Fig. 1: Single molecule FLIM setup and acquisition scheme.
From: Wide-field fluorescence lifetime imaging of single molecules with a gated single-photon camera
a Sketch of the experimental setup where a picosecond-pulsed green laser beam undergoes total internal reflection at the sample coverslip to excite single fluorescent molecules close to its surface. The emitted fluorescence is collected to form an image on the camera chip, which is synchronized with the excitation laser. b Typical images of single molecules obtained from 20 ms integration time for the first gate capturing the full fluorescence decay (top) and the second gate capturing only late photons reaching the camera after a gate delay T. The same molecule is shown by yellow circles in the two cases. c Sketch of the time-gated imaging scheme that enables retrieving single-molecule fluorescence lifetimes. In the first gate (top) the No detected photons can be decomposed into single-molecule fluorescent signal (S) and background (B). In the second gate (bottom) early fluorescence is rejected and fewer photons are measured (N1). We assumed Bo = B1 = B for simplicity on this sketch but in the general case we use Bo ≠ B1. The gate response is mapped in more details in Fig. S10
