We introduced a novel X-ray microscope system based on scanning electron Microscopy Using thin film, which enables the measurement Of unstained biological samples without damage. Ail unstained yeast sample was adsorbed under a titanium (Ti)-coated silicon nitride (Si3N4) film 90 nm thick. The X-ray signal from the film was detected by an X-ray photodiode (PD) placed below the sample. With ail electron beam at 2.6 kV acceleration and 6.75 nA current, the yeast image is obtained Using the X-ray PD. The image is created by soft X-rays from the Ti layer. The Ti layer is effective in generating the characteristic 2.7-nm wavelength X-rays by the irradiation of electrons. Furthermore, we investigated the electron trajectory and the generation of the characteristic X-rays within the Ti-coated Si3N4 film by Monte Carlo simulation. Our system can be easily utilized to observe various unstained biological samples of cells, bacteria, and viruses. (C) 2009 Elsevier Inc. All rights reserved.