Humidity-controlled preparation of frozen-hydrated biological samples for cryogenic coherent x-ray diffraction microscopy.
Coherent x-ray diffraction microscopy (CXDM) has the potential to visualize the structures of micro- to sub-micrometer-sized biological particles, such as cells and organelles, at high resolution. Toward advancing structural studies on the functional states of such particles, here, we developed a system for the preparation of frozen-hydrated biological samples for cryogenic CXDM experiments. The system, which comprised a moist air generator, microscope, micro-injector mounted on a micromanipulator, custom-made sample preparation chamber, and flash-cooling device, allowed for the manipulation of sample particles in the relative humidity range of 20%-94%rh at 293 K to maintain their hydrated and functional states. Here, we report the details of the system and the operation procedure, including its application to the preparation of a frozen-hydrated chloroplast sample. Sample quality was evaluated through a cryogenic CXDM experiment conducted at BL29XUL of SPring-8. Taking the performance of the system and the quality of the sample, the system was suitable to prepare frozen-hydrated biological samples for cryogenic CXDM experiments.
Department of Physics, Faculty of Science and Technology, Keio University, 3-14-1 Hiyoshi, Kanagawa 223-8522, Japan.
Yuki
Y
Takayama
Masayoshi
M
Nakasako
D000388:Air; D002736:Chloroplasts / Q000378:metabolism; D005615:Freezing; D006813:Humidity; D008853:Microscopy / Q000295:instrumentation*; D055616:Microtechnology; D018724:Spinacia oleracea / Q000166:cytology / Q000378:metabolism; D014867:Water / Q000378:metabolism; D014961:X-Ray Diffraction
D014867:Water