Supplementary Figure 4: Schematic designs of an objective-type and a prism-type TIRF setup.
From: Precision and accuracy of single-molecule FRET measurements—a multi-laboratory benchmark study
a, Objective. b, Prism. Green and red lasers are used to excite donor and acceptor dyes, respectively. M, mirror. L, lens. DM, dichroic mirror. Obj, objective. AD, achromatic doublet lens. Sl, tunable slit. F, filters. Det, detector (e.g. electron multiplying charge-coupled device camera, EMCCD). The inset shows a side view of the objective with the out-of-plane (45°) mirror below. SC, sample chamber. Ir, iris. St, translation stage, Pr, prism. The dashed black line in (a) indicates the on-axis path to the objective, in contrast to the displayed off-axis path for TIR illumination. Elements used for the correction factors in Table 2 (main text) were: Dichroic before objective: F53-534 (AHF), Dichroics in detection: F33-726 and F33-644 (AHF). Band pass filters in detection: BP F39-572 and BP F37-677 (AHF). SI: SP40 (Owis), Objective: CFI Apo TIRF 100x, NA 1.49 (Nikon). Camera: EMCCD, iXonUltra, Andor. Lasers: 532nm, Compass 215M (Coherent) and 635nm, Lasiris (Stoker Yale). Note that we have used a Dichroic in the fluorescence excitation and emission path that reflects the higher wavelength, but this does not have any effect on the FRET efficiency measurement and related determination of correction factors. For details on all used setups and analysis software, see Supplementary Note 8.
