Difference between revisions of "DNA Melting: Model function and parameter estimation by nonlinear regression"

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 I_Total = I_Ambient + I_LED, 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

$ \frac{\partial \bar{S}}{\partial t} = p f_{Total} $