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Soft x-ray microscopy in the water window ( ∼ 285 − 535 e V ) is an emerging and unique tool for 2D and 3D imaging of unstained intact cellular samples in their near-native state with few-10-nm detail. However, present microscopes rely on the high x-ray brightness of synchrotron-radiation sources. Having access to water-window microscopy in the home laboratory would increase the impact and extend the applicability of the method. In the present paper, we review three decades of efforts to build laboratory water-window microscopes and conclude that the method is now reaching the maturity to allow biological studies. The instruments as well as their key components are quantitatively and qualitatively compared. We find that the brightness and the reliability of the laboratory source are the most critical parameters, but that the optics as well as the sample preparation also must be optimized to enable high-resolution imaging with adequate exposure times. We then describe the two sister microscopes in Stockholm and Berlin, which allow reliable high-resolution biological imaging with short exposure times of a few tens of seconds in 2D and a few tens of minutes in 3D. They both rely on a liquid-jet laser-plasma source combined with high-reflectivity normal-incidence multilayer condenser optics, high-resolution zone-plate imaging optics, CCD detection, and cryogenic sample handling. Finally, we present several examples of biological imaging demonstrating the unique properties of these instruments.
Soft x-ray microscopy in the water window ( ∼ 285 − 535 e V ) is an emerging and unique tool for 2D and 3D imaging of unstained intact cellular samples in their near-native state with few-10-nm detail. However, present microscopes rely on the high x-ray brightness of synchrotron-radiation sources. Having access to water-window microscopy in the home laboratory would increase the impact and extend the applicability of the method. In the present paper, we review three decades of efforts to build laboratory water-window microscopes and conclude that the method is now reaching the maturity to allow biological studies. The instruments as well as their key components are quantitatively and qualitatively compared. We find that the brightness and the reliability of the laboratory source are the most critical parameters, but that the optics as well as the sample preparation also must be optimized to enable high-resolution imaging with adequate exposure times. We then describe the two sister microscopes in Stockholm and Berlin, which allow reliable high-resolution biological imaging with short exposure times of a few tens of seconds in 2D and a few tens of minutes in 3D. They both rely on a liquid-jet laser-plasma source combined with high-reflectivity normal-incidence multilayer condenser optics, high-resolution zone-plate imaging optics, CCD detection, and cryogenic sample handling. Finally, we present several examples of biological imaging demonstrating the unique properties of these instruments.
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