Table 2.

Selection criteria for P-TIRF traces with rationales

Criterion	Rationale
Clear anti-correlation in donor and acceptor signal when FRET states switch	In genuine FRET, only one signal is active at a time
Acceptor signal is present	Filters out traces with a bleached acceptor or no QP.
No multistep transitions	It is hard to distinguish genuine FRET transitions from noise in such traces
No signal drifting into the baseline	Worsening S/N renders some FRET states indistinguishable from noise
Very low high-FRET or very high low-FRET values and very weak S/N	These traces are susceptible to incorrect FRET assignments in HMM modeling
Only the longest segment or the one with the best S/N is chosen	Prevents data bias by a few traces with a large number of transitions
Low-FRET last events in segment accepted only if acceptor is present after that event.	Prevents acceptor photobleaching from tainting kinetic data
Traces must have distinction between signal and baseline	A static signal and an unusually intense baseline cannot be distinguished