Direct observation of unstained wet biological samples by scanning-electron generation X-ray microscopy

Abstract

Analytical tools of nanometre-scale resolution are indispensable in the fields of biology, physics and chemistry. One suitable tool, the soft X-ray microscope, provides high spatial resolution of visible light for wet specimens. For biological specimens, X-rays of water-window wavelength between carbon (284eV; 4.3nm) and oxygen (540eV; 2.3nm) absorption edges provide high-contrast imaging of biological samples in water. Among types of X-ray microscope, the transmission X-ray microscope using a synchrotron radiation source with diffractive zone plates offers the highest spatial resolution, approaching 15–10nm. However, even higher resolution is required to measure proteins and protein complexes in biological specimens; therefore, a new type of X-ray microscope with higher resolution that uses a simple light source is desirable. Here we report a novel scanning-electron generation X-ray microscope (SGXM) that demonstrates direct imaging of unstained wet biological specimens. We deposited wet yeasts in the space between two silicon nitride (Si3N4) films. A scanning electron beam of accelerating voltage 5keV and current 1.6nA irradiates the titanium (Ti)-coated Si3N4 film, and the soft X-ray signal from it is detected by an X-ray photodiode (PD) placed below the sample. The SGXM can theoretically achieve better than 5nm resolution. Our method can be utilized easily for various wet biological samples of bacteria, viruses, and protein complexes.