Modeling single molecule detection probabilities in microdroplets. Final report

Optimization of molecular detection efficiencies is important for analytical applications of single molecule detection methods. In microdroplets some experimental limitations can be reduced, primarily because the molecule cannot diffuse away from the excitation and collection volume. Digital molecular detection using a stream of microdroplets has been proposed as a method of reducing concentration detection limits by several orders of magnitude relative to conventional measurements. However, the bending and reflection of light at the microdroplet`s liquid-air interface cause the illumination intensity and fluorescence intensity collected to be strongly dependent on the position of the molecule within the droplet. The goal is to model the detection of single molecules in microdroplets so that one can better understand and optimize detection efficiencies. In the first year of this modeling effort the authors studied the collection of fluorescence from unit-amplitude dipoles inside of spheres. In this second year they modified their analysis to accurately model the effects of excitation inhomogeneities, including effects of molecular saturation, motion of the droplet, and phase variations between the two counter-propagating waves that illuminate the droplet. They showed that counter-propagating plane wave illumination can decrease the variations in the intensity which excites the molecules. Also in this second year they simulated (using a Monte Carlo method) the detection of fluorescence from many droplets, each of which may contain zero, or one (or at higher concentrations, a few) fluorescent molecules