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Histochem Cell Biol. 2014 Jul;142(1):5-17. doi: 10.1007/s00418-014-1217-y. Epub 2014 Apr 20.

Progress in quantitative single-molecule localization microscopy.

Author information

1
Laboratory of Nanoscale Biology, Institute of Bioengineering, School of Engineering, EPFL, Lausanne, Switzerland.

Abstract

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).

PMID:
24748502
PMCID:
PMC4072926
DOI:
10.1007/s00418-014-1217-y
[Indexed for MEDLINE]
Free PMC Article

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