Ensure That the Signal Actually Originates from a Single Molecule

Another experimental challenge in SMD is to ensure that the observed signal arises from a single molecule. Intensity fluctuations observed in single-molecule experiments could be due to changes in single-molecule dynamics or the molecular environment, but they could also originate from emissions of nearby molecules. It is crucial to eliminate this source of noise uncertainty. One way to accomplish this involves a combination of focusing the laser to a small excitation volume and working at an ultralow concentration of the molecule of interest so that the average number of fluorescent molecules residing in the probe volume is one or less [3,40]. Alternatively, a molecule-specific excitation and detection procedure can be used so that only the target molecule in the probed volume is in resonance with the laser. Even if the laser beam excites a large area, no molecules other than the target molecule in the excitation volume would be excited because they are out of resonance.

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