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.

本Python笔记本使用FRETbursts（FRETbursts，下载地址：https://github.com/tritemio/FRETBursts），演示如何对自由扩散单分子的共聚焦式微秒级交替激光激发（microsecond alternating laser excitation, usALEX）荧光共振能量转移（Fluorescence Resonance Energy Transfer, FRET）测量数据进行分析。脚本包含各类校正因子的分步计算与实现流程，具体包括：供体荧光向受体检测通道的泄露因子（Lk）、供体激发激光所诱导的受体直接荧光因子（Dir）、受体与供体荧光量子产率及检测效率的失衡因子（Gamma），以及供体与受体激发产率的失衡因子（Beta）。本脚本基于Lee等人2005年的研究方法，实现全局Gamma校正，假设所有FRET群体的Gamma校正因子均为恒定值。用于识别FRET群体的爆发搜索采用双通道爆发搜索算法。校正完成后，将展示校正后的FRET直方图（爆发选择阶段已考虑经Beta与Gamma校正后的爆发强度）。此外，本脚本还对重复出现分子的爆发事件进行分析，并展示了荧光相关光谱（Fluorescence Correlation Spectroscopy, FCS，因采用微秒级激光交替机制，此处相关性较弱）、2CDE与BVA绘图结果，用于判断某一FRET群体究竟是：其FRET状态的时间平均值（且该状态转换速率快于分子扩散时间），或是处于静态且仅对应单一构象状态。样本数据来源于Tsukanov等人2013年报道的3号发夹结构的测量结果，其浓度为50皮摩尔（pM），采用ATTO染料标记（供体染料为ATTO 550，受体染料为ATTO 647N），使用532 nm与640 nm连续波（continuous wave, cw）激光激发，激光交替周期为50微秒。