophysics , biological system , computational biology , fluorescence , chemistry , computer science , physics , biology , materials science , optics , artificial intelligence , biochemistry , image (mathematics)
Single molecule localization microscopy (SMLM), which can provide up to an order of magnitude improvement in spatial resolution over conventional fluorescence microscopy, has the potential to be a highly useful tool for quantitative biological experiments. It has already been used for this purpose in varied fields in biology, ranging from molecular biology to neuroscience. In this review article, we briefly review the applications of SMLM in quantitative biology, and also the challenges involved and some of the solutions that have been proposed. Due to its advantages in labeling specificity and the relatively low overcounting caused by photoblinking when photo‐activable fluorescent proteins (PA‐FPs) are used as labels, we focus specifically on Photo‐Activated Localization Microscopy (PALM), even though the ideas presented might be applicable to SMLM in general. Also, we focus on the following three quantitative measurements: single molecule counting, analysis of protein spatial distribution heterogeneity and co‐localization analysis.