Advanced Multiwavelength Detection in Analytical Ultracentrifugation

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Advanced Multiwavelength Detection in Analytical Ultracentrifugation Joseph Pearson, Johannes Walter, Wolfgang Peukert, and Helmut Cölfen Anal. Chem., Just Accepted Manuscript • DOI: 10.1021/acs.analchem.7b04056 • Publication Date (Web): 07 Dec 2017 Downloaded from http://pubs.acs.org on December 15, 2017

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Analytical Chemistry

Advanced Multiwavelength Ultracentrifugation

Detection

in

Analytical

Joseph Pearson,a,† Johannes Walter,b,† Wolfgang Peukert,b,* Helmut Cölfena,* †

J. Pearson and J. Walter contributed equally to the manuscript.

University of Konstanz, Physical Chemistry, Universitätsstraße 10, 78457 Konstanz, Germany. E-Mail: [email protected]

a)

b) Institute of Particle Technology (LFG), Friedrich-Alexander-Universität Erlangen-Nürnberg (FAU), Cauerstraße 4, 91058 Erlangen, Germany. E-Mail: [email protected]

Abstract This work highlights significant advancements in detector hardware and software for multiwavelength analytical ultracentrifugation (MWL-AUC) experiments, demonstrating improvement in both, the spectral performance and UV capabilities of the instrument. The hardware is an extension of the Open AUC MWL detector developed in academia and first introduced in 2006 by Bhattacharya et al. Additional modifications as well as new analytical methods available for MWL data have since been reported. The present work describes new and continuing improvements to the MWL detector, including mirror source and imaging optics, UV sensitive acquisition modes and revised data acquisition software. The marked improvement of experimental data promises to provide access to increasingly complex systems, especially semiconductor nanoparticles, synthetic polymers, biopolymers and other chromophores absorbing in the UV. Details of the detection system and components are examined to reveal the influences on data quality, and to guide further developments. The benchmark comparisons of data quality across platforms will also serve as a reference guide for evaluation of forthcoming commercial absorbance optics.

Introduction The open source multiwavelength (MWL) absorbance optics for analytical ultracentrifugation (AUC),1 first presented by Bhattacharya et al. and developed in academia in collaboration with an industrial partner,2 is typically referred to as the MWL detector.3 Since its introduction in 2006,2 the MWL optics have undergone continual development in the Cölfen lab,4-7 and since 2010 have been significantly advanced by contributions from the Peukert lab of FAU.8-10 A commercialized spin off version of the design has also been introduced by Nanolytics of Potsdam.7 Highlights of the generational developments in MWL detector technology have been recently reviewed by Pearson et al.3,7 The industry and academic standard for AUC instrumentation has been the XL-A from Beckman-Coulter, since its introduction in 1991.11 The XL-A platform has been the technology from which a renaissance of AUC use

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has come forth, significantly buoyed by advances in evaluation methods.12-30 Yet, the underlying hardware of the XL-A absorbance optics has remained essentially unchanged since its development in the early 1990s. With the exception of the MWL detection optics implemented by a few research groups, the XL-A has set the standard for AUC data quality worldwide. However, the next generation of AUC hardware is already emerging both from academic and commercial developments. The coming years are likely to see abundant enhancements in the performance and capability of AUC absorbance technology. While the commercial XL-A is technically capable of acquiring three wavelengths in a sedimentation velocity (SV) experiment, it is seldom used in this mode.7,31,32 A redesigned Optima AUC by Beckman Coulter presented early this year aims to overcome this, but is restricted to a limited number of wavelengths in SV experiments, as all wavelengths have to be acquired consecutively.33 Remarkably, the MWL architecture allows for the acquisition of full UV/Vis absorbance spectra (230 nm to 1000 nm, depending on the grating option of the CCD spectrometer) within a single experiment. The additional layer of spectral information combined with typical hydrodynamic information collected in an AUC experiment, has been shown to provide a wealth of new possibilities for analysis.30,34 Even without analysis, the raw data already contains information on the number of species and their spectral properties.7,8,35 The spectral data acquired will also compliment already developed multi-signal AUC experimental methods and evaluation techniques, such as those combining absorbance detector signals with the refractometric and fluorescence detector signals.32 Challenges of broad spectral absorbance detection have been documented in the continual development of the MWL system, including noise levels of the spectrometer based detection design, as well as chromatic aberration present in the lens based optics.5 Nevertheless, the effectiveness of the MWL detection system has been repeatedly demonstrated, allowing for an ever greater number of experimental investigations.7-10,30,34,36-43 The latest improvements in detector design, highlighted here, will set a new benchmark for broadband spectral absorbance signals in AUC. The MWL detector hardware can be obtained free of charge within the Open AUC project.1 Design drawings and a listing of necessary hardware components can be found at http://wiki.bcf2.uthscsa.edu/openAUC/. The data acquisition software is provided upon request: http://lfg.uni-erlangen.de/research/AUC/.

MWL detection in AUC – An overview of hardware concepts All MWL detector designs feature a xenon flash lamp with fiber optic cables delivering light into the vacuum chamber of the AUC. The various design concepts for MWL optics are summarized in Figure 1. The exit tip of the fiber optic cable is connected to the illumination optics on the lower assembly of the sliding arm. The illumination optics are


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mechanically fixed to the upper arm assembly holding the imaging optics and detector, such that the illumination and imaging optics scan in unison across the cell channel.7 The tip of the fiber optic cable is the point source approximation to the illumination optics. Typically 100 µm to 200 µm fibers are used. A secondary design modification uses a slit adapter fixed in front of the fiber tip to reduce the point source size, and has been successfully implemented with the lens focused and mirror focused illumination optics.8 Because the fiber tip or exit slit is always a point source approximation, the ability to focus or collimate the illumination beam is limited.

Figure 1 Optical design concepts for MWL-AUC hardware. (A) Lens focused illumination – lens imaging. (B) Lens collimated illumination – lens imaging. (C) Mirror collimated illumination – lens imaging. (D) Mirror focused illumination – lens imaging. (E) Mirror collimated illumination – mirror imaging. (F) Mirror focused illumination – mirror imaging

