High-density three-dimensional localization microscopy across large volumes

作者：Legant Wesley R; Shao Lin; Grimm Jonathan B; Brown Timothy A; Milkie Daniel E; Avants Brian B; Lavis Luke D; Betzig Eric

来源：Nature Methods, 2016, 13(4): 359-365.

DOI：10.1038/NMETH.3797

Extending three-dimensional (3D) single-molecule localization microscopy away from the coverslip and into thicker specimens will greatly broaden its biological utility. However, because of the limitations of both conventional imaging modalities and conventional labeling techniques, it is a challenge to localize molecules in three dimensions with high precision in such samples white simultaneously achieving the labeling densities required for high resolution of densely crowded structures. Here we combined lattice light-sheet microscopy with newly developed, freely diffusing, cell-permeable chemical probes with targeted affinity for DNA, intracellular membranes or the plasma membrane. We used this combination to perform high localization precision, ultrahigh-labeling density, multicolor localization microscopy in samples up to 20 pin thick, including dividing cells and the neuromast organ of a zebrafish embryo. We also demonstrate super-resolution correlative imaging with protein-specific photoactivable fluorophores, providing a mutually compatible, single-platform alternative to correlative light-electron microscopy over large volumes.

• 出版日期2016-4