We demonstrate beam scanning-stimulated emission depletion microscopy with in vivo labeled cells. A red emitting fluorescent dye is introduced into membrane protein fused to a multifunctional reporter protein (HaloTag, Promega, Madison, WI) in live cells. This approach allows superresolution stimulated emission depletion imaging without the limitations of immunofluorescence-based staining.
Noninvasive optical imaging is still an indispensable and elementary tool in modern life sciences. Unfortunately, the predominant obstacle of classical microscopes is limited spatial resolution due to the diffractive nature of light. Distinct technologies like electron and x-ray microscopy as well as near-field approaches evolved during the past decades to circumvent this major limitation. Furthermore, various techniques emerged in recent years breaking the diffraction resolution barrier of far-field optical microscopy. In particular, stimulated emission depletion (STED) microscopy became a breakthrough technologyfor fluorescence imaging and is now available commercially (see Supplemetary Material). Commercial systems are fully integrated in optimized multispectral, high sensitivity confocal scanning microscopes for all needs of modern life science research. Since STED microscopy has evolved from high-end technology in specialist's labs to a reliable and easy-to-use instrument for life science, the expected widespread use of STED certainly will push the technology as well as applications to a next level.