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This in situ tracking approach elucidated steady-state motions of the nanoparticles moving at a constant speed under the counter-balanced electrophoretic and viscous drag forces, which also allowed estimations of their surface charge densities. The present method can be utilized as a speedometer for nanoscale objects of virtually any size as long as they are able to be put through the sensing zones with potential applications for single-molecule time-of-flight mass spectrometry.The influence of the fluorination positions on the optical