Figure 4

Single molecule localisation microscopy of Alexa647 in SIM-dedicated mounting media. A549 human pulmonary adherent cells were labelled for Lamin B1 using Alexa647-conjugated antibody and imaged under exposure to high intensity exciting light using a microscope suited for single molecule detection. (A) Collective histograms of photon counts for single molecule signals of Alexa647 acquired in 5 independent measurements (movies) after embedding in different SIM-dedicated mounting media (see Figs 1–3 for more details) with respective average values presented in a bar-plot in (B). See Supplementary Fig. S14 for respective data for standard SMLM mounting media based on cysteamine. (C) Collective histograms of corresponding localisation uncertainties with respective average values in (D). SIM-dedicated mounting media containing 20% vol/vol Vectashield were also compared to mounting media comprising 40% vol/vol mounting media (H71VE40, S65VE40 in B and D). See Supplementary Fig. S13 for respective transmitted light images of the cells embedded in a variety of media. (E,F) Examples of SMLM reconstructions of Lamin B1 – Alexa647 labelled A549 cells embedded in S65VE (E, nucleus bottom) and Sorb70VE (F, equatorial part of cell’s nucleus). Conventional wide-field images are presented in grey whereas SMLM reconstructions are colour-coded using a “red hot” look-up table. 2.5 × 3.0 µm² region of interest (white dashed box) is enlarged and shown on the right-hand side of E and F.