The microfluidic electrokinetic sample holder for serial femtosecond X-ray crystallography
Abstract/Contents
- Abstract
- Serial femtosecond X-ray crystallography (SFX) is sitting at the frontier of a paradigm shift in structural biology. The intense X-rays with short femtosecond pulses available from free electron lasers (FELs) has allowed us to view biological structures with similar, or sometimes better, resolution than current techniques. Refinement of these methods will lead us to structures of macromolecules once not attainable at current generation lightsources or with other imaging techniques. The SFX technique can yield high resolution room temperature data of biological structures. It relies on gathering statistics on tens of thousands of protein crystals, typically on the order of a dozen microns or smaller as opposed to larger millimeter-sized crystals common at synchrotrons. The intense power of the X-rays destroys the sample and thus does not allow for multiple collections on the same crystal. This has required careful consideration on sample delivery techniques. The current sample environments for data collection are in mid-vacuum (10e-6 Torr) and need a constant supply of sample, while not consuming too much sample, remaining stable and minimizing background while keeping the proteins crystalline in solution. This multifaceted optimization has made sample delivery one of the cruxes of the SFX field. This dissertation will outline one of many techniques that have been developed in the past years to address many of the issues of sample injection: the microfluidic electrokinetic sample holder (MESH). This technique leverages the moderate viscosities of the mother liquor and common additives found in crystallization conditions in order to reduce the sample consumption and use an applied electric field in order to focus and stabilize the meniscus to better interact with the incident X-rays probing the sample. This technique, similar to the electrospin process, will be described along with some of the different apparatus designed to interface it at the Coherent X-ray Imaging (CXI) endstation at the Linac Coherent Light Source (LCLS) at the SLAC National Accelerator Lab (SLAC). Finally, a new concentric technique will be described, the coMESH, which attempts to address some shortcomings of the first technique, while delivering delicate crystals at low sample consumption. From solution scattering, to protein crystal standards, nanocrystals, large complexes, time-resolved experiments, and experimental phasing, the electrokinetic technique proves quite capable to move between experimental, geometric, and biological constraints in order to leverage the advantages of SFX to the larger structural biology community with it's minimal sample consumption (micro- to milligrams) and simplistic and modular design.
Description
| Type of resource | text |
|---|---|
| Form | electronic; electronic resource; remote |
| Extent | 1 online resource. |
| Publication date | 2016 |
| Issuance | monographic |
| Language | English |
Creators/Contributors
| Associated with | Sierra, Raymond George |
|---|---|
| Associated with | Stanford University, Department of Mechanical Engineering. |
| Primary advisor | Bucksbaum, Philip H |
| Primary advisor | Santiago, Juan G |
| Thesis advisor | Bucksbaum, Philip H |
| Thesis advisor | Santiago, Juan G |
| Thesis advisor | Bogan, Michael A |
| Advisor | Bogan, Michael A |
Subjects
| Genre | Theses |
|---|
Bibliographic information
| Statement of responsibility | Raymond George Sierra. |
|---|---|
| Note | Submitted to the Department of Mechanical Engineering. |
| Thesis | Thesis (Ph.D.)--Stanford University, 2016. |
| Location | electronic resource |
Access conditions
- Copyright
- © 2016 by Raymond George Sierra
- License
- This work is licensed under a Creative Commons Attribution Non Commercial 3.0 Unported license (CC BY-NC).
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