Ground state depletion microscopy followed by individual molecule return (GSDIM) is a super-resolution technique based on single molecule localization. To build a high-resolution image the ensemble of overlapping fluorophores in the diffraction-limited image has to be temporally “separated” to allow the high precision detection of single dyes. To achieve the optimal (super-)resolution the overall system, including the biological samples as well as optical, electronical and mechanical components, needs to be optimally integrated. Commercial solutions for GSD take care of the microscope components, and it is up to the researcher to prepare the sample in an optimal manner.
This video tutorial presents a reliable way to produce samples for super-resolution GSD imaging with a special focus on the mounting step. Stable and flat mounting of the coverslip increases the performance of the overall system, leading to an improved resolution of the GSD image.