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Single-molecule localization microscopy (SMLM) relies on the temporally separated emission of individual molecules. While the acquired image is still diffraction limited, due to their sparsity, each molecule can be localized at nanometer precision and their coordinates accumulated to form a super-resolved image. But the power of SMLM goes far beyond generating images. Computational analysis tools allow extracting the spatial organization, colocalization, clustering, and dynamics of individual molecules, which has transformed the study of nanoscale biology over the past decade. This wiki gives an introduction to SMLM and mostly practical information to get you started with your own experiments or data analysis.
This page describes how to perform a single-molecule calibration experiment and provides some scripts to extract quantitative information. I provide a test dataset of Alexa647-labelled antibodies blinking in COT STORM buffer.
Here you'll see how to perform a basic DBSCAN clustering of SMLM data.
The tracking page describes some simple analysis steps for sptPALM data.
A number of excellent open-source software platforms are available trough the SMLM community. While I provide some of my personal analysis scripts in this repository, I summarize what else is available and give main applications.