Superresolution and Single Molecule Imaging of Transcription by Reflected Light Sheet Microscopy

Understanding gene expression in mammalian cells requires techniques that allow to probe abundance, dynamics and distribution of the key molecular players such as transcription factors, RNA Polymerase and RNA inside the nucleus. To circumvent ensemble averaging of biomolecular dynamics, single molecule sensitivity inside the cell is essential, while superresolution imaging beyond the diffraction limit is needed to resolve the structural architecture of macromolecular assemblies involved in transcription.We achieve both high temporal and spatial resolution in single cells using a new variant of light sheet microscopy. This new technique, Reflected Light Sheet Microscopy, employs a small mirror near the cell to project a light sheet into the nucleus. This optical sectioning reduces out-of-focus background and allows video-rate detection of individual fluorescent proteins and superresolved STORM imaging in mammalian nuclei.In live cells, tracking of single transcription factors labeled with a fluorescent protein reveals the kinetics of free diffusion as well as non specific and specific binding to DNA. Furthermore, STORM imaging in fixed cells using fluorescently labeled antibodies against different phosphorylated forms of RNA polymerase II reveals a homogenous distribution of transcription throughout the nucleus. This superresolution view of mammalian transcription provides no evidence for transcription factories.