Fig. 2.

In vivo single-molecule fluorescence microscopy. A) Schematic of an example microscope setup for single-molecule microscopy. Photoactivation and excitation lasers are coupled into an optical fiber. Light from the fiber output is collimated and focused on the back focal plane of the objective. Translation of the fiber output, collimation and focusing lenses allows for control of the incident angle of the beam at the coverslip. The emission signal is filtered from the excitation light with a polychroic mirror and focused onto an EMCCD camera. Transmission light is provided by an LED above the sample, and autofocus is provided by an infrared LED and a position-sensitive photo detector. B) An example transmission image of a live E. coli cell. C) A single frame of a PALM movie showing the fluorescence image from a single labelled RNA polymerase molecule. D) A super-resolved image of RNAP generated from imaging and localizing all available RNAP molecules over 20,000 frames. E) Trajectories of RNAP; each color corresponds to a single track. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)