Fast automated energy changes at synchrotron radiation beamlines equipped with transfocator or focusing mirrors.

Algorithms and procedures to fully automate retuning of synchrotron radiation beamlines over wide energy ranges are discussed. The discussion is based on the implementation at the National Institute of General Medical Sciences and the National Cancer Institute Structural Biology Facility at the Advanced Photon Source. When a user selects a new beamline energy, software synchronously controls the beamline monochromator and undulator to maintain the X-ray beam flux after the monochromator, preserves beam attenuation by determining a new set of attenuator foils, changes, as needed, mirror reflecting stripes and the undulator harmonic, preserves beam focal distance of compound refractive lens focusing by changing the In/Out combination of lenses in the transfocator, and, finally, restores beam position at the sample by on-the-fly scanning of either the Kirkpatrick-Baez mirror angles or the transfocator up/down and inboard/outboard positions.

High-viscosity injector-based pink-beam serial crystallography of microcrystals at a synchrotron radiation source.

Since the first successful serial crystallography (SX) experiment at a synchrotron radiation source, the popularity of this approach has continued to grow showing that third-generation synchrotrons can be viable alternatives to scarce X-ray free-electron laser sources. Synchrotron radiation flux may be increased ∼100 times by a moderate increase in the bandwidth ('pink beam' conditions) at some cost to data analysis complexity. Here, we report the first high-viscosity injector-based pink-beam SX experiments.

Solving protein structure from sparse serial microcrystal diffraction data at a storage-ring synchrotron source.

In recent years, the success of serial femtosecond crystallography and the paucity of beamtime at X-ray free-electron lasers have motivated the development of serial microcrystallography experiments at storage-ring synchrotron sources. However, especially at storage-ring sources, if a crystal is too small it will have suffered significant radiation damage before diffracting a sufficient number of X-rays into Bragg peaks for peak-indexing software to determine the crystal orientation. As a consequence, the data frames of small crystals often cannot be indexed and are discarded.

High-throughput in situ X-ray screening of and data collection from protein crystals at room temperature and under cryogenic conditions.

Protein crystallography has significantly advanced in recent years, with in situ data collection, in which crystals are placed in the X-ray beam within their growth medium, being a major point of focus. In situ methods eliminate the need to harvest crystals, a previously unavoidable drawback, particularly for often small membrane-protein crystals. Here, we present a protocol for the high-throughput in situ X-ray screening of and data collection from soluble and membrane-protein crystals at room temperature (20-25°C) and under cryogenic conditions.

Pink-beam serial crystallography.

Serial X-ray crystallography allows macromolecular structure determination at both X-ray free electron lasers (XFELs) and, more recently, synchrotron sources. The time resolution for serial synchrotron crystallography experiments has been limited to millisecond timescales with monochromatic beams. The polychromatic, "pink", beam provides a more than two orders of magnitude increased photon flux and hence allows accessing much shorter timescales in diffraction experiments at synchrotron sources.

Serial millisecond crystallography of membrane and soluble protein microcrystals using synchrotron radiation.

Crystal structure determination of biological macromolecules using the novel technique of serial femtosecond crystallography (SFX) is severely limited by the scarcity of X-ray free-electron laser (XFEL) sources. However, recent and future upgrades render microfocus beamlines at synchrotron-radiation sources suitable for room-temperature serial crystallography data collection also. Owing to the longer exposure times that are needed at synchrotrons, serial data collection is termed serial millisecond crystallography (SMX).

Locating and Visualizing Crystals for X-Ray Diffraction Experiments.

Macromolecular crystallography has advanced from using macroscopic crystals, which might be >1 mm on a side, to crystals that are essentially invisible to the naked eye, or even under a standard laboratory microscope. As crystallography requires recognizing crystals when they are produced, and then placing them in an X-ray, electron, or neutron beam, this provides challenges, particularly in the case of advanced X-ray sources, where beams have very small cross sections and crystals may be vanishingly small. Methods for visualizing crystals are reviewed here, and examples of different types of cases are presented, including: standard crystals, crystals grown in mesophase, in situ crystallography, and crystals grown for X-ray Free Electron Laser or Micro Electron Diffraction experiments.

