Research

Femtosecond Crystallography


Gas Dynamic Virtual Nozzle

 

We have developed an injector nozzle for liquid injection into vacuum. The liquid jet diameter ranges from tens of microns to sub-microns. The smallest jet diameter so far achieved is 200nm. These nozzles produce a small jet without the associated clogging problems. This is achieved with a co-flowing gas stream which focuses the jet to a small diameter. Extensive characterization has been done with optical microscopy in vacuum using stroboscopic and single shot nanosecond pulse illumination. Submicron jets have been examined in an Environmental SEM.

These nozzle are used for hydrated injection of biomolecules and nano- crystals into an X-ray beam at conventional synchrotron sources and X-ray Free Electron Lasers (XFEL). Extensive experiments with Rayleigh jets, Electrospray and Drop on Demand sources have been performed to examine their usefulness for biomolecule injection.


Liquid injector system for XFEL’s

We have developed an injector nozzle for liquid injection into vacuum. The liquid jet diameter ranges from tens of microns to sub-microns. The smallest jet diameter so far achieved is 200nm. These nozzles produce a small jet without the associated clogging problems. This is achieved with a co-flowing gas stream which focuses the jet to a small diameter. Extensive characterization has been done with optical microscopy in vacuum using stroboscopic and single shot nanosecond pulse illumination. Submicron jets have been examined in an Environmental SEM.

These nozzle are used for hydrated injection of biomolecules and nano- crystals into an X-ray beam at conventional synchrotron sources and X-ray Free Electron Lasers (XFEL). Extensive experiments with Rayleigh jets, Electrospray and Drop on Demand sources have been performed to examine their usefulness for biomolecule injection.


Femtosecond X-ray structure determination of Photosystem I

 

X-ray crystallography provides the vast majority of macromolecular structures, but the success of the method relies on growing crystals of sufficient size. In conventional measurements, the necessary increase in X-ray dose to record data from crystals that are too small leads to extensive damage before a diffraction signal can be recorded. A method for structure determination has been developed where single-crystal X-ray diffraction ‘snapshots’ are collected from a fully hydrated stream of nanocrystals using femtosecond pulses from a hard-X-ray free-electron laser, the Linac Coherent Light Source. We proved this concept with nanocrystals of photosystem I, one of the largest membrane protein complexes. More than 3 million diffraction patterns were collected during our first beamtime in December 2009, and a three-dimensional data set was assembled from individual photosystem I nanocrystals (~200 nm to 2 μm in size). The software to merge all the diffraction patterns into a 3D diffraction volume was developed at ASU. We avoided the problem of radiation damage in crystallography by using pulses briefer than the timescale of most damage processes. This offers a new approach to structure determination of macromolecules that do not yield crystals of sufficient size for studies using conventional radiation sources or are particularly sensitive to radiation damage.


Femtosecond X-ray Diffraction Imaging of single viruses

X-ray lasers offer new capabilities in understanding the structure of biological systems, complex materials and matter under extreme conditions. Very short and extremely bright, coherent X-ray pulses can be used to outrun key damage processes and obtain a single diffraction pattern from a large macromolecule, a virus or a cell before the sample explodes and turns into plasma. The continuous diffraction pattern of non-crystalline objects permits oversampling and direct phase retrieval. In a collaboration with Uppsala University the first diffraction patterns of single Viruses have been recorded at the LCLS in December 2009. These Mimi viruses where injected with an aerosol injector developed at Uppsala. Calculations indicate that the energy deposited into the virus by the pulse heated the particle to over 100,000 K after the pulse had left the sample. The reconstructed exit wavefront yielded 32-nm full-period resolution in a single exposure and showed no measurable damage. The reconstruction indicates inhomogeneous arrangement of dense material inside the virion. We expect that significantly higher resolutions will be achieved in such experiments with shorter and brighter photon pulses focused to a smaller area.


X-ray Powder Diffraction from protein microcrystals in a liquid jet

Membrane proteins constitute more then 30% of the proteins in an average cell, and yet the number of currently known structures of unique membrane proteins is less then 300. To develop new concepts for membrane protein structure determination, we have explored a serial nano-crystallography method, in which fully hydrated protein nanocrystals are delivered to an x-ray beam within a liquid jet at room temperature. As a model system, we have collected x-ray powder diffraction data from the integral membrane protein Photosystem I, which consists of 36 subunits and 381 cofactors. Data were collected from crys- tals ranging in size from 100 nm to 2 mm. The results demonstrate that there are membrane protein crystals that contain less then 100 unit cells (200 total molecules) and that 3D crystals of membrane proteins, which contain less then 200 molecules, may be suitable for structural investigation. Serial nanocrystallography overcomes the problem of x-ray damage, which is currently one of the major limitations for x-ray structure determination of small crystals.


X-ray Diffraction imaging

The inversion problem of coherent scattering - the reconstruction of a potential (image) from measurements of scattered intensity in the far-field - has occupied physicists for over a century, and arises in fields as varied as optics, radar, X-ray crystallography, medical tomographic imaging, holography, electron microscopy and particle scattering generally. The scientific payoff from a solution to this phase problem is understood to be considerable, in view of the lensless imaging capability it would provide for the various radiations (such as coherent atom beams, neutrons, and X-rays) for which no lenses exist. Where imperfect lenses do exist, such a diffractive imaging method would offer the possibility of diffraction-limited image reconstruction without aberrations.

The hybrid input-output iterative algorithm (HIO), which solves the phase problem for scattering from non-periodic objects, has been used to phase X-ray and electron diffraction data and experimental results from applying the algorithm to coherent electron diffraction patterns were presented, using specially made e-beam lithographed support structures. This work has been preformed in collaboration with the Cornell Nanofabrication Facility.  Coherent laser-optical experiments where performed, which demonstrate the ability of the algorithm to recover phases from experimental diffraction patterns. In collaboration with Lawrence Livermore National Laboratory, the first 3D reconstruction of a fabricated test object has been achieved.