The exposure of subnuclear compartments of cells to ionizing radiation is currently not trivial. We describe here a collimator for micrometer-wide stripe irradiation designed to work with conventional high-voltage X-ray tubes and cells cultured on standard glass cover slips. The microcollimator was fabricated by high-precision silicon micromachining and consists of X-ray absorbing chips with grooves of highly controlled depths, between 0.5-10 microm, along their surfaces. These grooves form X-ray collimating slits when the chips are stacked against each other. The use of this device for radiation biology was examined by irradiating human cells with X rays having energies between 20-30 keV. After irradiation, p53 binding protein 1 (53BP1), a nuclear protein that is recruited at sites of DNA double-strand breaks, clustered in lines corresponding to the irradiated stripes.