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. 2018 Dec 7;116(2):319–329. doi: 10.1016/j.bpj.2018.11.3136

Figure 1.

Figure 1

Labeling and 3D SR imaging of the ciliary membrane. (a) SNAP-SMO proteins, where the SNAP protein is on the extracellular side, are covalently labeled with benzylguanine-Alexa647 along the ciliary membrane, which is usually found near the coverslip surface. PACT-YFP indicates the base of the cilium, and a nearby bright fiducial is used to correct for spatial drift. (b) Overlaid diffraction-limited images of the SNAP-SMO and PACT-YFP in chemically fixed control MEF cells that were treated with SAG. 3D SR microscopy using the double-helix point spread function (DH-PSF) was performed to obtain a localization map of SNAP-SMO molecules along one primary cilium, reconstructed as a (c) 2D histogram, where the bin size is 20 nm and the color bar represents the number of localizations per bin, and (d) 3D scatterplot. For SNAP-SMO distributions of other primary cilia, there is evidence of (e) kinking, (f) bulging, and (f) budding within the same control MEF cells. Scale bars, 1 μm. To see this figure in color, go online.