(Color online) (a) Schematic of the submicrometer fluidic channel and sequentially separated focal volumes used for single-molecule mobility measurements. The first focal volume was defined by a focused laser and was used to detect BODIPY molecules conjugated with the fluorescent labels. The second focal volume was defined by a focused laser and was used to detect conjugated Alexa Flour 488 fluorophores. (b) Schematic of photon burst detection in the two color channels. As a fluorescent label traveled through the fluidic channel, chronological red and green photon bursts were detected. The distance between the focal volumes was divided by the time between photon bursts to determine the speed of the molecule.
(a) Transit time distributions for overlapping focal volumes and 2.5-, 3.6-, and focal volume separations. When the laser spots were aligned to each other, photon bursts were detected simultaneously in the two color channels. As the laser spots were separated, the time between photon bursts in the two color channels increased. The histograms were skewed to the slow side, presumably because of surface interactions with the channel walls. (b) Transit time distributions were fitted to Gaussian or exponentially modified Gaussian distributions to extract the most probable transit times, which are shown as a function of focal volume separation, and were fitted to a line.
(a) Transit time distributions for a focal volume separation with the applied electric field varying from in the submicrometer fluidic channel. As the field increased, the labels spent less time between the two focal volumes. (b) Label speed as a function of electric field, shown fitted to a line. The speed was calculated by dividing the distance between the two focal volumes by the most probable transit time.
Transit time distributions for 1G1R and 4G4R labels. The histogram areas were normalized to 1 to account for different numbers of detected labels. 81% of 1G1R labels and 77% of 4G4R labels were identifiable by their measured mobility.
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