PIE/nsALEX FRET analysis notebook using FRETbursts - corrections, MFD, FCS, 2CDE & BVA

The herein Python notebook uses FRETbursts (download from here: https://github.com/tritemio/FRETBursts) to show how to analyze pulsed-interleaved excitation (PIE) / nanosecond alternating laser excitation (nsALEX) 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 Gamma correction per each FRET population, based on both the un-corrected mean of the FRET ratio, and the donor fluorescence mean nanotimes. Burst search for sohwing 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 the MFD, FCS, 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 33 pM dsDNA labeled with ATTO dyes (ATTO 488 & ATTO 647N as donor and acceptor dyes, respectively) - 470 & 640 nm pulsed excitation, operting at 20 MHz. Everything is dposited in Zenodo (https://doi.org/10.5281/zenodo.3569585) FRET liness were calculated using the code deposited by Thomas Peulen in https://github.com/Fluorescence-Tools/ChiSurf/blob/master/docs/notebooks/fret_lines.ipynb