Isotachophoresis (ITP) has wide applications in chemistry and life sciences due to its pre-concentration function.A simplified ITP system consists of a leading electrolyte (LE) and a terminating electrolyte (TE).For LE and TE to migrate at the same velocity (ν),the respective E in each zone must adapt to their electrophoretic mobility (μ) according to Eq.1.νTE=νLE=μE (1) The LE zone with a high electrophoretic mobility will have a low E,and the TE zone with a low electrophoretic mobility will have a high E (Scheme 1).Current literature commonly assumes that the adaption of E is fast and is not the rate-limiting step.However,actual adaption kinetics of ITP has not been studied yet.Moreover,it is not clear how electroosmotic flow affects the kinetics of ITP.By taking advantage of single molecule imaging,here we examined the changes in electric field and the resulting non-uniform motion of single DNA molecules in capillary isotachophoresis.The individual DNA molecules passing the detection window are consecutively imaged in real time at 50-milisecond intervals.Since the migration velocity of DNA is directly proportional to the applied electric field strength (E),imaging the movement of DNA provides necessary information for understanding the distribution of E throughout the capillary.On the basis of velocity-time curves and the EOF and μ values,we obtained the electric field-time curves (Fig.2),which show three discrete E zones: a zone of high E flanking with two zones of low E.The phenomenon of the three distinct E zones has not been reported previously.This work allows us to gain insights into the kinetics of varying-field ITP and the developed strategy is applicable to the focusing and detecting single DNA molecules and DNA damages.