The BioCAT Beamline 18ID
The BioCAT undulator beamline 18ID at the Advanced Photon Source, although now 15 years old, remains a state-of-the-art instrument for biological small and wide angle fiber diffraction and macromolecular solution scattering (Fischetti, et al., 2004 J. Synchrotron Rad. (2004). 11, 399-405). It is capable of delivering well over 1013 photons/s into focal spots as small as 60 x 150 µm2 with the conventional beamline optics. The BioCAT beamline was designed by Dr. Rosenbaum (Argonne National Laboratory) and shares the same overall mechanical design of the SBC undulator 19-ID (Rosenbaum et al. J Synchrotron. 2006 Jan;13(Pt 1):30-45). Undulator "A" provides very intense monochromatic radiation in the 3.2 - 14 keV range (first harmonic) with low angular divergence (<8.3 micro-radian vertical and <29.3 micro-radian horizontal FWHM) with a small source size (typically ~ 680 micrometers horizontal by ~22 micrometers vertical FWHM, specifications as of fall 2010). A differential pump separates the vacuum structure from the storage ring eliminating the need for any windows between the and the storage ring.
The first optical element is a moveable fixed mask with an aperture of 4.2 mm x 2.1 mm just upstream of two independent double-crystal monochromator assemblies. The upstream monochromator #1 has fixed Si(111) crystals to facilitate energy changes for the microfocus setup while he downstream monochromator #2 has a sagittal focusing second crystal assemblies that can provide horizontal focusing of the beam for the main SAXS camera. We have observed a FWHM of 150 &micor;m in intensity profiles of the sagittally focused 12.0 KeV X-ray beam at the focal point for a 2 meter SAXS camera (about 64 m from the source) which corresponds to a demagnification ratio of 4.4:1. The maximum horizontal demagnification ratios are 6.2:1 and 7.3:1 for monochromator #1 and monochromator #2, respectively. (The increased divergence due to the relatively high demagnification ratios is not generally a problem for the systems we are studying). The beamline mirror is used for harmonic rejection and vertical focusing but it easily can be withdrawn from the beam path when desired.
The current vertical focusing mirror is an adaptive, so-called bi-morph design from SESO. Typical vertical beam profiles when focused for 1.5-3.5 m SAXS camera configurations are about 65 µm FWHM. Downstream of the mirror are horizontal and vertical beam-defining slits for the monochromatic beam. These can be set to pass the entire beam or define a beam as small as 25 µm x 25 µm. A monochromatic photon shutter allows the monochromator optics to stay warm while allowing the user to enter the experimental enclosure.
The experimental enclosure is 14m long x 5m wide x 3.3m tall. The first 2 m are taken up by the
vertical collimation slits and the downstream support, which incorporates filter/shutter assemblies,
and an ion chamber for the primary beam (I0) monitor. Following the vertical collimation
slits is a beryllium window. All components upstream of this window are under high vacuum
(10-7 – 10-8 torr); thereafter, all components are in rough vacuum
(10-3 – 10-4torr). The downstream support moves all these components under
computer control to follow the beam as reflected off of the mirror. Downstream of this table is 6.4
x 1.5 m vibration-isolation table that is used for most small-angle diffraction and scattering experiments.
The beamline control software is based on the Experimental Physics and Industrial Control System
The beamline motors and data acquisition systems are controlled by four VME crates with Motorola
MVME162FX controllers. The beamline motors were chosen to be DC servos because of their advantages
in high torque, speed, and power consumption over stepper motors. Nine 8-channel Delta Tau PMAC-1
servo motor controllers control the beamline optics and XY positioning stages. These 15 year-old
motor controllers are in the process of being replaced by new Delta Tau Power PMAC motor controllers).
For most of the experimental hutch equipment, stepper motors are used controlled by five 8-channel
Oregon Microsystems OMS-58 stepper motor controllers.
For time resolved data acquisition there is a Struck 7201 multichannel scaler with 32 inputs and
4K memory arrays per each input.
The beamline control software is based on the Experimental Physics and Industrial Control System
The beamline motors and data acquisition systems are controlled by four VME crates with Motorola MVME162FX controllers. The beamline motors were chosen to be DC servos because of their advantages in high torque, speed, and power consumption over stepper motors. Nine 8-channel Delta Tau PMAC-1 servo motor controllers control the beamline optics and XY positioning stages. These 15 year-old motor controllers are in the process of being replaced by new Delta Tau Power PMAC motor controllers). For most of the experimental hutch equipment, stepper motors are used controlled by five 8-channel Oregon Microsystems OMS-58 stepper motor controllers.