A Versatile System for High-Throughput In Situ X-ray Screening and Data Collection of Soluble and Membrane-Protein Crystals.

In recent years, in situ data collection has been a major focus of progress in protein crystallography. Here, we introduce the Mylar in situ method using Mylar-based sandwich plates that are inexpensive, easy to make and handle, and show significantly less background scattering than other setups. A variety of cognate holders for patches of Mylar in situ sandwich films corresponding to one or more wells makes the method robust and versatile, allows for storage and shipping of entire wells, and enables automated crystal imaging, screening, and goniometer-based X-ray diffraction data-collection at room temperature and under cryogenic conditions for soluble and membrane-protein crystals grown in or transferred to these plates.

Synchrotron X-Ray Diffraction Dynamic Sampling for Protein Crystal Centering.

A supervised learning approach for dynamic sampling (SLADS) was developed to reduce X-ray exposure prior to data collection in protein structure determination. Implementation of this algorithm allowed reduction of the X-ray dose to the central core of the crystal by up to 20-fold compared to current raster scanning approaches. This dose reduction corresponds directly to a reduction on X-ray damage to the protein crystals prior to data collection for structure determination.

Dynamic X-ray diffraction sampling for protein crystal positioning.

A sparse supervised learning approach for dynamic sampling (SLADS) is described for dose reduction in diffraction-based protein crystal positioning. Crystal centering is typically a prerequisite for macromolecular diffraction at synchrotron facilities, with X-ray diffraction mapping growing in popularity as a mechanism for localization. In X-ray raster scanning, diffraction is used to identify the crystal positions based on the detection of Bragg-like peaks in the scattering patterns; however, this additional X-ray exposure may result in detectable damage to the crystal prior to data collection.

Rapid in situ X-ray position stabilization via extremum seeking feedback.

X-ray beam stability is crucial for acquiring high-quality data at synchrotron beamline facilities. When the X-ray beam and defining apertures are of similar dimensions, small misalignments driven by position instabilities give rise to large intensity fluctuations. This problem is solved using extremum seeking feedback control (ESFC) for in situ vertical beam position stabilization.

Classification of Large Cellular Populations and Discovery of Rare Cells Using Single Cell Matrix-Assisted Laser Desorption/Ionization Time-of-Flight Mass Spectrometry.

Cell-to-cell variability and functional heterogeneity are integral features of multicellular organisms. Chemical classification of cells into cell type is important for understanding cellular specialization as well as organismal function and organization. Assays to elucidate these chemical variations are best performed with single cell samples because tissue homogenates average the biochemical composition of many different cells and oftentimes include extracellular components.

Kinetic trapping of metastable amino acid polymorphs.

Second harmonic generation (SHG) microscopy measurements indicate that inkjet-printed racemic solutions of amino acids can produce nanocrystals trapped in metastable polymorph forms upon rapid solvent evaporation. Polymorphism impacts the composition, distribution, and physico-kinetic properties of organic solids, with energetic arguments favoring the most stable polymorph. In this study, unfavored noncentrosymmetric crystal forms were observed by SHG microscopy.

Towards protein-crystal centering using second-harmonic generation (SHG) microscopy.

The potential of second-harmonic generation (SHG) microscopy for automated crystal centering to guide synchrotron X-ray diffraction of protein crystals was explored. These studies included (i) comparison of microcrystal positions in cryoloops as determined by SHG imaging and by X-ray diffraction rastering and (ii) X-ray structure determinations of selected proteins to investigate the potential for laser-induced damage from SHG imaging. In studies using β2 adrenergic receptor membrane-protein crystals prepared in lipidic mesophase, the crystal locations identified by SHG images obtained in transmission mode were found to correlate well with the crystal locations identified by raster scanning using an X-ray minibeam.

