Proposed hardware and software components

New ultracentrifuge
A new analytical ultracentrifuge by Spin Analytical Inc. (Durham, NH), the centrifugal fluid analyzer (CFA), is being developed specifically for the Open AUC Project. This base instrument will facilitate significant improvements to the traditional AUC optical systems (absorbance, interference, and fluorescence) and, more importantly, stimulate the development of new optical detectors as outlined above. The CFA serves as a spinning sample holder (Fig. 2). All light sources and detectors are external to the rotor chamber. Three 50-mm diameter optical tracks are positioned at 120° intervals around the rotor chamber. The larger diameter optics will overcome fundamental accuracy limits in the XLI Rayleigh optics (Yphantis et al. 1994). In addition, optical systems, which require multiple lenses and other optical components and thus a long optical path like the ultrasensitive Schlieren optics (Cölfen and Borchard 1994) can be realized. Optical working distances to the sample cells are minimized (~8–10 cm, set by safety considerations) to optimize radial resolution.
Fig. 2 The analytical ultracentrifuge for the Open AUC Project is simply a rotating sample holder that sits in three optical paths simultaneously. Sketches are shown of the CFA (A, B) and fiber composite eight hole rotor (C) being developed for the Open AUC Project by Spin Analytical. The base centrifuge (A, B) simply consists of a vacuum containment chamber (a), a rotor (b), and high-speed motor (c). Since only analytical rotors will be used in this instrument, a shorter rotor chamber may be used. Three optical tracks are arranged at 120° intervals around the chamber. The optical tracks (B) have to use a periscope to avoid the drive motor. While complicating the optical path somewhat, using the periscopes means that the CFA can accommodate different drive motors, thus providing flexibility for future designs. The new rotor (C) being developed by Spin Analytical holds eight samples and may be operated at speeds up to 60,000 rpm. The sample holders are compatible with existing cell components
The CFA uses the newest high-speed drive and drive electronics from Beckman Coulter. However, a different high-speed drive may be used by changing the base plate and drive electronics. The vacuum system is isolated from the rest of the instrument to reduce vibrations and surface contamination that degrade optical performance. The CFA does not require special electrical power and is mounted on a bench with space above and below it for optics.
Resources are provided on the CFA to aid optics development. Power (5, 15, and 72 VDC) and a 48-bit external digital bus are provided on the CFA to operate the positioners, light sources, and detectors that comprise optical systems. A variety of specialized cards (described below) are being produced as part of the Open AUC Project to help interface optical systems to the signal bus. Since 48-bit digital I/O cards are widely available, developers can choose computers and operating systems that fit their requirements. There also is an internal data bus that provides the signals needed to synchronize external electronics to the spinning rotor (described below). Motion control may be provided by servos or by stepping motors. Descriptions, schematics, and timing diagrams for the bus signals will be published, as will the data bus and motion control driver software source code. While these resources are provided to help AUC developers, they are not constrained to use them.

New rotor
An eight-hole, fiber composite rotor that will operate at 60,000 rpm is under development (Spin Analytical). A fiber composite rotor offers three advantages over titanium rotors. First, due to its lower mass and greater strength, fiber composite offers a higher safety margin than titanium. Furthermore, should a rotor failure occur, much less kinetic energy is released by the fiber composite than by a titanium rotor. Second, by using a stainless steel hub, a fiber composite rotor has a lower center of mass and moment of inertia, thus reducing precession and vibration compared to a monolithic titanium rotor. Third, a temperature measurement system may be incorporated directly inside the rotor, thus improving temperature accuracy. The design for the new rotor is shown in Fig. 2c.

