TY - JOUR
T1 - Correction of multiple-blinking artifacts in photoactivated localization microscopy
AU - Jensen, Louis G.
AU - Hoh, Tjun Yee
AU - Williamson, David J.
AU - Griffié, Juliette
AU - Sage, Daniel
AU - Rubin-Delanchy, Patrick
AU - Owen, Dylan M.
N1 - Funding Information:
L.G.J. was supported by the Center for Stochastic Geometry and Advanced Bioimaging, funded by grant no. 8721 from the Villum Foundation. D.O. acknowledges funding from the Biotechnology and Biological Sciences Research Council (BBSRC) grant no. BB/R007365/1. P.R.D. acknowledges funding from the BBSRC grant no. BB/R007837/1. We also acknowledge the use of the King’s College London Nikon Imaging Center. We thank U. Hahn (Aarhus University) for motivating this project in initial discussions. We acknowledge J. Ries and the European Molecular Biology Laboratory for supplying NPC data.
Funding Information:
L.G.J. was supported by the Center for Stochastic Geometry and Advanced Bioimaging, funded by grant no. 8721 from the Villum Foundation. D.O. acknowledges funding from the Biotechnology and Biological Sciences Research Council (BBSRC) grant no. BB/R007365/1. P.R.D. acknowledges funding from the BBSRC grant no. BB/R007837/1. We also acknowledge the use of the King’s College London Nikon Imaging Center. We thank U. Hahn (Aarhus University) for motivating this project in initial discussions. We acknowledge J. Ries and the European Molecular Biology Laboratory for supplying NPC data.
Publisher Copyright:
© 2022, The Author(s), under exclusive licence to Springer Nature America, Inc.
PY - 2022/5
Y1 - 2022/5
N2 - Photoactivated localization microscopy (PALM) produces an array of localization coordinates by means of photoactivatable fluorescent proteins. However, observations are subject to fluorophore multiple blinking and each protein is included in the dataset an unknown number of times at different positions, due to localization error. This causes artificial clustering to be observed in the data. We present a ‘model-based correction’ (MBC) workflow using calibration-free estimation of blinking dynamics and model-based clustering to produce a corrected set of localization coordinates representing the true underlying fluorophore locations with enhanced localization precision, outperforming the state of the art. The corrected data can be reliably tested for spatial randomness or analyzed by other clustering approaches, and descriptors such as the absolute number of fluorophores per cluster are now quantifiable, which we validate with simulated data and experimental data with known ground truth. Using MBC, we confirm that the adapter protein, the linker for activation of T cells, is clustered at the T cell immunological synapse.
AB - Photoactivated localization microscopy (PALM) produces an array of localization coordinates by means of photoactivatable fluorescent proteins. However, observations are subject to fluorophore multiple blinking and each protein is included in the dataset an unknown number of times at different positions, due to localization error. This causes artificial clustering to be observed in the data. We present a ‘model-based correction’ (MBC) workflow using calibration-free estimation of blinking dynamics and model-based clustering to produce a corrected set of localization coordinates representing the true underlying fluorophore locations with enhanced localization precision, outperforming the state of the art. The corrected data can be reliably tested for spatial randomness or analyzed by other clustering approaches, and descriptors such as the absolute number of fluorophores per cluster are now quantifiable, which we validate with simulated data and experimental data with known ground truth. Using MBC, we confirm that the adapter protein, the linker for activation of T cells, is clustered at the T cell immunological synapse.
UR - http://www.scopus.com/inward/record.url?scp=85129858088&partnerID=8YFLogxK
U2 - 10.1038/s41592-022-01463-w
DO - 10.1038/s41592-022-01463-w
M3 - Article
AN - SCOPUS:85129858088
SN - 1548-7091
VL - 19
SP - 594
EP - 602
JO - NATURE METHODS
JF - NATURE METHODS
IS - 5
ER -