Real-time dynamic range and signal to noise enhancement in beam-scanning microscopy by integration of sensor characteristics, data acquisition hardware, and statistical methods.

Despite the ubiquitous use of multi-photon and confocal microscopy measurements in biology, the core techniques typically suffer from fundamental compromises between signal to noise (S/N) and linear dynamic range (LDR). In this study, direct synchronous digitization of voltage transients coupled with statistical analysis is shown to allow S/N approaching the theoretical maximum throughout an LDR spanning more than 8 decades, limited only by the dark counts of the detector on the low end and by the intrinsic nonlinearities of the photomultiplier tube (PMT) detector on the high end. Synchronous digitization of each voltage transient represents a fundamental departure from established methods in confocal/multi-photon imaging, which are currently based on either photon counting or signal averaging.

Statistical connection of binomial photon counting and photon averaging in high dynamic range beam-scanning microscopy.

Data from photomultiplier tubes are typically analyzed using either counting or averaging techniques, which are most accurate in the dim and bright signal limits, respectively. A statistical means of adjoining these two techniques is presented by recovering the Poisson parameter from averaged data and relating it to the statistics of binomial counting from Kissick et al. [Anal.

Second-order nonlinear optical imaging of chiral crystals.

Second-order nonlinear optical imaging of chiral crystals (SONICC) is an emerging technique for crystal imaging and characterization. We provide a brief overview of the origin of second harmonic generation signals in SONICC and discuss recent studies using SONICC for biological applications. Given that they provide near-complete suppression of any background, SONICC images can be used to determine the presence or absence of protein crystals through both manual inspection and automated analysis.

Detection of membrane protein two-dimensional crystals in living cells.

It is notoriously difficult to grow membrane protein crystals and solve membrane protein structures. Improved detection and screening of membrane protein crystals are needed. We have shown here that second-order nonlinear optical imaging of chiral crystals based on second harmonic generation can provide sensitive and selective detection of two-dimensional protein crystalline arrays with sufficiently low background to enable crystal detection within the membranes of live cells.

Statistical treatment of photon/electron counting: extending the linear dynamic range from the dark count rate to saturation.

An experimentally simple photon counting method is demonstrated providing 7 orders of magnitude in linear dynamic range (LDR) for a single photomultiplier tube (PMT) detector. In conventional photon/electron counting methods, the linear range is dictated by the agreement between the binomially distributed measurement of counted events and the underlying Poisson distribution of photons/electrons. By explicitly considering the log-normal probability distribution in voltage transients as a function of the number of photons present and the Poisson distribution of photons, observed counts for a given threshold can be related to the mean number of photons well beyond the conventional limit.

Selective detection and quantitation of organic molecule crystallization by second harmonic generation microscopy.

Second order nonlinear optical imaging of chiral crystals (SONICC) was applied to selectively detect crystal formation at early stages and characterize the kinetics of nucleation and growth. SONICC relies on second harmonic generation (SHG), a nonlinear optical effect that only arises from noncentosymmetric ordered domain structures, which include crystals of chiral molecules. The model systems studied include pharmaceutically relevant compounds: griseofulvin and chlorpropamide.

Nonlinear optical imaging of integral membrane protein crystals in lipidic mesophases.

Second-order nonlinear optical imaging of chiral crystals (SONICC) is explored for selective detection of integral membrane protein crystals grown in opaque and turbid environments. High turbidity is a hallmark of membrane protein crystallization due to the extensive use of detergent and/or lipids that often form various mesophases. Detection of crystals in such media by conventional optical methods (e.

Selective detection of protein crystals by second harmonic microscopy.

The unique symmetry properties of second harmonic generation (SHG) microscopy enabled sensitive and selective imaging of protein microcrystals with negligible contributions from solvated proteins or amorphous protein aggregates. In studies of microcrystallites of green fluorescent protein (GFP) prepared in 500 pL droplets, the SHG intensities rivaled those of fluorescence, but with superb selectivity for crystalline regions. GFP in amorphous aggregates and in solution produced substantial background fluorescence, but no detectable SHG.