Optical systems for the CFA platform
A number of optical systems should be adapted to the CFA platform. Traditional optical systems such as absorption and Rayleigh interference detectors should be improved to replace 20-year-old technology and take advantage of state-of-the-art equipment. In addition, several new detectors should be tested for suitability. The envisioned detectors and improvements for integration into the CFA are: Absorbance optics Optical absorbance is the most widely used detector for AUC. The XLI absorbance system requires at least a minute to scan a single sample, and has notoriously poor wavelength reproducibility, limiting its utility for many applications. The prototype absorbance system built for the XLI uses a continuous light source and software synchronization (Laue et al. 2006), acquires data from all samples simultaneously, has a 100-fold better wavelength reproducibility, has a tenfold higher sensitivity and precision, and acquires data at twice the radial resolution than the XLI.
Multi-wavelength absorbance optics The multi-wavelength system provides an absorbance spectrum at each radial position (Strauss et al. 2008; Bhattacharyya et al. 2006). Deconvolution of these spectra allows the discrimination of individual species sedimenting in complex mixtures of molecules. Cellular and molecular biology will benefit from this optical system since it will allow the size distributions of labeled molecules to be determined in a complex milieu (e.g. cell lysates). Molecules with different chromophores, such as DNA binding proteins, heme proteins or ligands and tagged molecules can be analyzed to study assemblies and complexes. For example, it will be possible to identify the components and characterize the cellular conditions under which molecules sediment as part of a macromolecular complex. Figure 3 shows the current multiwavelength detector and also the sedimentation of bovine serum albumin detected by two different operation modes: the time mode which detects the time dependent sedimentation of the sample with the detector set at a fixed radius and the radial mode which scans the cell radius at a given time. The optics will use a constant light source, a rapid scanning stepping motor, and a fast Andor ICCD camera capable of nanosecond integration time, allowing signal collection of all cell channels in each rotor revolution even at speeds as high as 60,000 rpm.
Fig. 3 The multiwavelength detector arm mounted in an XL-A AUC (left) as well as the typical experimental traces in time mode with radially fixed detector and radial mode scanning the AUC cell radially at a fixed time. The x-axis in these plots is the wavelength and the z-axis absorption. The data shown are for sedimenting bovine serum albumin (BSA)
High-precision interferometer Interference optics can detect solutes that have no convenient absorbance signal and have a higher precision than the absorbance optics. The XLI interferometer has a sensitivity of approximately 3.25 fringe mg−1 ml−1 and a precision of ±0.01 fringe (Yphantis et al. 1994). Changes to the source and detector will improve the sensitivity more than twofold and the precision more than 100-fold (see detector shown in Fig. 4). The changes will extend the useful concentration range for interference detection by two orders of magnitude, making it useful for characterizing trace quantities of materials and high-affinity interactions (Howlett et al. 2006).
Fig. 4 Advances for Rayleigh Interference optics: a high-precision Rayleigh interference optical system mounted on an Xl ultracentrifuge (Laserarm from Spin Analytical); b large format interference camera from Philips (3,000 × 2,000 pixels); c data quality from the stock Beckman Coulter interference camera; d data quality from a large format Philips camera (1,024 × 2,048 pixels); e comparison of data quality as the residual noise from ten successive scans taken approximately 1 min apart. The residuals were calculated using WinMatch (available from http://www.rasmb.org) to optimize the fringe displacements. In c the residuals from the large format camera (rms 0.00017 fringe) are superimposed on those from the stock camera (camera rms 0.009). The increased number of fringes provided by the large format camera reduces the magnitude of the residuals over 50-fold, thus making it possible to acquire data at lower concentrations and improving the precision of analysis. Additional improvements can be expected from the Philips or any other large format CCD camera. Data supplied courtesy of David Yphantis and Jeff Lary
Low angle light scattering and turbidity Turbidity detectors have been described for AUC for particle size distribution analysis (Mächtle 1999a; Müller 1989; Scholtan and Lange 1972). A small-angle laser light scattering detector was also described to analyze molar mass distributions (Bhattacharyya 2006). These optical systems typically operate at a fixed radial position and acquire data with every rotor turn in order to capture the sedimentation of fast-moving particles. In combination with speed ramps they reach a measurement range of 20–5,000 nm for polymer particles (Mächtle and Börger 2006).
Multi wavelength fluorescence Fluorescence detection for AUC offers exquisite sensitivity and unparalleled solute discrimination for characterizing high-affinity interactions (Kingsbury et al. 2008) and the association state of proteins in serum and cell lysates (Kingsbury et al. 2008; Kroe and Laue 2009). The XLI AU-FDS fluorescence system is constrained to a single excitation wavelength and long-pass emission filter (MacGregor et al. 2004). A fluorescence system will be built that provides multiple excitation sources and four emission filters. This system will allow fluorescence and fluorescence resonance energy transfer characterization by AUC of multiple components and their complexes in biological fluids (e.g. serum, sputum, and cell lysates). Applications for these capabilities include detection of antigens and antibodies in biological fluids for medical research, and the direct physical characterization of complexes in proteomics research (Kroe and Laue 2009).
Schlieren optics Schlieren optics provide “tunable sensitivity,” making them useful for working with extremely high-concentrations and with compressible fluids. The CFA version of Schlieren optics will be based on the prototype system built for the XLI (Mächtle 1999b), and will be useful for characterizing protein formulations (Shire et al. 2004). They also could be useful for the rapid, quantitative characterization of a variety of blood disorders (e.g. light chain amyloidosis, Sanchorawala 2006). However, the most important application of the Schlieren system will be analytical density gradients, which cannot be evaluated very well by any of the standard optical AUC systems (Mächtle and Börger 2006).

