Microsecond ALEX FRET analysis notebook using FRETbursts - corrections, FRET burst analysis of recurring molecules, FCS, 2CDE & BVA

The herein Python notebook uses FRETbursts (download from here: https://github.com/tritemio/FRETBursts) to show how to analyze microsecond alternating laser excitation (usALEX) confocal-based FRET measurements of freely diffusing single molecules. It includes a step-by-step calculation and implementation of correction factors, donor fluorescence leakage to the acceptor detection channel (Lk), acceptor fluorescence caused by acceptor excitation by the laser intended for donor excitation (Dir), the imbalance in acceptor/donor fluorecence quantum yields and detection efficiencies (Gamma) and the imbalance in donor/acceptor excitation yields (Beta). The notebook implments a global Gamma correction, assuming the Gamma correction factor is constant for all FRET populations, based on the procedure from Lee et al. 2005. Burst search for showing the FRET population is a dual-channel burst search. After correction, the corrected FRET histogram is presented (after burst selection takes into account Beta & Gamma corrected burst sizes). We also present analysis of bursts from recurring molecules, as well as the FCS (a bit irrelevant here, due to the lasr alternation in microeconds), 2CDE & BVA plots, helping in identifying whether a FRET population is a time average of FRET states, occurring faster then molecular diffusion time, or wheather the FRET population is static and represents a single conformational state. The sample data is a result of measurement of 50 pM of hairpin 3 presented in Tsukanov et al. 2013, labeled with ATTO dyes (ATTO 550 & ATTO 647N as donor and acceptor dyes, respectively) - 532 & 640 nm cw excitation, with an alternation period of 50 microseconds.