Supplementary Figure 10: Single molecule signal quality control across an entire 96-well plate for PALM imaging. | Nature Methods

Supplementary Figure 10: Single molecule signal quality control across an entire 96-well plate for PALM imaging.

From: Localization-based super-resolution imaging meets high-content screening

Supplementary Figure 10

(a) Example of single molecule localization quality control. Here, acquired positions whose photon distributions demonstrate a strong (above 10%) population of multi-emitter detections are automatically excluded. The limit between single and multi-emitter is defined on 10 distributions of isolated proteins. White arrows indicate multi-emitter signals due to a too high density of proteins inside the cell. (b) Plate layout of a control assay on p96-well plate with purified mEos3.2 protein adsorbed to the glass (4 dark grey wells on plate corners). Ten random positions per well were acquired and the histogram represents the cumulative diffusion coefficient metadata. The threshold between mobile and immobile molecules was set to 10-2 um²/sec (see Online Methods). Only a weak fraction of mobile tracks (10%) were measured, indicating good stability of the plate on the stage (no drift, no deformation) during the screening process. (c) Left: Plate layout of a control assay with living HeLa cells expressing SEP::GluA1::mEos2 across 60 wells of a p96-well plate. Three positions per well were acquired and the diffusion coefficients pooled in one histogram per well. Right: Histogram of 6 representative wells (corresponding to dark grey wells), showing similar DCoef distributions and fractions of immobile tracks (N=180 cells).

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