TY - JOUR
T1 - Progress in quantitative single-molecule localization microscopy
AU - Deschout, H.
AU - Shivanandan, A.
AU - Annibale, P.
AU - Scarselli, M.
AU - Radenovic, A.
N1 - Funding Information:
Acknowledgments This work was financially supported by FNS grants No. 200021–125319 and No. 20021–132206. A. Shivanandan was funded by a PhD fellowship grant from NCCBI.
PY - 2014/7
Y1 - 2014/7
N2 - With the advent of single-molecule localization microscopy (SMLM) techniques, intracellular proteins can be imaged at unprecedented resolution with high specificity and contrast. These techniques can lead to a better understanding of cell functioning, as they allow, among other applications, counting the number of molecules of a protein specie in a single cell, studying the heterogeneity in protein spatial organization, and probing the spatial interactions between different protein species. However, the use of these techniques for accurate quantitative measurements requires corrections for multiple inherent sources of error, including: overcounting due to multiple localizations of a single fluorophore (i.e., photoblinking), undercounting caused by incomplete photoconversion, uncertainty in the localization of single molecules, sample drift during the long imaging time, and inaccurate image registration in the case of dual-color imaging. In this paper, we review recent efforts that address some of these sources of error in quantitative SMLM and give examples in the context of photoactivated localization microscopy (PALM).
AB - With the advent of single-molecule localization microscopy (SMLM) techniques, intracellular proteins can be imaged at unprecedented resolution with high specificity and contrast. These techniques can lead to a better understanding of cell functioning, as they allow, among other applications, counting the number of molecules of a protein specie in a single cell, studying the heterogeneity in protein spatial organization, and probing the spatial interactions between different protein species. However, the use of these techniques for accurate quantitative measurements requires corrections for multiple inherent sources of error, including: overcounting due to multiple localizations of a single fluorophore (i.e., photoblinking), undercounting caused by incomplete photoconversion, uncertainty in the localization of single molecules, sample drift during the long imaging time, and inaccurate image registration in the case of dual-color imaging. In this paper, we review recent efforts that address some of these sources of error in quantitative SMLM and give examples in the context of photoactivated localization microscopy (PALM).
KW - Cluster analysis
KW - Co-localization
KW - Fluorescent protein
KW - Photoactivated localization microscopy (PALM)
KW - Quantitative microscopy
KW - Single-molecule counting
KW - Single-molecule localization microscopy (SMLM)
UR - http://www.scopus.com/inward/record.url?scp=84903750507&partnerID=8YFLogxK
U2 - 10.1007/s00418-014-1217-y
DO - 10.1007/s00418-014-1217-y
M3 - Review article
C2 - 24748502
AN - SCOPUS:84903750507
SN - 0948-6143
VL - 142
SP - 5
EP - 17
JO - Histochemistry and Cell Biology
JF - Histochemistry and Cell Biology
IS - 1
ER -