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Quantitative Multicolor Subdiffraction Imaging of Bacterial Protein Ultrastructures in Three Dimensions

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posted on 13.03.2013, 00:00 by Andreas Gahlmann, Jerod L. Ptacin, Ginni Grover, Sean Quirin, Alexander R. S. von Diezmann, Marissa K. Lee, Mikael P. Backlund, Lucy Shapiro, Rafael Piestun, W. E. Moerner
We demonstrate quantitative multicolor three-dimensional (3D) subdiffraction imaging of the structural arrangement of fluorescent protein fusions in living Caulobacter crescentus bacteria. Given single-molecule localization precisions of 20–40 nm, a flexible locally weighted image registration algorithm is critical to accurately combine the super-resolution data with <10 nm error. Surface-relief dielectric phase masks implement a double-helix response at two wavelengths to distinguish two different fluorescent labels and to quantitatively and precisely localize them relative to each other in 3D.