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howto:calculate_ratiometric_single_pair_fret_distributions_using_the_pie-fret_script [2014/06/18 15:58] – adminhowto:calculate_ratiometric_single_pair_fret_distributions_using_the_pie-fret_script [2015/11/03 14:12] (current) – admin
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 {{tag> tutorial howto FRET PIE analysis time-trace SymPhoTime}} {{tag> tutorial howto FRET PIE analysis time-trace SymPhoTime}}
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 ====== Calculate Ratiometric Single Pair FRET Distributions Using PIE-FRET ====== ====== Calculate Ratiometric Single Pair FRET Distributions Using PIE-FRET ======
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 This tutorial shows step-by-step, how to calculate a FRET histogram from single [[glossary:fret|FRET]]pairs detected under single molecule conditions using pulsed interleaved excitation (PIE). The script requires a spectrally resolved fluorescence time trace, in which donor and acceptor have been excited alternately. As a result, the distribution of FRET efficiencies of different molecules is shown which can be used to detect subpopulations.  This tutorial shows step-by-step, how to calculate a FRET histogram from single [[glossary:fret|FRET]]pairs detected under single molecule conditions using pulsed interleaved excitation (PIE). The script requires a spectrally resolved fluorescence time trace, in which donor and acceptor have been excited alternately. As a result, the distribution of FRET efficiencies of different molecules is shown which can be used to detect subpopulations. 
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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.
  
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 **Note:** Further information about the sample used in this measurement can be found under: J.L. Fiore et. al., Enthalpy-driven RNA-folding: single-molecule thermodynamics of tetraloop-receptor tertiary interaction,  Biochemistry (2009), 48(11), 2550-8. ([[http://pubs.acs.org/doi/abs/10.1021/bi8019788]]). **Note:** Further information about the sample used in this measurement can be found under: J.L. Fiore et. al., Enthalpy-driven RNA-folding: single-molecule thermodynamics of tetraloop-receptor tertiary interaction,  Biochemistry (2009), 48(11), 2550-8. ([[http://pubs.acs.org/doi/abs/10.1021/bi8019788]]).
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howto/calculate_ratiometric_single_pair_fret_distributions_using_the_pie-fret_script.txt · Last modified: 2015/11/03 14:12 by admin