Synaptic FUS Localization During Motoneuron Development and Its Accumulation in Human ALS Synapses

In 1873, Ernst Abbe discovered what was to become a well-known paradigm: the inability of a lens-based optical microscope to discern details that are closer together than half of the wavelength of light. However, for its most popular imaging mode, fluorescence microscopy, the diffraction barrier is crumbling. Here, I discuss the physical concepts that have pushed fluorescence microscopy to the nanoscale, once the prerogative of electron and scanning probe microscopes. Initial applications indicate that emergent far-field optical nanoscopy will have a strong impact in the life sciences and in other areas benefiting from nanoscale visualization.

While super-resolution fluorescence microscopy is a powerful tool for biological research, obtaining multiplexed images for a large number of distinct target species remains challenging. Here we use the transient binding of short fluorescently labeled oligonucleotides (DNA-PAINT, point accumulation for imaging in nanoscale topography) for simple and easy-to-implement multiplexed 3D super-resolution imaging inside fixed cells and achieve sub-10 nm spatial resolution in vitro using synthetic DNA structures. We also report a novel approach for multiplexing (Exchange-PAINT) that allows sequential imaging of multiple targets using only a single dye and a single laser source. We experimentally demonstrate ten-“color” super-resolution imaging in vitro on synthetic DNA structures and four-“color” imaging of proteins in a fixed cell.

In DNA-PAINT, transient binding of dye-labeled oligonucleotides to their target strands creates the ‘blinking’ required for stochastic nanoscopy. This protocol describes how to apply DNA-PAINT, from sample preparation to data processing.