With lens focused illumination optics, shown in Figure 1 (A), a 10 mm bi-convex lens is positioned to image the fiber optic tip source slit to a spot at the mid-plane of the cell sector. The imaging optics is subsequently brought to focus on the same illumination spot in a confocal type arrangement. The image distance (i.e. from the principle planes of the illumination focusing lens to the mid plane of the cell), is approximately 50 mm. With this geometry, the point source (i.e. fiber tip) must be positioned 12.5 mm behind the lens to bring the light to focus. The 100 µm fibers from OZ optics have a numerical aperture (NA) of 0.22. However, the stop radius of the 10 mm lens is 4.5 mm. Therefore, a 100 µm fiber tip or slit width will result in an object space NA of 0.34, and NA of ~0.09



in image space with -4x magnification, and thereby produce a spot size of approximately 400 µm at the mid plane of the cell. A focused beam lens based source serves to provide significantly higher source luminance on the region of measurement, and therefore improved signal quality. However, a focused beam is more susceptible to chromatic aberration, and should be used cautiously when broad spectrum signals are recorded. Lens collimated illumination optics, schematically depicted in Figure 1 (B), will still be subject to chromatic aberration, although to a lesser degree. While the chromatic aberration of refractive optical elements, i.e. lenses, is a recognized problem in the MWL instruments, it can also be used advantageously to selectively enhance the light power from spectral regions. That is, by adjusting the focus of the lens, the generally lower UV sensitive regions of the spectra can be intensified. Various modifications have been applied to improve the MWL detector design. These include using the aforementioned collimated illumination beam, changing the focal length of the imaging optics or position of the detector, adjusting the aperture of the imaging optics and using lamps with different power ratings. Typically changing one optical component requires subsequent changes to another component and involves a concerted alignment focusing and optimization routine. Both lens focused and collimated illumination optics have been used successfully with 40 mm and 50 mm focal length lenses and without modification to the basic architecture of the instrument. Moreover, both setups have been shown to provide adequate imaging over narrow spectral regions.7-10,30,34-41 The concerns of chromatic aberration in a broadband optical system are most readily addressed by exchanging lenses for reflective mirror optical elements. The introduction of mirrors presents unique challenges to the design of an optical system, especially with the space constraints of the centrifuge vacuum chamber. Here we describe the use of offaxis parabolic (OAP) mirrors in our optical system. In section 0, the performance of the mirror design is compared to the other setups, while the challenges of this particular setup are detailed in section 0. Up to now, Thorlabs is the only manufacturer offering preassembled reflective collimators. Four different models with effective focal lengths of 7 mm, 15 mm, 33 mm and 50.8 mm are available, all equipped with an UV-enhanced aluminum coating, and adapt to standard SMA optical connectors, being the “SubMiniature version A”. The OAP mirror collimators treat the fiber optical tip as a point source and reflect the emittance cone rays along a collimated path, producing a circular beam front with an approximately uniform intensity cross-section. The reflective elements, being free of chromatic aberration, produce good light collimation across the spectrum. This design implementation of the mirror collimation optics is illustrated in Figure 1 (C).


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As mentioned previously, the source illumination lens optics use a short focal length element. The focal length is approximately inversely proportional to the NA for a given entrance pupil, therefore short focal length elements will allow maximum light capture, while producing a narrower beam diameter with higher luminance (luminous intensity per m2). In contrast to lens source optics, a single reflective OAP mirror element does not provide the option of illumination with a focused beam, because an OAP mirror will collimate rays from a point source but does not have the geometry to effectively refocus to a spot. Thus, spot focusing requires an optical system with a second OAP mirror. This OAP mirror replaces the 90° plane mirror directly below the rotor. A stock 33 mm reflected focal length (RFL), also available from Thorlabs, is the correct dimension to refocus the collimated beam to the mid-plane of the centerpiece. This design has been implemented, in order to gain the benefit of increased signal, while minimizing an apparent stray light phenomenon. Mirror focused illumination combined with a lens imaging setup is depicted in Figure 1 (D). Again, a comparison of the performance of the different setups is given in section 0. Next, the basic concept for the design of an OAP mirror based imaging system will be presented. Two OAP mirrors can be implemented to effectively image the centrifuge channel. A second plane mirror is required to allow the path length of the optics to fit within the vacuum chamber. The layouts for the assemblies with collimated and focused illumination path are shown in Figure 1 (E) and (F), respectively. In addition to the optical developments, new spectrometer and flash lamp hardware has recently become available. Ocean Optics last year released the Flame spectrometer series to replace the USB2000+, and has the option of interchangeable entrance slits. The advantage of slit size is discussed at the end of section 0. However, the Flame spectrometer uses the same CCD chip as the USB2000+, the Sony ILX-511, and therefore is expected to have similar performance. Hamamatsu has also recently released a new Xenon flash module, the 20 W L12745. The L12745 is reported in the specifications to have a similar noise level to the previously used L9455, but has the advantage of offering significantly higher energies per flash (maximum input energy per flash is 320 mJ compared to 50.4 mJ) and much higher flash rates. In fact, with the smallest capacitor model, the L12745 is as powerful as the highest capacitor model L9455, but has the possibility to flash at up to 1000 Hz, which is exactly equal to 1 flash per rotor revolution at the highest rpm of the AUC. Testing thus far with the new lamp has shown the noise level to be superior to the L9455. One drawback is the L12745 does not currently come with an SMA905 fiber adapter, and therefore requires a custom coupler. The final contribution to the hardware improvements is the addition of flash to flash correction, also referred to as flash referencing, which is discussed in section 0. Here a small portion of light is decoupled from the incident light beam and sensed by a secondary detection unit. Either an additional spectrometer or a photodiode can be used



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for this. However, the latter can only record the integral signal over all wavelengths. A number of apparatus were tested for sampling the intensity fluctuations, including a photodiode placed next to a section of fiber optic cable that was stripped of the outer sheathing, a UV beam splitter prism, and a bifurcated optical fiber splitter. A three legged fiber splitter provided by far the best results and is convenient to implement. A solarization resistance fiber splitter from Ocean Optics was acquired. The first leg of the fiber splitter is coupled to the lamp and serves to mix any inconsistencies across the flash arc. The first fiber leg then feeds the “homogenized” light to the bifurcated splitter. One leg of the splitter goes to the vacuum chamber fiber feed-through, and the other to the second spectrometer for the flash referencing. The “homogenizing” of the first fiber leg was found to give a significant improvement over the bifurcated fiber directly coupled to the lamp.

Mirror optics Challenges and opportunities arising from mirror optics The beam diameter generated by an optical element can be directly related to its effective focal length EFL as it is given by: ݀௕௘௔௠ = 2 ∙ ܰ‫ܣ‬௙௜௕௘௥ ∙ ‫ܮܨܧ‬

(1)

NAfiber denotes the NA of the fiber. Hence, the beam diameter is directly proportional to the EFL. In turn, a larger beam diameter means luminance is lower and results in diminished signal quality. However, beam collimation also assumes a point source approximation. As the MWL setup uses optical fibers for the illumination point source, a dependency on the fiber core diameter is observed. In general, the luminance increases for a larger core diameter but the beam quality is significantly affected by divergence from collimation. This holds true not only for the lens but also for the mirror based illumination optics. The divergence is obtained by tracing a ray from the bottom edge of the fiber core through the top edge of the OAP mirror. Applying the thin lens approximation yields the following correlation for the divergence angle: ߆ = tanିଵ ቀ

ௗ್೐ೌ೘ ாி௅

ቁ

(2)

A larger core diameter will thus not only increase the beam diameter dbeam and intensity but also the divergence angle. For the experimental setup, an optimum has to be identified between intensity (large fiber) and sufficient radial resolution (small fiber). Core diameters between 100 µm and 200 µm were found to be reasonable for the mirror and lens based setups. The best choice identified for a mirror based design is a 15 mm OAP mirror and a 100 µm fiber. Thorlabs 15 mm OAP mirrors specify a 4 mm beam


