3d structured illumination microscopy (sim) system  (Applied Precision Inc)

Journal: Nucleic Acids Research

Article Title: Mutational analysis of the F plasmid partitioning protein ParA reveals residues required for oligomerization and plasmid maintenance

doi: 10.1093/nar/gkaf537

Figure Lengend Snippet: Residues Q351 and W362 influence nsDNA binding by ParA F . ( A ) Filaments formed by ParA F Q351H–GFP and ParA F W362E–GFP are not nucleoid associated. The ParA F Q351H–GFP and ParA F W362E–GFP filaments appeared to be localized in the space between the nucleoid and the inner membrane, while ParA F –GFP localized over the nucleoids. Nucleoids were stained with DAPI, and the plasma membrane was stained using FM4-64. Scale bars represent 3 μm. ( B ) Super-resolution imaging using 3D-SIM showing that the filaments assembled by (i) ParA F Q351H–GFP and (ii) ParA F W362E–GFP are not associated with the nucleoid. DNA was stained with DAPI and pseudo-coloured red. Cell outlines were obtained from the phase contrast images. Scale bars represent 1 μm. ( C ) Filaments formed by ParA F Q351H–GFP and ParA F W362E–GFP are found in the nucleoid-free spaces in the cytoplasm of E. coli . WT ParA F –GFP localized to the nucleoid. Nucleoids were visualized by staining with DAPI and pseudo-coloured red in the images. Cells were treated with 25 μg/ml cephalexin for 30 min to prevent cell division and 100 μg/ml chloramphenicol for 10 min to condense the nucleoids so that the nucleoid-free space appears pronounced. Scale bars represent 3 μm. Expression of ParA F –GFP or its variants (from pDSW210) for all the above experiments was achieved by adding 100 μM IPTG to the cultures.

Article Snippet: 3D structured illumination microscopy (3D-SIM) imaging was carried out using a DeltaVision OMX-SR Blaze microscope equipped with a PCO Edge 4.2 sCMOS camera, and a ×60, NA 1.42 oil-immersion objective lens was used to acquire raw images.

Techniques: Binding Assay, Membrane, Staining, Clinical Proteomics, Imaging, Expressing