Advances in accelerator technology enabling radiotherapy performing conformal irradiations with charged hadronic species has brought benefits to patients but also, potentially, new risks. For a better understanding of the effects of ionizing radiation on tumor and surrounding tissue investigating and quantifying the relation between energy deposition at nanometric scale and the initial biological events is a key issue. Monte Carlo track structure simulation codes provide a powerful tool for investigating this relation; however, their success and reliability rely on their improvement and development accordingly to the dedicated biological data to which they are challenged. For this aim, microbeam facility that allows the controlling of the fluence down to one ion per cell nucleus was used to evaluate relative frequencies of DNA damage following the interaction between the incoming ion and DNA according to radiation quality. Primary human cells were exposed to α particles of three different energies with respective LETs of about 36, 85 or 170 keV·μm-1 at the cells’ center position, or to protons (19 keV·μm-1). Statistical evaluation of nuclear foci formation (53BP1/γ-H2AX) observed by immunofluorescence and related to a particle traversal was undertaken in a large population of cell nuclei. The biological results were adjusted considering the factors that drive the experimental uncertainties and, then challenged with results using Geant4-DNA code modeling the ionizing particle interactions on a virtual phantom of the cell nucleus with the same mean geometry and DNA density as the cells used in our experiments. Both results show an increase of relative frequencies of foci (or simulated DNA damage) in cell nuclei as a function of increasing LET of the traversing particles, reaching a quasi-plateau when the LET exceeds 80-90 keV·μm-1. For the LET of an α particle ranging from 80-90 to 170 keV·μm-1, 10-30% of the particle hits do not lead to DNA damage inducing 53BP1 or γ-H2AX foci formation.