Multicolor Super-Resolution Imaging using a Laser Line Scanning Hyperspectral Microscope

Single-molecule super-resolution imaging techniques have proven to be successful in observing cellular structures with a resolution below the diffraction limit. However, these experiments often restrict observations to a region of shallow z-depth near the cover slip due to TIRF imaging or else suffer from high backgrounds due to wide field excitation. Confocal microscopy, which offers good background rejection, is prohibitively slow and cannot be used effectively for single-molecule localization based super-resolution. Here we introduce a new method that uses a fast, laser line scanning system that can be used to image a z-section anywhere within a cell and is not restricted to regions near the cover slip. The line scanning system allows a trade-off between acquisition speed and background rejection. Emission light is passed through a prism spectrometer, and a complete spectrum is collected for each spatial pixel. We exploit this hyperspectral emission path to make simultaneous measurements of multiple fluorophore species in the same sample.