Registration and Visualization of Correlative Super-Resolution Microscopy Data

We introduce a method for registration and visualization of correlative super-resolution microscopy images from different microscopy techniques. We established an automated registration procedure based on the generalized Hough transform. We developed a software tool to apply this algorithm and visualize correlated images from structured illumination microscopy (SIM) and direct stochastic optical reconstruction microscopy (dSTORM). To demonstrate the potential of this super-resolution correlator, we visualize the distribution of the presynaptic protein bassoon in the active zones of synapses in the molecular layer of the mouse cerebellum. First, a multiple labeled sample is imaged by SIM, followed by imaging of one of the fluorescent labels by dSTORM. To avoid the use of artificial fiducial markers, we used the signal of Alexa Fluor 647 recorded in switching buffer on the two microscopes for image superposition. We recorded multicolor SIM images in 20-μm thick brain slices to identify synapses in the dendritic system of Purkinje cells and put higher-resolved dSTORM images of the synaptic distribution of bassoon in registry.