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Excitation Schemes in Single-Molecule FRET: Single vs. Two-Color Alternating Excitation

About excitation schemes

iSMS currently supports the two most common excitation schemes: Single-color excitation and two-color alternating laser excitation (ALEX/PIE). In single color excitation, only D excitation is used. In ALEX, a direct acceptor excitation is additionally used in order to monitor the acceptor reference intensity independent of FRET.

  • In single color excitation the excitation order of the frames in the raw movie is all donor excitations: 'DDDDDD...'s
  • In ALEX, the excitation order of consecutive frames in the raw movie is '...DADADADA...'. iSMS automatically registers the correct order when loading the data based on the default ROI positions. Thus, make sure the default ROI positions properly match the emission channels of the raw image.

While single-color excitation is the easiest experimentally, ALEX has noteworthy advantages, some of which are:

  1. Inspection of acceptor photophysics irrespective of FRET.
  2. Quicker and more robust determination of quantitative single-molecule FRET parameters.
  3. Calculation of the stoichiometry factor which is related to the ratio of fluorophores in the molecule spot.
  4. Quick determination and correction of direct acceptor excitation.
  5. Easier determination of acceptor bleaching.
  6. More possibilities for automated analysis - the automated algorithms implemented in iSMS often exploit the direct acceptor reference trace.

Single color excitation

Two-color alternating laser excitation (ALEX)