Time-resolved crystallography for the masses NatureComms
. Briefly: via a piezo-actuator, 75 or 150 picoliter-sized droplets are shot from a 50 or 70 µm glass capillary with a velocity of 2 m sonto the target mesh. The nozzle is brought close to the target via manual, rail-mounted translation stages that enable precise lateral and vertical alignment of the nozzles to adjust for differences in sample holder length and also to correct for bent meshes. Nozzle alignment is aided by two perpendicularly aligned cameras, which focus on the target mesh .
To obtain accurate vitrification times we used a thermocouple with similar dimensions to the crystal size. For comparison to previous studies, which mainly used larger thermocouples we thus recorded vitrification times using two temperature sensors of different size.
To determine the actual vitrification time, we also recorded the temperature decrease independent of the spitrobot. Here the glass-transition temperature is reached within 7.5 ms and a 90–10% analysis of the temperature drop resulted in a fall time of 7.5 ms. Thus, the total delay time of theconsists of the intrinsic delay of the device , and the actual vitrification process ~7.5 ms corresponding to a cooling rate of 2.3 × 10.
Characterization of the processes demonstrates a vitrification time of ~7.5 ms, for samples on the order of 10 µm, and that physiological conditions can be maintained within the sample area and . The minimal delay time currently achievable with thesetup is ~50 ms. This constraint is the result of the mechanical piston motion , air-valve delay , and vitrification time —the deposition of the substrate solution occurs during the air-valve delay and is thus accounted for.
is equipped with two cameras from different viewing directions, 90° apart. The primary purpose of the camera system is sample alignment and LAMA-nozzle positioning. The axis of the LAMA nozzle is at an angle of 60° relative to the surface of the mesh. The LAMA nozzle can be positioned using the aforementioned 3-axis stage to optimally deposit the substrate on the micromesh and ensure reproducible results between different samples.
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