Extended Data Fig. 1: Imaging platform for p-TIRF smFRET.
From: Parallel stopped-flow interrogation of diverse biological systems at the single-molecule scale
(a) Schematic layout of the single-molecule imaging setup with prism-based flat-field total internal reflection (TIRF) excitation. BD, beam dump; BP, emission bandpass filter; CLT, cylindrical lens beam expanding telescope; CU, laser clean up filter; D, dichroic mirror; FL, achromatic doublet TIRF focusing lens; GTH, Gaussian to top hat refractive beam shaping lens; L, singlet lens; λ/2, half wave plate; λ/4, quarter wave plate; PBS, polarizing beam splitter; M, mirror. (b) TIRF illumination profiles (640 nm), obtained using a 20x air objective imaging a slide filled with LD655 fluorophores free in solution: Gaussian illumination (left), flat top illumination without (middle), and with (right) cylindrical lens telescope. Dashed square overlay shows the 60x magnification microscope field of view used for single molecule imaging. (c) Distribution of fluorescent molecules by their average photon count per 100 ms exposure time demonstrating more homogeneous illumination compared to Gaussian illumination profile.