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diameter with a 0.13 NA fiber. The 100 µm fibers from OZ optics have a NA of 0.22, resulting in a beam diameter of 6.6 mm and maximum divergence angle of 0.4° in contrast to collimation with a 10 mm lens, where the beam diameter is 4.4 mm with the same divergence angle only in dependence of the fiber NA. It is also important to note that point source imperfections, and mirror distortions in general will be significantly worse for short focal length mirrors. In addition, beam collimation quality becomes increasingly sensitive to fiber tip positioning. Fiber optic cables with adjustable SMA adapters available from OZ optics, allow for precise positioning of the tip in the OAP collimator housing. By replacing the short focal length lens of the illumination optics with an OAP mirror, the most significant cause of chromatic aberration is mitigated. As indicated in section 0, using collimated illumination has advantages and disadvantages. The advantage of collimated light is improved radial resolution. By illumination with rays parallel to the optical axis, the light passing through the sample column will be effectively confined to the radial increment selected for imaging, typically 50 μm. Whereas, the convergent illumination of focused optics will enter the sample column at an angle of ~2° from the optical axis, and therefore pass through regions of the solution immediately adjacent to the region of measurement. That is, if the selected measurement region is a 50 μm segment of the solution, then the light rays imaged from that region, have passed through regions of solution on either side, beginning at points next to the cell window set 200 μm away from the measurement segment. This is further complicated by the fact that the segment of the measurement region will also possess a refractive gradient, and therefore be subject to skewing of the illumination beams.44 Such behavior will of course be present in both focused and collimated illumination optics. Focused illumination has the direct advantage of providing increased intensity compared to collimated illumination. Therefore, mirror focused illumination was implemented into the lower mechanical assembly of the scanning arm as a subsequent design attempt. Here a stock OAP mirror from Thorlabs was selected that provided a RFL (33 mm) to refocus the collimated illumination beam onto the mid-plane of the cell, in a confocal type arrangement. The basic design concept of this focused illumination assembly is depicted in Figure 1 (D) and (F). Mirror focused illumination, similar to lens focused illumination, has the significant added benefit of better signal to noise ratio (SNR) due to higher luminance while requiring lower lamp power. This makes the adaptation of flash referencing simpler, described in section 0, as there is plenty of available light that may be sampled by a secondary detector. The introduction of mirror imaging optics presents the latest significant advancement in MWL-AUC optical design. Here two OAP mirrors are implemented into the scanning arm mechanics as illustrated in Figure 1 (F). Optical design parameters were determined as



part of a parallel study examining the basic optical principles of UV/Vis imaging in AUC. The resulting mirror optic models, as adapted for use with the MWL mechanical system, will be described in detail within the context of our forthcoming publication. The results of the different design concepts are compared in section 0. Unfortunately, the percent reflectance has large wavelength dependence, and is especially reduced in the deep UV or NIR (see Thorlabs documentation). Furthermore, the mirror imaging system uses three optical elements. Therefore the losses will be a sum from each element. In consequence, less light will be available in the UV and NIR when comparing the mirror imaging with the lens imaging setup. However, multi-flash acquisition, further emphasized in section 0, can help to overcome this drawback of the mirror imaging system.

Performance of mirror optics Several methods have been proposed to assess the resolution performance of the absorbance optical systems. Including, the edge of a counter balance, the edge of a cell, radial calibration masks and optical slit masks.5,7,45 While each method provides a relative indication of the radial resolution of the optical system, each is also subject to limitations of interpretation. For example, the counter balance mask edges are positioned at a plane that is equivalent to the lower window-centerpiece plane when installed in the rotor (see Figure S1 in the SI). This would be near the exit window for the XL-A/I optics, but is conversely the entrance window for the MWL optics, since the MWL imaging optics are positioned above the rotor. This is an imaging plane that is not relevant for either the XL-A or MWL optics, and is therefore nonsensical as a resolution metric. In other words, an optical system that is focused to give sharp counterbalance edges is out of focus on the plane of interest within the solution column. The radial magnification correction masks previously reported,45,46 use two windows to fix the thin metal mask at the mid-plane of the cell, and would therefore be a more meaningful indicator of optical focus. However, the authors did not attempt to include a measure of this metric in their study. A 200 μm slit mask was reported by Strauss et al.5 This device also includes the possibility of additionally positioning a cell window at the centerpiece exit plane, which will extend the optical focusing path length. The edge of a channel bottom is suggested by Pearson et al. as a more meaningful indicator of the radial resolution, since the channel extends the entire 12 mm of the solution column, and includes any effects of the cell windows.7 This is only possible with centerpieces that have a short channel length, and are therefore not blocked by the cell screw ring. The channel bottom also has the added complication of inducing reflections from any light rays that do not pass vertically through the solution column, and can produce optical artifacts. We also include the scan of a high absorbing liquid as alternative indicating device. In this case the aromatic molecule toluene, measured in a titanium centerpiece spinning at 20 krpm, absorbs nearly all light below 280 nm. This effectively


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creates a “liquid edge” that will have a vertical plane exactly parallel to the axis of rotation, and will follow the arc of the rotation of the cell. Alternatively, a simple mixture of food coloring dyes provides an opaque aqueous solution and exhibits no sedimentation at moderate rotor speeds. It is important to note that the meniscus will sharpen with increased rotor speed, as the centrifugal field overcomes the capillary forces and the downward effect of earth’s gravity on the solution column. Above approximately 15 krpm, the meniscus shape is observed remaining constant (data not shown). normalized intensity at 270 nm / -

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1.0 0.8 0.6 0.4

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0.2 0.0 0

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Figure 2 Comparison of the sharpness of several edge devices scanned with the mirror focused illumination – mirror imaging optics. The liquid edge is taken from a high absorbing, non-sedimenting sample scanned at 20 krpm and is the most realistic indicator of stray light.

While none of these methods described are true metrics of radial resolution, they can be useful indicators of optical alignment and focus. The “liquid edge” method in particular provides a nice indication of total stray light present, even if the source of the unwanted light is not clear. Figure 2 depicts the edge resolution for the mirror focused illumination – mirror imaging optics using several tools. It is an example of the typical scan quality possible with this detector setup. However, it is also important to note that the edge effects do not translate to a direct metric of overall system accuracy as observed by the evaluation of well-known standards as will be shown in section 0. As discussed in section 0, the lens based illumination optics of the MWL detector use a short focal length refractive element (10 mm lens) to provide a narrow collimated or focused illumination beam. Lens based setups are therefore significantly influenced by spherical and chromatic aberration. Spherical aberration is known to often be a limiting factor for imaging optics. However with broadband lens optics, chromatic aberration is likely to be a dominant contributor to the overall aberration profile of the optical system, and will be significantly influential on both the imaging and the illumination elements. The effects of chromatic aberration can be seen in several artifacts of experimental data.



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Figure 3 Radial intensity scans of a reference channel meniscus at representative wavelengths of 250 nm (blue lines) to 600 nm (red lines) in 50 nm increments. (A) Lens focused illumination – lens imaging. (B) Lens collimated illumination – lens imaging. (C) Mirror collimated illumination – lens imaging. D) Mirror focused illumination – lens imaging. (E) Mirror collimated illumination – mirror imaging. (F) Mirror focused illumination – mirror imaging. All plots show a 1.2 mm radial scan segment with scans step size of 10 μm. The relative intensities of the wavelength signals are normalized and artificially offset for clarity. The letters correspond to the optical system design concepts of Figure 1.

