Super-Resolution STORM Microscopy
STochastic Optical Reconstruction Microscopy (STORM) uses the ability to identify single fluorophores with ~20nm spatial resolution in order to effectively improve the resolution of the light microscope 10-fold. As an example, compare the STORM and TIRF images of the same field of actin filaments, labeled with fluorescent phalloidin and adhered to a glass surface. To reconstruct the super-resolution image, fluorophores are forced into the dark state from which they recover stochastically and fluoresce. As they fluoresce, their positions (i.e. center of point spread functions) are identified with sub-pixel resolution and the positions for all fluorophores on a single actin filament are then used to reconstruct the super-resolution image.
By placing a cylindrical lens in the optical path, an optically-induced astigmatism can be used to resolve the Z-position of a fluorophore within the focal plane. As an example, the height of fluorescently-labeled actin filaments suspended from 3µm beads can be resolved with 50nm resolution.