Fig. 1: The smfBox and smFRET.
From: The smfBox is an open-source platform for single-molecule FRET
a Schematic of the smfBox with parts labelled according to Supplementary Tables 1, 2 and 4. Lasers are collimated (L1), cropped (Iris) and steered by two mirrors (M1, M2) onto a 10:90 beam splitter (BS1). 10% of the beam is focused on to a photodiode for continuous power measurement and alternation cycle monitoring. 90% of the beam is directed via dichroic mirror 1 (DC1) to the objective. Light from the back reflection is reflected by DC1 and BS1 onto a CCD camera for accurate focusing. Fluorescence emission from the sample passes through the excitation dichroic (DC1) and is focused onto a pinhole (P1) to remove out of focus light, before being split by colour (DC2) onto one of two avalanche photodiodes (APD0, APD1). b 3D model of the completed smfBox, with the front panel of the microscope body removed. A CAD file of the assembled instrument is provided (Supplementary Data 1) along with an animated build sequence (Supplementary Movie 1). c A flowchart of the smfBox platform showing the functionality of the acquisition software. d Schematic: single molecules diffuse through a confocal volume (residence time ~1 ms) constructed by focusing the lasers into a near-diffraction-limited spot and using a pinhole to section the emission light in a thin focal plane. Lasers (515 nm and 638 nm) are alternated (20 kHz) to ensure multiple excitations of the donor and acceptor dyes for each molecule. e A typical time trace for an smFRET experiment. Fluorescently-labelled molecules diffuse through the confocal volume emitting bursts of fluorescence. Individual photons generated by emission from the donor under donor excitation (DD—green) are recorded on APD0. APD1 records photons emitted by the acceptor either under donor excitation (DA—red) or direct acceptor excitation (AA—purple). f 2D ES histogram showing uncorrected FRET efficiency (E*) and stoichiometry (S). Donor-only molecules appear with low E* but high S, and acceptor-only molecules appear with low S. Doubly labelled molecules appear with intermediate S.
