The Importance of Photon Arrival Times in STED Microscopy

Lens-based or far-field fluorescence microscopy is a very popular technique for investigating the living cell. However, the spatial resolution of its standard versions is limited to about 200 nm due to diffraction, impeding the imaging of molecular assemblies at smaller scales. The turn of the twenty-first century has witnessed the advent of far-field fluorescence super-resolution microscopy or nanoscopy, a fluorescence microscopy featuring a spatial resolution down to molecular scales. STED microscopy was the first of such nanoscopy techniques, but was for a long time considered as a very complex technique, hard to apply in everyday biological research. Based on developments in label and laser technology, recent years have however seen major improvements of the STED nanoscopy approach, one of which is gated continuous-wave STED (gCW-STED) microscopy. gCW-STED microscopy reduces complexity by combining STED laser operating in CW with pulsed excitation and time-gated photon detection. Here, we describe the physical principles of gCW-STED, formulate the theoretical framework which characterizes its main benefits and limitations, as well as show experimental data.