Difference between revisions of "DNA Melting: Model function and parameter estimation by nonlinear regression"
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Revision as of 19:26, 13 November 2012
This subject matter will be explained in lecture, in tutorials, and in the lab.
Photobleaching model
Assumptions
- The dye is divided into two populations, bleached and unbleached, with concentrations S and S
- The initial dye concentration is S + S
- Bleached and unbleached molecules bind to dsDNA with the same affinity.
- Only bound dye molecules may be excited.
- An excited fluorophore will either bleach with probability p or return to the ground state with probability 1-p.
- The constant 1-p includes all cases in which a molecule returns to the ground state unbleached.
- Mechanisms for returning to the ground state include fluorescence, phosphorescence, and non-radiative relaxation.
- Only excited fluorophore molecules bleach
- Bleaching is irreversible
- Magnitude of the illumination is equal to ITotal = IAmbient + ILED, the intensities of ambient light and LED illumination, respectively.
- Total fluorescence, $ f_{total} $ is proportional to $ I_{total} $
- The lock-in amplifier only detects the portion of the fluorescence proportional to $ I_{LED} $
- $ I_{Ambient} $ is approximately constant
Model
