Matching Items (2)

Sample delivery enabled by 3D printing for reduced sample consumption and mix-and-inject serial crystallography at x-ray free electron lasers

Description

Serial femtosecond crystallography (SFX) with X-ray free electron lasers (XFELs) has enabled the determination of damage-free protein structures at ambient temperatures and of reaction intermediate species with time resolution on

Serial femtosecond crystallography (SFX) with X-ray free electron lasers (XFELs) has enabled the determination of damage-free protein structures at ambient temperatures and of reaction intermediate species with time resolution on the order of hundreds of femtoseconds. However, currently available XFEL facility X-ray pulse structures waste the majority of continuously injected crystal sample, requiring a large quantity (up to grams) of crystal sample to solve a protein structure. Furthermore, mix-and-inject serial crystallography (MISC) at XFEL facilities requires fast mixing for short (millisecond) reaction time points (𝑡"), and current sample delivery methods have complex fabrication and assembly requirements.

To reduce sample consumption during SFX, a 3D printed T-junction for generating segmented aqueous-in-oil droplets was developed. The device surface properties were characterized both with and without a surface coating for improved droplet generation stability. Additionally, the droplet generation frequency was characterized. The 3D printed device interfaced with gas dynamic virtual nozzles (GDVNs) at the Linac Coherent Light Source (LCLS), and a relationship between the aqueous phase volume and the resulting crystal hit rate was developed. Furthermore, at the European XFEL (EuXFEL) a similar quantity and quality of diffraction data was collected for segmented sample delivery using ~60% less sample volume than continuous injection, and a structure of 3-deoxy-D-manno- octulosonate 8-phosphate synthase (KDO8PS) delivered by segmented injection was solved that revealed new structural details to a resolution of 2.8 Å.

For MISC, a 3D printed hydrodynamic focusing mixer for fast mixing by diffusion was developed to automate device fabrication and simplify device assembly. The mixer was characterized with numerical models and fluorescence microscopy. A variety of devices were developed to reach reaction intermediate time points, 𝑡", on the order of 100 – 103 ms. These devices include 3D printed mixers coupled to glass or 3D printed GDVNs and two designs of mixers with GDVNs integrated into the one device. A 3D printed mixer coupled to a glass GDVN was utilized at LCLS to study the oxidation of cytochrome c oxidase (CcO), and a structure of the CcO Pr intermediate was determined at 𝑡" = 8 s.

Contributors

Agent

Created

Date Created
  • 2019

154214-Thumbnail Image.png

Method for generation of pendant drops through localized condensation for contact angle measurements in conditions deviating from standard environment

Description

Contact angle goniometer is one of the most common tools in surfaces science. Since the introduction of this instrument by Fox and Zisman1 in 1950, dispensing the liquid using

Contact angle goniometer is one of the most common tools in surfaces science. Since the introduction of this instrument by Fox and Zisman1 in 1950, dispensing the liquid using a syringe has generated pendant drops. However, using such approach at conditions significantly deviating from standard pressure and temperature would require an elaborate and costly fluidic system. To this end, this thesis work introduces alternative design of a goniometer capable of contact angle measurement at wide pressure and temperature range. In this design, pendant droplets are not dispensed through a pipette but are generated through localized condensation on a tip of a preferentially cooled small metal wire encapsulated within a thick thermal insulator layer. This thesis work covers experimental study of the relation between the geometry of the condensation-based pendant drop generator geometry and subcooling, and growth rate of drops of representative high (water) and low (pentane) surface tension liquids. Several routes that the generated pendant drops can be used to measure static and dynamic contact angles of the two liquids on common substrates well as nanoengineered superhydrophobic and omniphobic surfaces are demonstrated.

Contributors

Agent

Created

Date Created
  • 2015