Direct observation of unstained wet biological samples by scanning-electron generation X-ray microscopy.
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 (284 eV; 4.3 nm) and oxygen (540 eV; 2.3 nm) 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 (Si(3)N(4)) films. A scanning electron beam of accelerating voltage 5 keV and current 1.6 nA irradiates the titanium (Ti)-coated Si(3)N(4) 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 5 nm resolution. Our method can be utilized easily for various wet biological samples of bacteria, viruses, and protein complexes.
Ogura
Toshihiko
T
Neuroscience Research Institute, National Institute of Advanced Industrial Science and Technology, Central 2, Tsukuba, Ibaraki 305-8568, Japan. t-ogura@aist.go.jp
D001419:Bacteria / Q000648:ultrastructure; D002244:Carbon / Q000737:chemistry; D008855:Microscopy, Electron, Scanning / Q000295:instrumentation* / Q000379:methods*; D010100:Oxygen / Q000737:chemistry; D011506:Proteins / Q000648:ultrastructure; D014025:Titanium / Q000737:chemistry; D014780:Viruses / Q000648:ultrastructure; D014867:Water / Q000737:chemistry; D014961:X-Ray Diffraction / Q000295:instrumentation* / Q000379:methods*; D015003:Yeasts / Q000648:ultrastructure
D011506:Proteins; D014867:Water; D002244:Carbon; D014025:Titanium; D010100:Oxygen