BioCAT supports a number of techniques for analysis of partially-ordered and
disordered biological materials. Below is a description of the techniques we
currently support as well as a HOWTO document detailing the procedure for
performing each experiment at the BioCAT facility.
These descriptions should help you to decide if our facility can support your research. However, it is strongly recommended that---prior to visiting BioCAT---you discuss your experiment with the user liaison for your technique after reading these descriptions. In this way, your experiment can be tailored to our facility so that you spend less time assembling and debugging your set-up and more time taking high-quality data.
Small- and Wide-Angle Fiber Diffraction
The design features of the BioCAT beam-line 18 ID and the unique source properties of the APS allow collection of fiber diffraction patterns of exceptional quality from complex, weakly diffracting biological systems within very short exposure times. The small focal spots achievable with this instrument (~50 x 150 μm2) have allowed excellent discrimination of fine detail in fiber patterns from muscle, connective tissue, and filamentous viruses as well as detection of weak diffraction features in the presence of large backgrounds. The low divergence of the undulator source and the independent horizontal and vertical focusing of our optics simultaneously allows small beam spots at the sample and at the detector that can be varied over a wide range. These beam sizes are very well matched to the resolution of our high sensitivity CCD detectors. The high X-ray flux of this instrument (~2.0 x 1013 photons/s) permits dynamic experiments on these systems with a high time-resolution (sub-milli-second).
The available sample-to-detector lengths range from 0.2 to 5.7 m to cover a very large range of reciprocal space and the CCD detector has very flexible binning and streak-camera modes for time-resolved experiments. We are currently developing micro-diffraction capabilities that will allow examination of ordered structures in regions as small as 5 x 5 μm2. Applications include, for example, finding small ordered regions in connective tissue for high resolution studies and mapping amyloid structures in brain tissues that may be associated with neurodegenerative disease.
Macromolecular Small-Angle Solution Scattering (SAXS)
Small-angle x-ray scattering (SAXS) is a widely used technique for the measurement of the radius of gyration (Rg) and the electron pair distance distribution function (P(r)), as well as for ab initio modeling of low-resolution molecular envelopes of macromolecules in solution.
The BioCAT standard experimental set-up includes a Pilatus 1M detector from Dectris (Switzerland) and a camera of 3.5 m sample-to-detector distance to access a range of momentum transfer, q, from ~0.04 to 3.3 nm-1. This range of q allows not only accurate determination of radius of gyration, but also detailed modeling using ab initio and rigid body approaches.
For static or equilibrium SAXS, a quartz capillary flow cell which is temperature controlled through a water bath can be used. It takes usually about 50-100 microliter of sample. However, in some cases smaller volume (< 20 microliter) can also yield usable SAXS data. In order to reduce radiation damage, the sample flows through the X-ray beam using a Hamilton programmable dual-syringe pump.
We also offer SAXS data collection coupled with online Size Exclusion Chromatography (SEC). The sample runs through a size exclusion column to separate potential aggregates or different oligomeric states intermediately before flowing through the capillary for X-ray exposure.
The high flux and efficient detectors at BioCAT allow collection of scattering patterns and permits time-resolved experiments on the ms time scale using the stopped-flow apparatus. We are also developing advanced microfluidic mixers, including the chaotic mixer and laminar flow mixer, to collect SAXS data on the tens of ms and sub-ms time regime. Friendly users are welcome to submit proposals to conduct pilot time-resolved SAXS experiments on their systems.
The utility of SAXS in no more limited to the structural characterization of biological macromolecules in equilibrium. We are actively developing technologies to investigate the dynamic behavior of the macromolecules using SAXS during processes such as protein and RNA folding, and enzyme-substrate/co-factor binding. Studying structural changes in time regimes ranging from ~50µs to seconds requires the use of a variety of mixers that can interface with the SAXS instrument. Stopped flow mixers have been used by several groups and have shown dead-times less than 1ms (Jacob, Krantz et al. 2004, Roh, Guo et al. 2010). However, continuous flow mixers have emerged as the best option for SAXS in sub-millisecond time-scales.
Rapid mixing devices for SAXS have fallen into two broad categories -- turbulent and laminar (Pollack and Doniach 2009, Kathuria, Guo et al. 2011, Kathuria, Chan et al. 2013). These devices facilitate rapid and efficient mixing events between multiple fluid streams containing the biological macromolecule of interest and small solutes that engender structural changes in the macromolecule. Laminar mixing utilizes hydrodynamic focusing to reduce the central flow channel to a narrow (typically ~0.1-10 µm) sheath. A version of this mixer is currently under development at BioCAT (See Figure). For turbulent mixing, turbulent flow that breaks the solution into eddies small enough for reactants to diffuse rapidly. Turbulent flow mixers have the advantage of being able to use all of the delivered photon flux in the X-ray beam for the best signal to noise ratio. The plug flow in this kind of mixer also makes the reaction time uniform orthogonally to the flow-direction thus making its incorporation into a SAXS camera quite straight forward. The progress we have made in terms of time resolution and sample consumption are well documented (Wu, Kondrashkina et al. 2008, Graceffa, Nobrega et al. 2014, Kathuria, Kayatekin et al. 2014, and Nobrega, Arora et al. 2014). In its' current iteration, we can access time regimes as low as 50µs and a complete experiment can be performed with as little as 10mgs of sample.
X-ray Micro-Diffraction and X-ray Florescence Microscopy (XFM)
The BioCAT micro-Diffraction and X-ray Florescent Microscopy(XFM) instruments are highly efficient tools for X-ray elemental mapping and micro-X-ray Diffraction studies of biological samples. (Please note that at this time XFM is not available through the GU system and is only available to collaborators.)
Areas of interest up to a few mm2 in tissue sections can be scanned to determine metal distributions with 2-3 µm resolution in XFM configuration. Several metals (10 or more) can be probed simultaneously with a minimum of sample preparation. Low-resolution maps may be performed initially over larger areas to determine smaller areas of interest that will subsequently be scanned at higher resolutions. This approach maximizes the efficiency of the microprobe, spending beamtime selectively where it is most needed. A 4 element, large active-area silicon drift detector system combined with the fast scanning capabilities of the BioCAT beamline allow use of fast scanning protocols to determine elemental maps of large area samples in relatively short periods of time.
The useful d-space range from the micro-Diffraction instrument is 1/200 - 1/3.4 Å-1 at 12 keV and 1/900-1/20 Å-1 at 8 keV with the modified Mar 165 detector with 40 mm pixels (60 mm psf function for phosphor) for very high-resolution data when the microbeam is focused at the detector.
Samples may be freeze- or air-dried and our Linkam cryostage can maintain frozen hydrated samples at liquid nitrogen temperatures.