For time resolved data acquisition there is a Struck 7201 multichannel scaler with 32 inputs and 4K memory arrays per each input.For conventional scans there is also a Joerger VCS16 scaler with 16 inputs and a voltage to frequency converter (Hytec VFC 2504, Hytec Electronics Ltd). External equipment (CCD detectors, shutters) can be interfaced to the control sytem using a digital I/O board (Acromag-9440) with 16 input and 16 output channels. To implement PID feedback control loops there is also an Acromag IP330 ADC with 16 inputs Systran DAC with 8 outputs. There are 8 current amplifiers (Keithley, model 42) which are interfaced through the RS232 ports on the beamline workstations. We have designed simulated EPICS servers for these devices so that they can be accessed from other computers in exactly the same way as the VME modules. CAMAC modules can also be accommodated via Kinetic Systems KS-2917 VME to CAMAC interface board and a Kinetic Systems CAMAC crate and model 3922 controller interfaced with EPICS.
To take full advantage of the high x-ray flux at the Advanced Photon Source and to reduce radiation damage to labile biological samples, considerable effort has been devoted to implementing fast on-the-fly scans and thus reducing the exposure times and, consequently, dose delivered to the sample. This was a challenging task since the distributed control system imposes network latencies on the communication between different VME crates and the control workstations. Currently two types of scans have been implemented – a "generic" fast scan and an "energy" fast scan. With the generic scan we can scan any servo or stepper motor at the beamline while recording the output into any of 16 inputs of the Joerger scaler. The scan may use one of three different algorithms but all of them have the same lower limit to the time resolution of ~150 ms/point imposed by the network latencies. As a result, the typical generic scan time is ~15-60 seconds.
These scanning protocols have been very useful in beamline diagnosis and alignment and have found use in various experimental protocols. The fast energy scan, as implemented for both the beamline monochromators is simplified by the fact that the synchronization of three different motions in the double crystal monochromator is handled by the PMAC controllers (which were designed for simultaneous multi-axis motions), so that the scan software program need only deal with one combined pseudo-motor called "energy." This scan makes use of the 32 memory arrays in the Struck scaler, which simultaneously records the monochromator encoder outputs and the x-ray intensities. The minimum time per point for this scan is ~1 ms and the total scan time is typically 1-10 s. We have also incorporated synchronous motion of monochromator and the beamline undulator into the energy scans so that the energy of the two devices can be changed simultaneously. Finally, all the energy and generic scans provide for two or three dimensional scans with step-wise motion of the second and the third motors respectively retaining continuous scanning in the first dimension.
Two XIA model PF2S2 filter assemblies contain a series of aluminum filters that allow at least 3 decades of beam attenuation (at 12 kev) as well as two pneumatically activated shutters. By using these in series, exposure times of less than 500 ms can be achieved. We have recently implemented a shutter capable of <500 micro-second minimum exposure time consisting of two electrically-activated, inclined blade-type shutters in series (Model LS500, NM laser products Inc) with ca. 1ms latency. A Kinetic Systems model 3655-LIA timing-pulse generator is used to control these fast shutter systems. Accurate exposure times are user-selectable via a simple GUI interface.
Detectors for Fiber Diffraction and Scattering
Fully developed and commissioned detectors for the beamline include a high sensitivity, high spatial resolution Aviex PCCD1680 (80 x 160mm, 39 µm pixels, 5 ADU/12 keV X-ray photon), and MAR165 CCD (165 mm diameter active area, standard mode 2k x 2k 80 µm pixels, ~1 ADU/ 12 keV X-ray photon) detectors. The MAR detector has been upgraded with baseline stabilization electronics and a thinner phosphor (~65 µm psf function) so it can be used in unbinned mode with 40 µm pixels. We have two Pilatus 100k pixel array detectors (84 x 33.5 mm active area, 170 µm pixel, photon counting) and a Fuji Bas2500 image plate scanner. For equilibrium and time resolved macromolecular SAXS we have a Pilatus 3 1M detector that is 981 x 1043 \, 172 x 172 µm2 pixels for a 170 x 180 mm2 active area, photon-counting detector able to read at 500 fps.