A convenient indicator of chromatic aberration is the meniscus appearance changing with wavelength. With lens focused illumination - lens imaging optics the chromatic aberration effects, as observed by the meniscus artifacts, are quite extreme, as shown in Figure 3 (A). In contrast, it is apparent from Figure 3 (B) and (C) that one can expect fewer optical artifacts with a collimated illumination beam (both lens and mirror) and lens imaging. The meniscus and cell edges will typically appear sharper, with less chromatic aberration. However, a strange result was observed when mirror collimated illumination was tested with a mirror imaging system, where stray light was apparently


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amplified by the imaging optics. While the meniscus appearance was quite sharp, see Figure 3 (E), a general indicator of good focus, and did not change with wavelength, the cell edges showed a large broadness (see Figure S2 in the SI). The edge broadness, as described previously is only useful as a relative indicator of imaging quality. However, the comparison with a lens imaging optical arrangement is clearly apparent. The origin of the stray light is difficult to ascribe with certainty, but we suspect it is a result of the baseline surface quality of the mirrors, and the fact that there are three reflectance surfaces in the optical path. Further tests were made with both lens imaging and mirror imaging. The dramatic effects of chromatic aberration are observed with lens imaging, Figure 3 (D), as revealed by the meniscus shifts. This observation is an indicator of the contributions of chromatic aberration from the imaging optics, irrespective of illumination contributions. In particular, when comparing results to Figure 3 (F), where mirror focused illumination and mirror imaging are implemented. Similar to the mirror collimated illumination – mirror imaging design concept, Figure 3 (E), but also using focused illumination, Figure 3 (F) shows the absence of chromatic aberration, but does not have the apparent stray light problem that was observed at the cell edges with collimated illumination. The edge imaging scans with the mirror focused illumination – mirror imaging optics are consistent with measurements that have been reported previously with AUC optics,5,7 but do not have a chromatic dependence, see Figure 2 and Figure 4. The stray light effect noted with mirror collimated illumination – mirror imaging, is somehow mitigated by the use of focused illumination. We suspect that this is due to the illumination beam being concentrated onto the measurement region of interest, whereby, the apertures of the imaging optics are more effectively able to cut off the rays that pass through neighboring regions of the solution column. While the meniscus shape in Figure 3 (F) is not the single sharp peak that is often expected in AUC absorbance scans, the peak artifact is still relatively narrow and the position remains consistent with wavelength. The often-observed sharp peak of the meniscus is a result of the infinite refractive gradient across the air-liquid phase boundary that will bend the illumination rays completely out of the imaging aperture. With focused illumination, the incoming rays have the possibility to be deflected at many angles and may subsequently make unpredictable reflections. This further emphasizes the point that the meniscus appearance is not a meaningful metric of overall imaging quality, and only serves as a qualitative indication of chromatic aberration. For the interested reader, a plot of meniscus shape versus wavelength was obtained with a commercial XL-A and is included in the Supporting Information (Figure S5), where chromatic aberration effects are also observed in the UV region. The primary advantage of focused illumination optics is much higher luminance compared to the collimated design, as all the radiant power emitted from the fiber is



concentrated to the segment of solution being measured. In contrast, collimation optics spread out the radiant power to the area of the circular beam. The higher signal intensity available with focused illumination improves the SNR of the experimental data and has been shown to produce excellent results with both lens and mirror designs. However, mirror optics have the added benefit of no chromatic aberrations, which significantly improves wavelength reproducibility as can be seen when comparing the lens and mirror setup, see Figure 3 (A) and (F). The higher light throughput with focused illumination also allows for the use of lower powered lamps, which has the benefit of reduced noise, higher flash rates, and less solarization of the fiber optic cables. Furthermore, issues with the focused illumination could be reduced by minimizing the NA of the illumination beam. A narrower cone of light entering or exiting the solution column will encounter less of the above mentioned features of focused illumination. For the present implementation, available stock mirrors were used that have focal lengths which most easily accommodate the geometry and space requirements of the AUC. Still, it is important to note that lens focused illumination optics have been shown to successfully produce high quality data,7-10,30,39,41 when properly adjusted on the spectral region of interest. However, when conducting broad spectrum analysis, the spectral resolution of the evaluation will be compromised, and a coarser solute discrimination will be implied. Finally, chromatic aberration was also previously characterized by use of a 200 μm slit mask 5 and, as noted above, is not a true radial resolution metric but can be a useful indicator of relative imaging quality and more importantly chromatic aberration. In Figure 4, the effect of spectrometer entrance slit size is probed by examining the scan of the 200 μm slit mask at several wavelengths using mirror collimated illumination and lens imaging. This slit mask assembly is identical to that reported previously, where a comparison of commercial instruments is also included.5 Also included is a scan using the latest mirror focused illumination - mirror imaging optics. Entrance slit width is typically varied in order to tune the wavelength resolution and sensitivity of a spectrometer. Here the entrance slit width also serves to limit the radial increment discretization of scan points. That is, assuming imaging magnification of 1, a scan will collect radial intensity points at the scan step increment set with the stepper motor, but each point will be an average intensity across the radial region of the spectrometer entrance slit width, typically 25 μm. Figure 4 illustrates the effect of spectrometer entrance slit width in broadening the edges of the slit mask target. From these results, the standard 25 μm spectrometer entrance slit appears adequate for the present optical setups. Figure 4 (A-E) also serves to illustrate the effects of chromatic aberration when scanning with lens imaging optics, as compared to mirror imaging optics (F).


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relative transmission / -

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1.0

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Figure 4 Radial intensity scans of a 200 μm slit mask at representative wavelengths of 250 nm (straight line / square), 350 nm (long dashed line / circle), 450 nm (short dashed line / triangle) and 550 nm (short-long dashed line / flipped triangle). Panels (A-D) are from an Ocean Optics Flame spectrometer with an XR grating, incorporating entrances slits of 10, 25, 50 and 100 μm, respectively, and with a mirror collimated illumination – lens imaging assembly. Panel (E) and (F) are from an Ocean Optics USB2000+ spectrometer with XR grating and 25 μm slit and mirror collimated illumination – lens imaging assembly (E) and mirror focused illumination – mirror imaging assembly (F), respectively.

