We report here the development of a device for single-molecule biochip readout using fast alternating excitation. The technology extends standard imaging cytometry by offering additional color channels in excitation. To enable the study of mobile objects, e.g. actively transported vesicles in living cells or freely diffusing lipids in a lipid bilayer, the frequency of the illumination pulses was chosen high enough to virtually freeze the motion of the biomolecules, as they are shifted through the illuminated area. The synchronization of sample illumination, scanning and line-camera readout yield two quasi-simultaneously recorded images covering the same sample region. Diffraction-limited resolution and high localization precision for point-light sources down to approximately 10 nm was shown by scanning immobilized 100 nm fluorescence latex beads. Ultra-sensitivity was demonstrated by imaging single fluorescent streptavidin molecules diffusing in a fluid lipid bilayer. Two-color streptavidin labeled with Cy3 and Cy5 could be easily identified in the two respective excitation channels; high accordance in the dye positions confirms the applicability for colocalization studies of moving objects. Finally, scans of antibody-receptor interactions in large populations of live cells illustrate the feasibility of this method for biochip application.