Counting
Counting means estimating absolute molecule numbers and can basically be performed with many different SMLM techniques, but with varying precision. Below I describe a simple counting procedure using STORM. An important pre-requisite for all counting approaches in SMLM is the calibration of the label. Since our protein of interest is labeled with a fluorescent molecule in one way or the other, it is critical to precisely know how such a molecule is recorded using our method. This is important to separate the behavior of the dye from the protein of interest. The simplest way to do this is to image the fluorescent reporter without the protein of interest and in known numbers, as described in the Analyze Photophysics section.
Analyzing the dye photophysics will result in two parameters - the gap time and the localization precision - required to group/merge our experimental data (see A and B below). The procedure will merge a number of localizations into a single position, which can be counted. A grouped/merged dataset can then be further processed for example by performing a clustering operation to retrieve molecule cluster, now with absolute molecule estimates.
The described analysis was performed here on EGFR STORM data and here on CD4 STORM data.
under construction