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- All Subjects: serial femtosecond crystallography
- Creators: Liu, Wei
Description
The understanding of normal human physiology and disease pathogenesis shows great promise for progress with increasing ability to profile genomic loci and transcripts in single cells in situ. Using biorthogonal cleavable fluorescent oligonucleotides, a highly multiplexed single-cell in situ RNA and DNA analysis is reported. In this report, azide-based cleavable linker connects oligonucleotides to fluorophores to show nucleic acids through in situ hybridization. Post-imaging, the fluorophores are effectively cleaved off in half an hour without loss of RNA or DNA integrity. Through multiple cycles of hybridization, imaging, and cleavage this approach proves to quantify thousands of different RNA species or genomic loci because of single-molecule sensitivity in single cells in situ. Different nucleic acids can be imaged by shown by multi-color staining in each hybridization cycle, and that multiple hybridization cycles can be run on the same specimen. It is shown that in situ analysis of DNA, RNA and protein can be accomplished using both cleavable fluorescent antibodies and oligonucleotides. The highly multiplexed imaging platforms will have the potential for wide applications in both systems biology and biomedical research. Thus, proving to be cost effective and time effective.
ContributorsSamuel, Adam David (Author) / Guo, Jia (Thesis director) / Liu, Wei (Committee member) / Wang, Xu (Committee member) / School of Molecular Sciences (Contributor) / Barrett, The Honors College (Contributor)
Created2018-05
Description
Serial femtosecond crystallography (SFX) uses diffraction patterns from crystals delivered in a serial fashion to an X-Ray Free Electron Laser (XFEL) for structure determination. Typically, each diffraction pattern is a snapshot from a different crystal. SFX limits the effect of radiation damage and enables the use of nano/micro crystals for structure determination. However, analysis of SFX data is challenging since each snapshot is processed individually.
Many photosystem II (PSII) dataset have been collected at XFELs, several of which are time-resolved (containing both dark and laser illuminated frames). Comparison of light and dark datasets requires understanding systematic errors that can be introduced during data analysis. This dissertation describes data analysis of PSII datasets with a focus on the effect of parameters on later results. The influence of the subset of data used in the analysis is also examined and several criteria are screened for their utility in creating better subsets of data. Subsets are compared with Bragg data analysis and continuous diffuse scattering data analysis.
A new tool, DatView aids in the creation of subsets and visualization of statistics. DatView was developed to improve the loading speed to visualize statistics of large SFX datasets and simplify the creation of subsets based on the statistics. It combines the functionality of several existing visualization tools into a single interface, improving the exploratory power of the tool. In addition, it has comparison features that allow a pattern-by-pattern analysis of the effect of processing parameters. \emph{DatView} improves the efficiency of SFX data analysis by reducing loading time and providing novel visualization tools.
Many photosystem II (PSII) dataset have been collected at XFELs, several of which are time-resolved (containing both dark and laser illuminated frames). Comparison of light and dark datasets requires understanding systematic errors that can be introduced during data analysis. This dissertation describes data analysis of PSII datasets with a focus on the effect of parameters on later results. The influence of the subset of data used in the analysis is also examined and several criteria are screened for their utility in creating better subsets of data. Subsets are compared with Bragg data analysis and continuous diffuse scattering data analysis.
A new tool, DatView aids in the creation of subsets and visualization of statistics. DatView was developed to improve the loading speed to visualize statistics of large SFX datasets and simplify the creation of subsets based on the statistics. It combines the functionality of several existing visualization tools into a single interface, improving the exploratory power of the tool. In addition, it has comparison features that allow a pattern-by-pattern analysis of the effect of processing parameters. \emph{DatView} improves the efficiency of SFX data analysis by reducing loading time and providing novel visualization tools.
ContributorsStander, Natasha (Author) / Fromme, Petra (Thesis advisor) / Zatsepin, Nadia (Thesis advisor) / Kirian, Richard (Committee member) / Liu, Wei (Committee member) / Arizona State University (Publisher)
Created2019
Description
This thesis focuses on serial crystallography studies with X-ray free electron lasers
(XFEL) with a special emphasis on data analysis to investigate important processes
in bioenergy conversion and medicinal applications.
First, the work on photosynthesis focuses on time-resolved femtosecond crystallography
studies of Photosystem II (PSII). The structural-dynamic studies of the water
splitting reaction centering on PSII is a current hot topic of interest in the field, the
goal of which is to capture snapshots of the structural changes during the Kok cycle.
This thesis presents results from time-resolved serial femtosecond (fs) crystallography
experiments (TR-SFX) where data sets are collected at room temperature from a
stream of crystals that intersect with the ultrashort femtosecond X-ray pulses at an
XFEL with the goal to obtain structural information from the transient state (S4)
state of the cycle where the O=O bond is formed, and oxygen is released. The most
current techniques available in SFX/TR-SFX to handle hundreds of millions of raw
diffraction patterns are discussed, including selection of the best diffraction patterns,
allowing for their indexing and further data processing. The results include two 4.0 Å
resolution structures of the ground S1 state and triple excited S4 transient state.
Second, this thesis reports on the first international XFEL user experiments in
South Korea at the Pohang Accelerator Laboratory (PAL-XFEL). The usability of this
new XFEL in a proof-of-principle experiment for the study of microcrystals of human
taspase1 (an important cancer target) by SFX has been tested. The descriptions of
experiments and discussions of specific data evaluation challenges of this project in
light of the taspase1 crystals’ high anisotropy, which limited the resolution to 4.5 Å,
are included in this report
In summary, this thesis examines current techniques that are available in the
SFX/TR-SFX domain to study crystal structures from microcrystals damage-free,
with the future potential of making movies of biological processes.
(XFEL) with a special emphasis on data analysis to investigate important processes
in bioenergy conversion and medicinal applications.
First, the work on photosynthesis focuses on time-resolved femtosecond crystallography
studies of Photosystem II (PSII). The structural-dynamic studies of the water
splitting reaction centering on PSII is a current hot topic of interest in the field, the
goal of which is to capture snapshots of the structural changes during the Kok cycle.
This thesis presents results from time-resolved serial femtosecond (fs) crystallography
experiments (TR-SFX) where data sets are collected at room temperature from a
stream of crystals that intersect with the ultrashort femtosecond X-ray pulses at an
XFEL with the goal to obtain structural information from the transient state (S4)
state of the cycle where the O=O bond is formed, and oxygen is released. The most
current techniques available in SFX/TR-SFX to handle hundreds of millions of raw
diffraction patterns are discussed, including selection of the best diffraction patterns,
allowing for their indexing and further data processing. The results include two 4.0 Å
resolution structures of the ground S1 state and triple excited S4 transient state.
Second, this thesis reports on the first international XFEL user experiments in
South Korea at the Pohang Accelerator Laboratory (PAL-XFEL). The usability of this
new XFEL in a proof-of-principle experiment for the study of microcrystals of human
taspase1 (an important cancer target) by SFX has been tested. The descriptions of
experiments and discussions of specific data evaluation challenges of this project in
light of the taspase1 crystals’ high anisotropy, which limited the resolution to 4.5 Å,
are included in this report
In summary, this thesis examines current techniques that are available in the
SFX/TR-SFX domain to study crystal structures from microcrystals damage-free,
with the future potential of making movies of biological processes.
ContributorsKetawala, Gihan Kaushyal (Author) / Fromme, Petra (Thesis advisor) / Liu, Wei (Committee member) / Kirian, Richard (Committee member) / Arizona State University (Publisher)
Created2020