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howto:calculate_ratiometric_single_pair_fret_distributions_using_the_pie-fret_script

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howto:calculate_ratiometric_single_pair_fret_distributions_using_the_pie-fret_script [2014/06/18 18:06]
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howto:calculate_ratiometric_single_pair_fret_distributions_using_the_pie-fret_script [2015/11/03 15:12] (current)
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 **Note:** The script requires a time trace containing the fluorescence of two spectrally separated channels, one channel mainly detecting the fluorescence of the donor dye and the other channel detecting mainly the fluorescence of the acceptor dye.\\ **Note:** The script requires a time trace containing the fluorescence of two spectrally separated channels, one channel mainly detecting the fluorescence of the donor dye and the other channel detecting mainly the fluorescence of the acceptor dye.\\
 The time traces can be acquired from single molecule events in a diluted solution, where the passages of single molecules are registered as "​bursts"​. Alternatively,​ traces obtained from a stationary single FRET pair can be analyzed, e.g. to observe conformational changes.\\ The time traces can be acquired from single molecule events in a diluted solution, where the passages of single molecules are registered as "​bursts"​. Alternatively,​ traces obtained from a stationary single FRET pair can be analyzed, e.g. to observe conformational changes.\\
-To record such traces, usually single molecule sensitive detectors as [[glossary:​spad|SPAD]] or Hybrid-PMA are necessary to successfully detect single molecule fluorescence.\\+To record such traces, usually single molecule sensitive detectors as [[glossary:​spad|SPAD]] or [[glossary: ​Hybrid ​PMT]]s ​are necessary to successfully detect single molecule fluorescence.\\
 For the pulsed interleaved excitation ([[glossary:​pie|PIE]]) analysis of the script, pulsed excitation using two lasers is required, one laser exciting the donor, the other the acceptor dye. Usually, this is achieved using the multichannel laser driver PDL828 “Sepia II” in combination with two pulsed diode lasers. For the pulsed interleaved excitation ([[glossary:​pie|PIE]]) analysis of the script, pulsed excitation using two lasers is required, one laser exciting the donor, the other the acceptor dye. Usually, this is achieved using the multichannel laser driver PDL828 “Sepia II” in combination with two pulsed diode lasers.
  
howto/calculate_ratiometric_single_pair_fret_distributions_using_the_pie-fret_script.txt · Last modified: 2015/11/03 15:12 by admin