Signal to noise ratio and random noise contributions Source to acquisition The detector noise will always be a combination of the source and acquisition device fluctuations. Signal intensity, registered as 0 to 65,535 counts by the 16 bit spectrometer, will be a function of the source luminance, absorbance by the sample or optical elements and sensitivity of the spectrometer chip. All of which will have a nonlinear and uncorrelated wavelength dependence. The USB2000+ Ocean Optics



spectrometers and L9455 Hamamatsu flash lamps have a manufacturer specified SNR, or coefficient of variation (CV) that will be considered: CV = rel. StdDev = 1/SNR. First the spectrometer noise profile will be considered. When using a continuous source lamp, the SNR of acquisitions scales approximately linearly with the relative illumination intensity. The data in Figure 5 show the SNR of a USB-DT lamp, as the intensity approaches the 65,535 count saturation of the detector, the SNR approaches 250:1 at 400 nm. This is in exact accordance with the specification provided by the manufacturer.47 However, when the same type of test is made with a 5 W xenon flash lamp module, the SNR at 400 nm is significantly lower for all but the lowest intensities, and does not scale considerably with intensity. The flash lamp noise is specified by Hamamatsu to be 0.41 % CV (SNR = 244:1).48 If we consider a single flash at 100% intensity, with one spectrometer acquisition per flash, then the combined expected error, by summing in quadrature, yields 0.57 % CV, or a SNR of 175:1. However, the experimental results show a maximum SNR of only approximately 80:1 at the highest intensity. signal to noise ratio at 400 nm / -

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250

USB-DT L9455-11

200 150 100 50 0 0

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Figure 5 SNR of an Ocean Optics USB2000+ spectrometer with XR grating as a function of measured signal intensity at 400 nm. A plot of the wavelength dependent SNR is depicted in Figure 7. Signal intensity was adjusted by an opto-mechanical iris. The Ocean Optics USB-DT continuous lamp was recorded with a spectrometer integration time of 80 ms, The Hamamatsu L9455-11 xenon flash lamp was recorded with synchronized external TTL triggers at 10 Hz.

Flash lamp contributions Flash to flash variation is inherent in the normal operation of a Xenon flash lamp. The L9455 flash lamps from Hamamatsu, used in the MWL system, are specified to have a CV less than 0.5 %. However, Figure 6 shows the relative spectral output and SNR variability of the lamp output intensity at 450 nm for different xenon flash lamp models. It is readily apparent that none of the available lamps produces a noise level meeting the device specifications. The three lamp models in the L9455 series contain the three discharge capacitor models available from Hamamatsu, and demonstrate that the


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A

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L9455-13 SNR = 50.4:1

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300 450 600 750 900

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L9455-11 SNR = 33.0:1

4 3 2 1 0



inherent SNR of the lamps does not scale with capacitor size, i.e. light output. Furthermore, it should be noted that significant variation in lamp noise has been observed by users for identical lamps of the same model L9455 series. The data shown in Figure 6 are from lamps which have been subjected to use for various amounts of time. Nevertheless, it illustrates the general trend that has been observed. The variation due to lamp age is similar to the random inherent noise levels that are observed with identical new lamps, presumably due to variations in the quality of the electronics and bulb manufacturing. It is important to mention that the newest model L12745 lamps from Hamamatsu show a significant increase in SNR, see Figure 6. Furthermore, the L12745 noise level does not scale with flash frequency as significantly as the L9455 lamp (see Figure S3 in the SI), and is therefore much better suited to high flash rate experiments.


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6 5

D

L12745-03 SNR = 73.5:1

4 3 2 1 0

300 450 600 750 900

300 450 600 750 900

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wavelength / nm

Figure 6 Relative spectral output and SNR variability for different xenon flash lamp models. The lamps and spectrometers were fixed in an optical track with approximately 20 cm of free space between the arc position and spectrometer entrance slit. The three spectral profiles from each lamp are recorded with an internal trimmer setting at the lowest (black), highest (green) and middle (blue) intensity position. The SNR from the 450 nm signal (red) was calculated from the average of 6000 flashes collected with 100 Hz flash pulses and synchronized spectrometer acquisition, with the internal trimmer at middle intensity. Note: The L12745 was measured with a 5 mm biconvex lens directly after the lamp window. This distorts the light throughput from the lamp, relative to the L9455 models with fixed SMA ball lens couplers. In practice, the L12745-03 model is seen to have similar light output to the brightest (L9455-11) lamp when coupled to the fiber optic assembly.

The results indicate that the extent of the fluctuations depends to a large degree on the lamp model and the repetition rate. The SNR has been observed to scale with internal trimmer setting to approximately half maximum output. However, above the low light



regime of approximately 10,000 counts, additional intensity trimmer adjustment does not improve SNR. For potential noise treatment, it is important to identify if and to what extent the lamp fluctuations are wavelength dependent. Our studies revealed that the relative noise contribution can be assumed to be wavelength independent in good approximation for the Hamamatsu light source used in the MWL setups. This has meaningful implications for how the flash variability can be corrected, as is discussed in the following sections. Spectrometer contributions The second source of noise is the spectrometer. Dark current is corrected for during the measurements by subtracting an acquisition with no illumination. However, read-out noise contributes to every acquisition and will be the most significant source of the 250:1 SNR inherent to the spectrometers. Due to its random but wavelength dependent profile it can only be reduced by averaging multiple wavelengths or acquisitions. Wavelength averaging is a valid procedure as the number of pixels/nm is typically higher than the spectral resolution of the detector. The spectral resolution will be dependent on the grating type and entrance slit size for the given path lengths and CCD chip of the spectrometer. E.g., for a broadband XR-type grating together with a 25 µm slit, the Ocean Optics USB2000+ or FLAME-S, resolution is 1.9 nm. The spectral range for this system is 925 nm, while the CCD has 2048 pixels. Hence, four wavelengths can be averaged without losing any spectral information. This simple procedure has the result of improving the SNR by 50% on average.

Strategies for flash lamp noise compensation Flash averaging The simplest approach to reduce random noise during measurements is to average multiple acquisitions. In such a case, the noise will scale approximately with the square root of the number of averages. An immediate trade-off is the increased scan time needed to record a radial extinction profile. At high rotor speeds the period of rotation is very short, therefore subsequent flashes can be quickly acquired for averaging, without a significant increase in overall scan time. However, for large, fast sedimenting particles that must be measured at lower rotor speeds, limited averaging may be applied without increasing the overall scan time too much. Flash averaging is most effective when sufficient light is available. Whereas, for low light levels, such as with a reduced illumination point source dimension or collimated illumination optics, the read-out noise contribution will outweigh the contribution from the flash lamp and averaging will no longer provide meaningful SNR improvement. This is demonstrated in Figure 7, where SNR is plotted as a function of wavelength when averaging multiple flashes.


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Multi-flash acquisition Often acquisitions are desired with spectral regions where there is limited signal intensity. These low light regions may be the result of optical designs, where the Xenon lamp has low emission, or where the CCD chip has a low sensitivity, such as in the UV or NIR. Multi-flash acquisition is a newly adapted operating mode especially useful for such low light conditions. Herein, the integration time of the spectrometer is extended and multiple flashes are accumulated in a single spectrometer acquisition. As a result, it doesn’t require recording and averaging the detector read-out noise multiple times. Hence, a much better SNR can be expected from multi-flash operation.