Standard SAXS camera configurations
SAXS cameras are used for both Small-Angle X-ray Solution Scattering (SAXS) and fiber diffraction studies of muscle and connective tissue. While cameras as short as 0.2 m and as long as 5.5 m are possible with the beamline optics, we have standardized on a few selected camera lengths that match our available detectors and commonly required Q-ranges. These camera lengths and Q-ranges (detector offset) at 12 keV X-ray energy are given in Table 1. At the current stage of development there is no great benefit in using cameras longer than 3.5 m. If the developments described in Technical R&D project 2 are successful this can be revisited. Various combinations of stainless steel flight tube sections allow a range of sample to detector distances. The flight tubes can be moved in and out of position using overhead cranes. Mica windows are used throughout except for the rear exit window, which is composed of 0.127 mm thick Kapton film. The setup routinely uses a 4 mm diameter backstop with an integrated single-element PIN photodiode for transmission measurements. For SAXS experiments, the beam is defined by the collimator slits described above. Three meters downstream of these slits are the guard slits Xenocs "scatterless" slits) mounted on motorized horizontal and vertical translation stages. This allows for accurate positioning and profiling of the beam for diagnostic purposes. A number of different sample holders, either supplied by the user or by BioCAT, can be mounted on crossed-roller horizontal and vertical translation slides with 100 mm of travel and capable of carrying up to 258 kg loads. Control of the sample holder position is integrated with the detector control and data acquisition systems.
Capabilities for X-ray Fluorescence Microcopy
The BioCAT X-ray microprobe instrument has been developed to the point where it is now a robust, high performance instrument for micron scale x-ray fluorescence microscopy (Barrea, et al. 2010). It is available on a collaborative basis with BioCAT staff (not available through APS General User Program). The basic beamline and microprobe instrument parameters are given in Table 1. The heart of the instrument is a Kirkpatrick-Baez mirror bender system of the University of Chicago design (Eng, et al., 1998) with two Rh-coated silicon mirrors each 200 mm long.
The shortest focal lengths of the horizontal and vertical KB mirrors are 220 mm and 420 mm respectively, which deliver a minimum beam size of 3 mm (V) x 5 µm (H) FWHM with a flux of 1.3 x 1012 photons/s. The mirrors can be dynamically bent under remote control via EPICS to focus at longer distances for different applications such as micro-diffraction or micro-SAXS. Total delivered flux can be increased ~4-fold by pre-focussing the beam with the main beamline optics at the expense of minimal focal spot size (Huang et al., 2010, Table 2). The incoming beam is defined by a set of slits located immediately upstream of the mirror box while a set of apertures (50-100-200 microns diameter each) downstream of the mirrors are available and can be selected to act as a guard aperture. Two ion chambers are located upstream and downstream of the KB mirror box, used for monitoring the flux of the incoming and delivered microbeam, respectively. The whole system is mounted on a remotely controllable table that is located at the end of the experimental hutch D, 70 m downstream from the undulator source, allowing for maximum demagnification of the source (the table is proposed to be upgraded in the upcoming grant cycle).
Three silicon drift detectors (SDD's) are available for fluorescence signal measurement. The first is a four-element Vortex SDD, with each element having 50 mm2 active area. The other two are single element SDDs one with a 10 mm2 and the other with a 80 mm2 active area, both with energy resolution of 170 eV at the Mn Ka line. The small area detector has a thin Moxtek Ap3.3 polymer window that allows low-energy X-ray photons from light elements to be measured. The 80 mm2 area detector with a 25 mm-thick Be window is now used as a backup to the Vortex. The fluorescence signal from the single element is collected and analyzed by two digital Saturn DXP spectrometers (XIA) that can handle up to 700,000 counts/s with very good stability, although showing a large deviation from linearity at these very high count rates due to a very high detector dead time. The Vortex detector uses the XIA DXP-XMAP electronics for collection and analysis of fluorescent signals. These electronics support sophisticated mapping modes, allowing for full spectrum or multi-SCA acquisition at sub-millisecond dwell times. The DXP-XMAP system consists of four Digital X-ray Processor (DXP) channels, a Digital Signal Processor (DSP), a System FPGA, SRAM memory and a PCI interface. Each of the four DXP channels accepts a preamplified signal input and produces a 16-bit pipelined output stream of x-ray energies. Each channel has up to 1,000,000 counts/sec throughput with peaking time range of 0.1 to 100 microseconds. Multi-channel analysis for each channel allows optimal use of data. Data is collected in HDF5 file format and processed using custom MatLAB program and program the MAPS (Vogt, 2003).
BioCAT's scanning software allows fast continuous scans to be performed while acquiring and storing full multichannel analyzer spectra per pixel on-the-fly with minimal overhead time (<20 ms per pixel). Together, the high-flux X-ray microbeam and the rapid-scanning capabilities of the BioCAT beamline allow the collection of XFM measurements from as many as 48 tissue sections per day.
