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| Supported Techniques |
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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 DiffractionThe 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 dynamical experiments on these systems with 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- and Wide-Angle Solution Scattering (SAXS/WAXS)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 3 m sample-to-detector length camera to access a range of scattering wave vectors, q, from ~0.05 to 3.2 nm-1 and a 0.3 m sample-to-detector length camera for range of q from ~0.4 to 16 nm-1 with the high-sensitivity Aviex CCD detector. This range of q allows reconstruction of molecular envelopes using any of a number of algorithms developed by the Svergun group and others. The standard sample chamber is a water-jacketed flow cell which takes about 50--100 microliters of sample and flows it through the beam to reduce radiation damage using a Hamilton programmable dual syringe pump. This cell design also accommodates mixing experiments. The high flux and efficient detectors at BioCAT allow collection of scattering patterns in a matter of seconds. This permits both high throughput modes of data collection and time-resolved experiments on the millisecond time scale using BioCAT's stopped-flow apparatus. Unstable or polydispersed systems may also be studied simultaneously with on-line liquid chromatography.
X-ray Microprobe and Micro-XASThe BioCAT microprobe is a highly efficient tool for x-ray elemental mapping and micro-x-ray absorption studies of biological samples. Areas of interest up to a few mm2 in tissue sections can be scanned to determine metal distributions with 2--3 μm resolution. Several metals (10 or more) can be probed simultaneously with a minimum of sample preparation.Samples may be freeze- or air-dried and our Linkam cryostage can maintain frozen hydrated samples at liquid nitrogen temperatures. The high flexibility of the microprobe optics allows scans at resolutions from 50--100 μm down to 2--3 μm. 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 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 speciation of selected elements such as Cu and Fe can be done immediately after the map is complete using XANES spectroscopy at user-selected sites. XANES spectroscopy is a powerful tool that allows measuring oxidation state of a given element (e.g. Cu II vs. Cu I) by scanning the excitation energy at the absorption edge of that element. XAFS spectroscopy may also be used to determine the local structure around selected metal sites.
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