Operating software
The Open AUC Project includes software for operating the analytical ultracentrifuge. In order to accommodate diverse optical systems, the operating software is divided into “building block” modules that are interconnected using TCP/IP communications. The four building block modules that make up the operating software are: (1) machine services, (2) protocol services, (3) sample services, and (4) optical system operations (OSO). These modules are collectively called Analytical Ultracentrifuge-Advanced Operating System (AU-AOS).The machine services module (MSM) controls centrifuge operation using ASCII commands that follow the XML-RPC standard. The draft standard for the commands is available http://www.rasmb.bbri.org/rasmb/AOS/Open_AUC_Project/MachineServices/. Any computer with network connectivity may connect to and operate the CFA through TCP/IP communications.
The protocol services module (PSM) provides the information necessary to conduct an experiment, including the hardware description needed to operate optical detectors (sample holder angles and radii) and the method used (e.g. rotor speed, temperature, temperature tolerance, acceleration, braking, duration at each speed, any delay after reaching speed prior to data acquisition, time between data acquisition events and duration of the experiment) to collect the data. The identities of the protocol developer and user are kept with this information and saved in a database. A draft standard database structure is available (Langhorst 2008) and is described in “Proposed data analysis and data management infrastructure”.
The sample services module (SSM) includes information about the solvent and solute properties, and the calculations needed to interpret AUC results (Langhorst 2008; Laue et al. 1992). Both a desktop and a web-served version of this module will be available.
Optical system operations: Separate modules are required to run the hardware for the different optical systems. These will be written using the Open AUC Project standards for communications with the Protocol services and Data handling services. The TCP/IP communications provide relatively low-speed communications, meaning that any time-critical code must be contained within the OSO modules for a specific optical system.

Optical system interfaces
The CFA and the Open AUC Project make rapid optical system development possible. The following specialized circuits and driver software will be available for optics developers. With these tools new optical systems (e.g. Raman, low angle X-ray scattering) can be developed for AUC. Rotor timing pulse and master clock All data acquisition must be synchronized to the spinning rotor, and a rotor timing pulse with jitter <1 part in 4,000 is critical (Laue et al. 1984). The CFA meets this specification, and a digital rotor timing pulse is provided on the CFA signal bus. The master clock automatically provides 500 kHz pulses below 7,000 rpm and 4 MHz above 7,000 rpm (with hysteresis to avoid switching back until 4,000 rpm). After each rotation, the period of rotation is latched and made accessible on the CFA signal bus, as is whether the low frequency or high frequency clock is being used (Laue et al. 1984).
Synchronizer The synchronizer uses the master clock to modulate either a light source or the detector so that data acquisition starts at a constant fraction of a revolution (the delay) and occurs over some fraction of a revolution (the duration), while compensating for propagation delays. The Open AUC Project synchronizer handles these functions using settings supplied by an external computer on the CFA signal bus (Laue et al. 1984).
Digital data acquisition and data storage A board with a 14-bit 5 MHz A/D with an addressable 16-bit by 4 Mb buffer storage is available for the CFA signal bus. Digitizing may be gated externally (e.g. by synchronizer delay/duration functions). Data are stored sequentially in memory. Additional memory boards may be cascaded to provide up to 15 banks of memory to allow continuous data acquisition over long periods, such as required by the light scattering detectors (Mächtle 1999a). Memory may be accessed from the external computer over the CFA signal bus.