120

25 1 flash 1 flash / 2 aver. 1 flash / 3 aver. 20 2 flashes 3 flashes 15

80

10

40

5

200

160


Figure 7 depicts the wavelength dependent mean standard deviations obtained for single-flash and multi-flash data acquisition. A significantly better SNR is obtained when comparing the results to single-flash acquisitions. The difference is most pronounced where signal intensity is low. The large xenon emission spikes result in regions of the spectrum having low signal, while the spikes may reach near the saturation limit of the detector in the VIS. Therefore, care must be taken when selecting the number of flash accumulations so the recorded signal remains below 65,535 counts. For many applications it is still reasonable to choose a high number of flashes, even though this will result in unusable data in the VIS. Examples, are samples that only show extinction in the UV or NIR. Here, the large benefit of MWL detection can be exploited by just using the wavelengths of interest. In line with Figure 7, baseline noise data for the commercial XL-A machine is included in the Supporting Information as a plot of SNR versus wavelength relative to signal intensity (Figure S6).

signal to noise ratio / -

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0

0 300

450

600

750

900

wavelength / nm

Figure 7 Comparison of wavelength dependent SNR obtained for single-flash, multiple averages and multi-flash data acquisition, plotted on left axis. The reference intensity spectrum is shown in red plotted on right axis. Recorded with a L9455-11 Hamamatsu flash lamp and Ocean Optics USB2000+ spectrometer with XR grating and 25 μm slit.

Multi-flash acquisition has been integrated into the latest version of the data acquisition software and can also be coupled with flash averaging to improve the SNR further.



Unfortunately, multi-flash acquisition is not available for gravitational sweep data where the rotor speed is continuously increased. Such operation would require the integration time to dynamically adapt to the rotor frequency, which is not feasible with the CCD detector currently applied in the setup due to the time required to programmatically change the integration time. More information on the latest data acquisition software for the fiber coupled MWL AUC can be acquired from the author upon request. Flash referencing Flash referencing, also referred to as flash to flash correction, is an alternate way to improve the SNR. In contrast to the previous techniques, no additional time is required to either average or integrate multiple flashes. As described above, a fiber splitter in conjunction with a secondary detector records the relative intensity fluctuations of the flash lamp. By recording the fluctuations in the intensity profile it becomes possible to correct for the light source induced noise. This technique is routinely applied by the commercial AUCs, type XL-A, equipped with an absorbance detector. The results of flash referencing are illustrated in Figure 8, showing an approximate doubling of the SNR when implemented. As is shown, a scan setting with five averages would be required to achieve the same SNR as a single acquisition with flash referencing. When flash referencing and averaging are applied together, the SNR increases further, as expected. Two modes for flash referencing have been implemented and tested, normalization and integral normalization. Flash normalization works by first smoothing the spectra of the reference spectrometer, then applying the correction factor to each respective wavelength of the scanning spectrometer. In this way, the grating parameters of the spectrometers do not need to match perfectly, but any wavelength dependent noise will still be cancelled out. Integral normalization works by taking the average noise increment from a spectral range, and applying the same correction to all wavelengths of the scanning spectrometer. This mode is similar to what would be achieved if a single photodiode was used instead of a second spectrometer.


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180

sin signal to noise ratio at 400 nm / gle no f las rm ho ali za pe t rat i o int n( ion eg 2 30 ral 70 no 0n rm no . (2 m) rm 30 aliz 70 ati 0n on int m) eg (30 ral 0-4 no 50 rm nm . (3 ) 00 -45 0n 5a m) ve r. + 5a no ve rm rag . (2 es 5a 30 -70 ve r. + 0n m) int eg r a (23 l 0-7 norm . 00 nm )

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150 120

90 60 30

0

Figure 8 Comparison of SNR improvements when using variations of flash referencing acquistion modes. Recorded with a L9455-11 Hamamatsu flash lamp and Ocean Optics USB2000+ spectrometer with XR grating and 25 μm slit.

Wavelengths invariant noise correction In principle, multi-flash operation or flash averaging can be combined with wavelength invariant noise correction. Here, a non-absorbing region of the spectra is selected and used to correct for flash to flash fluctuations. However, for low light levels applicability has to be questioned because of the wavelength dependent nature of the spectrometer read-out noise and the possibility to implement the aforementioned flash referencing. For a user seeking to build a system without the additional cost and complexity of flash referencing hardware, this could offer a simple means of signal quality improvement for many applications.

Comparative MWL study In the previous pages, hardware and software modifications have been presented for the MWL detector. In this section, the lens focused illumination – lens imaging setup will be compared to the latest mirror focused illumination – mirror imaging detector by investigating the standard protein BSA. The MWL analysis methods now available in UltraScan3 allow for Lamm equation fitting of the full spectrum data sets.34 The resulting wavelength dependent sedimentation coefficient distributions provide a very sensitive indicator of chromatic aberration present in the data. Figure 9 shows data collected with lens focused illumination – lens imaging and mirror focused illumination mirror imaging optics using the latest data acquisition software. The mirror optics provided a spectral profile that matches very well with that expected for the BSA



monomer. Whereas data from lens focused illumination and imaging optics, subjected to chromatic aberration, show distinctly wavelength dependent artifacts in the resulting fits Figure 9 (A). The spectral discrepancies seen in the 3D plots with lens optics appear more extreme than the effective results. That is to say, when an integral peak value is extracted over the entire monomer species, the spectral discrepancies are largely cancelled out and a typical BSA monomer spectrum is recovered. Nevertheless, the spectral consistency of the mirror illumination - mirror imaging data is qualitatively impressive. The improvement in spectral performance of the optics will also provide increased reliability for other MWL data evaluation methods. For example, the spectral deconvolution described in 30 and 34.

Figure 9 SV data evaluation results of BSA recorded with the lens focused illumination - lens imaging optics (A and C) and with mirror focused illumination - mirror imaging optics (B and D). Panels (A) and (B) show 3D plots of sedimentation coefficient versus wavelength as obtained by parallel processing of the individual wavelength datasets with the 2DSA-MC analysis method in UltraScan3. Panels (C) and (D) are pseudo 3D projection views of the same results.

The results of the evaluations in UltraScan3 show excellent fits to the experimental data for both optical systems evaluated at the peak 280 nm wavelength. The lens based system data was fit with an RMSD of 0.00217 and giving a BSA monomer s20w = 4.392 S. The mirror based system was fit with an RMSD of 0.0034 giving a BSA monomer s20w = 4.468 S. The lens based system was tested with fatty acid free, ≥98 % pure BSA from Sigma-Aldrich® CAS-Nr. [9048-46-8], diluted in 100 mM NaCl to a concentration of 0.5 g/L (~0.5 OD at 280 nm) and performed at 20 °C and 50 krpm, with acquisition settings for three flash accumulations and four averages, with 50 μm radial step size providing a channel scan time of ~1 minute. The mirror based system was tested with ACS Paragon Plus Environment

