Charged particle microbeams provide unique features to study targeted and non-targeted radiation response and have recently emerged as a powerful tool to investigate radiation-induced DNA damage and repair. We have developed a charged particle microbeam delivering protons and alpha particles in the MeV energy range equipped with online time-lapse imaging capabilities. The beam is focused to a sub-micrometer beam spot under vacuum by means of a triplet of magnetic quadrupoles and extracted in air through a 200 nm Si3N4 window. The end-station is equipped with an automated fluorescence microscope used for single cell targeting and online time-lapse imaging. Cells are kept in their medium during the irradiation procedure and the sample temperature is regulated to 37 C. An overall targeting accuracy of 2.0 ± 0.7 lm has been measured by tracking the re-localization of the XRCC1 protein. First measurements of this re-localization shows the ability of our system to follow online the radiationinduced re-localization of proteins in the first minutes after irradiation.