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).

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