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fatty acid free, ≥98 % pure BSA from Carl Roth® Albumin Fraction V CAS-Nr. [90604-298], diluted in PBS buffer (5.60 mM Na2HPO4, 1.06 mM KH2PO4, 154 mM NaCl) to 1 g/L (~1.0 OD at 280 nm) and performed at 20 °C and 50 krpm, with acquisition settings for three flash accumulations and three averages, with 50 μm radial step size providing a channel scan time of ~1 minute. The BSA in PBS sample closely matches that used in the 2015 multilaboratory study,46 and the run conditions are identical. The results of the BSA monomer sedimentation coefficient are ~1.5 % and ~0.5 % lower than the multilaboratory study reported, for the lens based and mirror based optics respectively. However, the comparison is subject to variability due to precision of the solvent density and viscosity estimations in the conversion to s20w, being the sedimentation coefficient corrected for water at standard conditions, and can be considered to be within the basic precision of the instrument. Moreover, charge effects might still play a small role as a slightly lower salt concentration was used for the measurement in the lens setup. Discrepancies in the fit quality, as observed by the RMSD of the 2DSA evaluations, are due to the relationship of sample concentration and reference light intensity. Figure S4 in the SI shows how RMSD varies with sample optical density and light intensity for the two test samples of Figure 9. For a comparison of the chromatic aberration effects also present in commercial instrumentation, experimental data was collected at three wavelengths in a Beckman-Coulter XL-A. The results are provided in the Supporting Information, Figure S7 and Table S1. As a final example a Myoglobin sample and a mixture of BSA and Myoglobin were tested at 60 krpm and 20 °C with the mirror optics detector. The measurement cells, BSA, buffer and all other acquisition parameters are identical to that previously shown in Figure 9 for the mirror optics assembly. The myoglobin sample was from SigmaAldrich® CAS-Nr. [100684-32-0]. Figure 10 shows the results from analysis of the Myoglobin sample alone, and the mixture of BSA and Myoglobin. In a previous study,7 raw data of a BSA - Hemoglobin mixture was shown, demonstrating qualitatively the spectral possibilities of the MWL optics. In a corresponding study,34 a BSA - DNA mixture was evaluated showing the capabilities of analytical methods to process MWL data and recover spectral data from the resolved mixture. The results of Figure 10 show that the same analytical methods applied to a sample mixture extending over a much broader spectral bandwidth achieve significantly better sedimentation coefficient and spectral resolution than was previously possible.



Figure 10 SV data evaluation results of Myoglobin (Panels A and C) and a BSA/Myoglobin mixture (Panels B and D) recorded with mirror focused illumination - mirror imaging optics. Panels (A) and (B) show 3D plots of sedimentation coefficient versus wavelength as obtained by parallel processing of the individual wavelength datasets with the 2DSA-MC analysis method in UltraScan3. Panels (C) and (D) are pseudo 3D projection views of the same results. Panel (E) shows the absorbance spectra of the mixture, and the spectra of the AUC resolved solutes extracted from the 2DSA data. Panel (F) shows the extracted spectra along with the corresponding spectra of the pure samples collected in a normal spectrophotometer.

Concluding remarks In the preceding pages we have described the evolution of optical designs through the Open AUC MWL detector developments, leading to the successful implementation of a complete mirror optic hardware design, free of chromatic aberration and providing excellent data quality across the UV-VIS spectrum. The hardware advances have been complimented by new improvements in the software acquisition modes, greatly


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reducing the random noise in the data and extending the useable range of the spectra. These developments directly translate into improved MWL characterization as was shown exemplarily for the proteins BSA and Myoglobin. The application of the new acquisition modes should always be chosen with attention to specifics of the sample under investigation, such as: What region of the spectra is of interest and what is the reference intensity signal across that region? Is this a fast sedimenting or slow sedimenting particle, i.e. to measure at low rotor speeds or high, and what will be the resulting scan time be for an acquisition mode selection? When setting up an acquisition mode, a user must always consider the basic trade-off of scan time versus acquisition repetitions. For example, with an acquisition mode using three flash accumulations, three averages and two sample channels, the acquisition repetitions will be the resulting product. Here 18 flashes are required for each radial position of a scan, which leads to a scan time of approximately 1 minute at rotor speeds of 40 krpm to 60 krpm. As a rule of thumb, acquisition modes are selected to provide a scan time of 1 to 3 minutes, depending on the details a user hopes to extract from the sample under investigation. Typical scan times for this instrument and commercial devices have been reported previously.7

Outlook The basic architecture of the Open AUC MWL detector has centered around the compact Ocean Optics spectrometers, Hamamatsu xenon flash lamps and fiber optic vacuum feedthroughs. The spectrometers use a Sony CCD chip that is representative of the general capability of linear CCD based detector devices. Newly available compact spectrometers have begun implementing CMOS chips, but a preliminary test has shown the improvement in quality to be negligible. While faster, the CMOS chips still do not have an acquisition rate fast enough to synchronize with the high speed rotor, and therefore give no alternative to the flash lamp. Therefore it is unlikely that improvement in spectrometer possibilities will arrive in the foreseeable future. In addition, the newest generation of flash lamps from Hamamatsu has recently arrived and provides a generous improvement in data quality. In perspective of the past development cycles for xenon flash lamps, we do not anticipate new advances in flash lamp technology to become available again soon. Without a complete redesign of the optics hardware, reliance on fiber optic cables to feed light into the vacuum chamber will continue. New developments in fiber optic materials are continually emerging. However, to date, the newest available solarization resistant fiber optic cables have shown only marginal improvement in UV transmission and stability.10 In light of these observations, we anticipate the present design to remain as the mature state of this technology over the coming years.



The results presented set a new benchmark in data quality from absorbance based detection in AUC. With new commercial instrumentation coming on the market this year, we expect these results to be especially pertinent, and provide a framework for MWL-AUC performance testing going forward. It is worth mentioning that our developments can be directly transferred to the commercial implementation of the Open AUC MWL detector as well.7

Acknowledgements JP and HC acknowledge financial support by the Center for Applied Photonics (CAP) at the University of Konstanz, and computer allocations through grant HKN00-12009 from the Jülich Supercomputing Center. HC also acknowledges the Deutsche Forschungsgemeinschaft (DFG) for financial support through project B6 of the SFB 1214. JW and WP acknowledge funding of the DFG through the Cluster of Excellence ‘‘Engineering of Advanced Materials’’. Moreover, the DFG is gratefully thanked for financial support through project PE 427/28-2.

