Fig. 3: Live cell dual colour SMLM with a protein and the membrane marker DiI to identify which clusters are caused by variations in membrane topography in HT29 cells. | Communications Biology

Fig. 3: Live cell dual colour SMLM with a protein and the membrane marker DiI to identify which clusters are caused by variations in membrane topography in HT29 cells.

From: Membrane topography and the overestimation of protein clustering in single molecule localisation microscopy – identification and correction

Fig. 3

a 2.5 × 2.5 μm SMLM 7 s snapshots of DiI and CD59 or the TfR with the proteins identified with Alexa Fluor-647 conjugated antibodies. Localizations of the protein are shown in red and the DiI in blue. Note areas of overlap appear black. b Nearest neighbour analysis and pair correlation analysis of the protein molecule to itself, to DiI and DiI to DiI. The nearest neighbour analysis is from the entire dataset. For pair correlation analysis representative results from 10 out of 25 snapshots from the entire dataset are displayed. Note that five of the six pair correlation graphs use the same y-axis range but the TfR prot-prot uses a much wider range. c 1 × 1 μm regions extracted from the larger images (marked with squares in a). Original and Gaussian filter (sigma 8 pixels) versions of all four images were normalised to the same mean intensity and displayed with the same intensity range. The final pair of images shows the result of subtraction of the DiI image from that of the relevant protein. The two Difference images use the same intensity range without any contrast alteration and the intensity range shows negative values in a greyscale.

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