Capabilities for X-ray Fiber Crystallography
We now have two mature, high-performance, instruments for fiber crystallography, one based on the KB mirror micro-focusing optics described above, and the other based on the collimated main beam. 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. Beam sizes and divergences for different focal positions are given in Table 3. The beam can be focused at the detector for maximum X-ray pattern data quality, the sample for maximum spatial resolution or somewhere in between when compromises are necessary. The second instrument focuses the main beam at a slit to give a well collimated 60 x 60 mm beam, which although is larger than with the micro-diffraction instrument, is still small enough to find the ordered domains in many samples. The advantage of this instrument is that it gives us useful and simple setup for near small angle through to wide angle (1/160 Å-1 to 1/2 Å-1) with a low background and high signal to noise ratio. Another advantage of this instrument is that it is more straightforward to change camera lengths from 250 mm to 1m allowing for ranges of d spacing from 1/125 – 1/3.5 Å-1 (shortest camera) and 1/500-1/13 Å (longest camera) at 12 keV. The delicate crystallinity of user samples coupled with the high intensity of the X-ray beam necessitates cryo capabilities. This is less straightforward then for crystallography beamlines due to the sample sizes and nature (tissue dissections, preparations, sections). To maintain fibrous samples at liquid nitrogen temperature we have a custom cold nitrogen stream system made to our specifications (it lacks the warm shield gas layer used in conventional crystallography cryo-jets) by Cryo Industries of America. Leica CPC plunge-freezing station for sample cryo-freezing / manipulation before being placed in the cryo-jet stream.
Auxiliary Instrumentation for muscle diffraction
For online muscle mechanics and X-ray measurements we have a horizontal muscle mechanics rig equipped with a laser diffraction system including a linear CCD sarcomere length computer system (Dexela Inc., UK), motors (Aurora Scientific 308B or Guth Scientific Instruments linear motor Si-MOTDB) and various force transducers for different force levels, and a high power muscle stimulator (Aurora Scientific 701B). For higher throughput measurements on transgenic mouse cardiac muscle samples, many users prefer to use small individual sample cells that allow length adjustment with sarcomere length being monitored by an offline video microscope and image analysis system (Aurora Scientific model 900B). For whole muscle studies we have an Aurora 300B muscle lever system custom modified for fast step response. Also available are fast shutters, capable of 0.5 ms exposures, storage oscilloscopes and a National Instruments based A/D, D/A system.
Auxiliary Instrumentation for SAXS
For static SAXS We have a custom flow cell with a water-jacketed 1.5 mm diameter quartz capillary for sample observation. This can either be connected to one of several Hamilton Microlab 500 series programmable syringe pumps or to the output of one of the FPLC’s. We have an AKTA and Akta Pure FPLC apparatus for liquid chromatography and a Malvern Zetasizer Dynamic Light Scattering apparatus. We have two ISCO model 500D and four Harvard Instrument model PHD 4400 programmable, high-pressure pumps for the continuous flow mixer project. We have two Biologic SFM-400 stopped flow mixers, one with an MEC 22998 microvolume mixer allowing 0.5 ms dead time and an x-ray observation cell. We have recently re-commissioned our autosampler system that can provide automated sample handling for systems that do not require inline size exclusion chromatography. The system consists of a Sparks-Holland sample robot that is used to select particular samples from 96 well plates (with temperature control) and deliver them to a Hamilton Microlab ML560 dispenser system that can inject them into the standard sample chamber. Labview based software controls the sequence of operations required for unattended data acquisition including cleaning the sample cell, loading samples, flowing samples and triggering the CCD and recovering the sample after exposure.
Other Support Facilities
The BioCAT facility has a ~550 square foot wet laboratory with sinks, hoods, analytical and platform balances, water de-ionizer, chromatography refrigerator, -20 and -80°C freezers, micro-centrifuge, high-speed centrifuge ultracentrifuge, platform rocker, as well as, a Cary 50 UV/VIS spectrophotometer, an EON plate reader and an AKTA FPLC. It is possible to do a complete on-site purification of proteins from cell-pellets. We have several Zeiss stereomicroscopes and a Nikon inverted microscope with epi-flourescence capability. The BioCAT instrumentation lab is also ~550 square feet and contains electronic testing equipment, fabrication and testing areas and a large granite table for assembling optics. We have access to a well-equipped machine shop at IIT with one machinist. Argonne Central Shops (with a satellite shop on site at the APS) can handle larger jobs. The Advanced Photon Source has a well-equippedmetrology shop which we use to characterize mirror and multilayer optics. For animal care we have access to the Animal Housing Facility in the Engineering Research Building at IIT.