References (1) Cölfen, H.; Laue, T. M.; Wohlleben, W.; Schilling, K.; Karabudak, E.; Langhorst, B. W.; Brookes, E.; Dubbs, B.; Zollars, D.; Rocco, M.; et al. Eur. Biophys. J. 2010, 39, 347-359. (2) Bhattacharyya, S. K.; Maciejewska, P.; Börger, L.; Stadler, M.; Gülsün, A. M.; Cicek, H. B.; Cölfen, H. In Analytical Ultracentrifugation VIII, Wandrey, C.; Cölfen, H., Eds.; Springer-Verlag: Berlin, Heidelberg, 2006, pp 9-22. (3) Karabudak, E.; Cölfen, H. In Analytical Ultracentrifugation: Instrumentation, Software, and Applications, Uchiyama, S.; Arisaka, F.; Stafford, W. F.; Laue, T., Eds.; Springer Japan: Tokyo, 2016, pp 63-80. (4) Bhattacharyya, S. K. Development of Detector for Analytical Ultracentrifuge. PhD thesis, University of Potsdam, Potsdam, 2006. (5) Strauss, H. M.; Karabudak, E.; Bhattacharyya, S.; Kretzschmar, A.; Wohlleben, W.; Cölfen, H. Colloid Polym. Sci. 2008, 286, 121-128. (6) Karabudak , E. Development of MWL-AUC / CCD-C-AUC / SLS-AUC detectors for the analytical ultracentrifuge. PhD thesis, University of Potsdam, Germany, 2009. (7) Pearson, J. Z.; Krause, F.; Haffke, D.; Demeler, B.; Schilling, K.; Cölfen, H. In Methods Enzymol., Cole, J. L., Ed.; Academic Press, 2015, pp 1-26. (8) Walter, J.; Löhr, K.; Karabudak, E.; Reis, W.; Mikhael, J.; Peukert, W.; Wohlleben, W.; Cölfen, H. ACS Nano 2014, 8, 8871-8886. (9) Walter, J.; Peukert, W. Nanoscale 2016, 8, 7484-7495. (10) Walter, J.; Segets, D.; Peukert, W. Part. Part. Syst. Charact. 2016, 33, 184-189. (11) Giebeler, R. In Analytical ultracentrifugation in biochemistry and polymer science, Harding, S. E.; Rowe, A. J.; Horton, J. C., Eds.; Royal Society of Chemistry: Cambridge, England, 1992, pp 16-25. (12) Stafford, W. F. Anal. Biochem. 1992, 203, 295-301. (13) Stafford, W. F. Methods Enzymol. 1994, 240, 478-501.


Page 24 of 27

Page 25 of 27 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


(14) Philo, J. S. Biophys. J. 1997, 72, 435-444. (15) Schuck, P. Biophys. J. 1998, 75, 1503-1512. (16) Behlke, J.; Ristau, O. Biophys. Chem. 1998, 70, 133-146. (17) Schuck, P.; Demeler, B. Biophys. J. 1999, 76, 2288-2296. (18) Behlke, J.; Ristau, O. Biophys. Chem. 1999, 76, 13-23. (19) Stafford, W. F. Methods Enzymol. 2000, 323, 302-325. (20) Solovyova, A.; Schuck, P.; Costenaro, L.; Ebel, C. Biophys. J. 2001, 81, 1868-1880. (21) Stafford, W. F.; Braswell, E. H. Biophys. Chem. 2004, 108, 273-279. (22) Stafford, W. F.; Sherwood, P. J. Biophys. Chem. 2004, 108, 231-243. (23) Cao, W.; Demeler, B. Biophys. J. 2005, 89, 1589-1602. (24) Brookes, E.; Demeler, B. Prog. Colloid Polym. Sci. 2006, 131, 33-40. (25) Brown, P. H.; Schuck, P. Biophys. J. 2006, 90, 4651-4661. (26) Demeler, B.; Brookes, E.; Nagel-Stegert, L. Methods Enzymol. 2009, 454, 87-113. (27) Philo, J. S. Anal. Biochem. 2011, 412, 189-202. (28) Gorbet, G.; Devlin, T.; Hernandez Uribe, B. I.; Demeler, A. K.; Lindsey, Z. L.; Ganji, S.; Breton, S.; Weise-Cross, L.; Lafer, E. M.; Brookes, E. H.; et al. Biophys. J. 2014, 106, 17411750. (29) Stafford, W. F. In Analytical ultracentrifugationinstrumentation, analysis and applications, Uchiyama, S.; Arisaka, F.; Laue, T.; Stafford, W., Eds.; Springer: Tokyo, 2015. (30) Walter, J.; Sherwood, P. J.; Lin, W.; Segets, D.; Stafford, W. F.; Peukert, W. Anal. Chem. 2015, 87, 3396-3403. (31) Wong, C. J.; Launer-Felty, K.; Cole, J. L. Methods Enzymol. 2011, 488, 59-79. (32) Brautigam, C. A.; Padrick, S. B.; Schuck, P. PloS one 2013, 8, e62694. (33) https://www.beckman.com/centrifuges/analytical-ultracentrifuges/optima-auc (accessed: 28.09.2017). (34) Gorbet, G. E.; Pearson, J. Z.; Demeler, A. K.; Cölfen, H.; Demeler, B. In Methods Enzymol., James, L. C., Ed.; Academic Press, 2015, pp 27-47. (35) Karabudak, E.; Wohlleben, W.; Cölfen, H. Eur. Biophys. J. 2010, 39, 397-403. (36) Karabudak, E.; Backes, C.; Hauke, F.; Schmidt, C. D.; Cölfen, H.; Hirsch, A.; Wohlleben, W. ChemPhysChem 2010, 11, 3224-3227. (37) Lin, W.; Walter, J.; Burger, A.; Maid, H.; Hirsch, A.; Peukert, W.; Segets, D. Chem. Mater. 2015, 27, 358-369. (38) Schindler, T.; Walter, J.; Peukert, W.; Segets, D.; Unruh, T. J. Phys. Chem. B 2015, 119, 15370–15380. (39) Karabudak, E.; Brookes, E.; Lesnyak, V.; Gaponik, N.; Eychmüller, A.; Walter, J.; Segets, D.; Peukert, W.; Wohlleben, W.; Demeler, B.; et al. Angew. Chem. Int. Ed. 2016, 55, 11770–11774. (40) Lin, W.; Haderlein, M.; Walter, J.; Peukert, W.; Segets, D. Angew. Chem. Int. Ed. 2016, 55, 932-935. (41) Zhang, J.; Pearson, J. Z.; Gorbet, G. E.; Cölfen, H.; Germann, M. W.; Brinton, M. A.; Demeler, B. Anal. Chem. 2017, 89, 862-870. (42) Thajudeen, T.; Walter, J.; Srikantharajah, R.; Lübbert, C.; Peukert, W. Nanoscale Horiz. 2017, 2, 253-260. (43) Walter, J.; Gorbet, G.; Akdas, T.; Segets, D.; Demeler, B.; Peukert, W. Analyst 2017, 142, 206-217. (44) Svensson, H. Opt. Acta 1954, 1, 25-32.



(45) Ghirlando, R.; Balbo, A.; Piszczek, G.; Brown, P. H.; Lewis, M. S.; Brautigam, C. A.; Schuck, P.; Zhao, H. Anal. Biochem. 2013, 440, 81-95. (46) Zhao, H.; Ghirlando, R.; Alfonso, C.; Arisaka, F.; Attali, I.; Bain, D. L.; Bakhtina, M. M.; Becker, D. F.; Bedwell, G. J.; Bekdemir, A.; et al. PloS one 2015, 10, e0126420. (47) https://oceanoptics.com/wp-content/uploads/USB2000-OperatingInstructions1.pdf (accessed: 06.07.2017). (48) http://www.hamamatsu.com/resources/pdf/etd/5W_Xe-F_TLSZ1006E.pdf (accessed: 06.07.2017).

Supporting Information Additional test data further illustrating the spatial resolution limits of the test targets and optics, SNR of lamp models as a function of repetition rate, relationship of model fitting (RMSD) to sample concentration and illumination intensity, comparison data from a commercial Beckman-Coulter XL-A.

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High27Resolution S - Spectra Page of Analytical 27 Chemistry 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25

Monomer Dimer Trimer


Advanced Multiwavelength Detection in Analytical Ultracentrifugation

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