For studying spFRET, a single color confocal setup, i.e. only exciting the donor, has the disadvantage that blinking or bleaching of one dye changes the FRET efficiency distribution. When the donor bleaches, the acceptor cannot be detected anymore, and when the acceptor bleaches, the detected signal is the same as for a donor only. To get more information and to count for blinking and bleaching, excitation is done by two lasers. The two lasers are alternating one after the other. We can distinguish between two different approaches: On the one hand, 'alternating laser excitation' (ALEX) [1] and, on the other hand, 'pulsed interleaved excitation' (PIE) [2]. In the case of ALEX, the two lasers alternate on a timescale between 25 and 3000 μs. This means that the switching between the excitation lasers is on a timescale faster than the diffusion. The difference to PIE is the alternating timescale. For PIE it is in the range of nanoseconds, e.g. for our two color setup 37.5 ns.
To maximize the information we can get from a single measurement, we use a multiparameter fluorescence detection (MFD) approach instead of a single detector. With this concept, we can collect the information of fundamental anisotropy, fluorescence lifetime, fluorescence intensity, microtime, excitation spectrum, fluorescence spectrum, fluorescence quantum yield and the distance between the fluorophores [3].

[1] Kapanidis AN, Laurence TA, Lee NK, Margeat E, Kong X, Weiss, S
Alternating-laser excitation of single molecules.
Accounts of Chemical Research. 2005 38(7):523-533.

[2] Müller BK, Zaychikov E, Bräuchle C, Lamb DC
Pulsed interleaved excitation
Biophysical Journal. 2005 89(5):3508-3522.

[3] Widengren J, Kudryavtsev V, Antonik M, Berger S, Gerken M, Seidel CAM
Single-molecule detection and identification of multiple species by multiparameter fluorescence detection.
Analytical Chemistry. 2006 78(6):2039-2050.

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