High density three-dimensional localization microscopy across large volumes

Extending three-dimensional (3D) single molecule localization microscopy away from the coverslip and into thicker specimens will greatly broaden its biological utility. However, localizing molecules in 3D with high precision in such samples, while simultaneously achieving the extreme labeling densities required for high resolution of densely crowded structures is challenging due to the limitations both of conventional imaging modalities and of conventional labeling techniques. Here, we combine lattice light sheet microscopy with newly developed, freely diffusing, cell permeable chemical probes with targeted affinity towards either DNA, intracellular membranes, or the plasma membrane. We use this combination to perform high localization precision, ultra-high labeling density, multicolor localization microscopy in samples up to 20 microns 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.