Microfluidic isotachophoresis (ITP) is an electrophoretic technique that can be used to co-focus reactants and thereby accelerate chemical reactions. The fluorescence images provided here (fluorescenceImages1.zip to fluorescenceImages3.zip) show the isotachophoretic transport of Alexa fluor 647 carboxylic acid (AF647) and Fluorescein using an oscillating electric field with a DC offset. The two fluorophores are focused in sample zones separated by a spacer. Different amplitudes, frequencies and offsets were applied with a HVS448 6000 D high voltage sequencer (Labsmith). The fluorescence images were obtained with a Nikon Eclipse Ti microscope and an Andor iXon+ 897 EMCCD camera. A 10x Plan Fluor objective (NA = 0.3, Nikon) and a Intensilight C-HGFI mercury fiber illuminator (Nikon) were used. The exposure time was set to 23 ms and the frame rate to 40 fps. More information on the experimental procedure can be found in the paper that will be linked below. The folder contains a description of the amplitude, frequency, DC offset, and spacer concentration that correspond to each image file. The code (code.zip) was used to quantify the spatial overlap of the sample zones separated by 500 µM of spacer.