3D-Printing for Analytical Ultracentrifugation

Analytical ultracentrifugation (AUC) is a classical technique of physical biochemistry providing information on size, shape, and interactions of macromolecules from the analysis of their migration in centrifugal fields while free in solution. A key mechanical element in AUC is the centerpiece, a component of the sample cell assembly that is mounted between the optical windows to allow imaging and to seal the sample solution column against high vacuum while exposed to gravitational forces in excess of 300,000 g. For sedimentation velocity it needs to be precisely sector-shaped to allow unimpeded radial macromolecular migration. During the history of AUC a great variety of centerpiece designs have been developed for different types of experiments. Here, we report that centerpieces can now be readily fabricated by 3D printing at low cost, from a variety of materials, and with customized designs. The new centerpieces can exhibit sufficient mechanical stability to withstand the gravitational forces at the highest rotor speeds and be sufficiently precise for sedimentation equilibrium and sedimentation velocity experiments. Sedimentation velocity experiments with bovine serum albumin as a reference molecule in 3D printed centerpieces with standard double-sector design result in sedimentation boundaries virtually indistinguishable from those in commercial double-sector epoxy centerpieces, with sedimentation coefficients well within the range of published values. The statistical error of the measurement is slightly above that obtained with commercial epoxy, but still below 1%. Facilitated by modern open-source design and fabrication paradigms, we believe 3D printed centerpieces and AUC accessories can spawn a variety of improvements in AUC experimental design, efficiency and resource allocation.Comment: 25 pages, 6 figure

Rodent arena tracker (RAT): A machine vision rodent tracking camera and closed loop control system

Video tracking is an essential tool in rodent research. Here, we demonstrate a machine vision rodent tracking camera based on a low-cost, open-source, machine vision camera, the OpenMV Cam M7. We call our device the rodent arena tracker (RAT), and it is a pocket-sized machine vision-based position tracker. The RAT does not require a tethered computer to operate and costs about $120 per device to build. These features make the RAT scalable to large installations and accessible to research institutions and educational settings where budgets may be limited. The RAT processes incoming video in real-time at 15 Hz and save

Temporal evolution of radial concentration profiles in a sedimentation velocity experiment with bovine serum albumin in a “prime gray” photopolymer centerpiece.

<p>Panel A: Absorbance data acquired at a rotor speeds of 50,000 rpm at a series of time points (symbols, only every 3^rd data point of every 2^nd scan shown, with color temperature indicating progress of time). The <i>c</i>(<i>s</i>) fit (lines) results in an rmsd of 0.0065 OD₂₈₀, with the residuals shown in the small plots as residuals bitmap and superposition. Panel B: The corresponding <i>c</i>(<i>s</i>) distribution (magenta), and for comparison the <i>c</i>(<i>s</i>) distribution from a control in the same run using a standard Epon centerpiece (black); microgreen (green); Xtreme white (blue dashed); in-house clear (cyan dotted).</p

Fluorescence optical data in a 3D printed carbonate centerpiece.

<p>A centerpiece featuring a 3 mm deep sector-shaped well at the top was used, with filling and venting holes, and an embossed seal. The focal depth of the fluorescence optics was 2.0 mm. (A) Shown are sedimentation profiles acquired with 561 nm excitation for 46 nM mCherry [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0155201#pone.0155201.ref057" target="_blank">57</a>] dissolved in phosphate buffered saline (dots), and best-fit c(s) sedimentation coefficient distribution with adjustments for characteristic signals of fluorescence detection [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0155201#pone.0155201.ref056" target="_blank">56</a>] (solid lines). The plot appended below shows the residuals of the fit. (B) Corresponding sedimentation coefficient distribution showing a main peak at 2.68 S and diffusional boundary broadening corresponding to a species of 26.9 kDa.</p

Sedimentation velocity experiment in rectangular cell.

<p>Sedimentation velocity analysis of bovine serum albumin sedimenting at 50,000 rpm in acrylic centerpieces with a sector-shaped (A) and rectangular shaped (B) solution column. The protein sample was identical in both. The upper panel shows the sedimentation boundaries (points, for clarity, only every 2^nd data point of every 2^nd scan is shown), along with the best-fit <i>c</i>(<i>s</i>) profiles (solid lines). Below are the residuals of the fit as bitmap and overlay plot. The <i>c</i>(<i>s</i>) distribution for both data sets are shown in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0155201#pone.0155201.g005" target="_blank">Fig 5</a>.</p

Radial concentration distribution in a sedimentation equilibrium experiment with enhanced green fluorescent protein in a “prime gray” photopolymer centerpiece.

<p>Data were acquired with the absorbance detection sequentially at rotor speeds of 15,000 rpm (purple), 24,000 rpm (blue), and 35,000 rpm (cyan) (symbols, only every 5^th data point shown). A global model (lines) results in an apparent molar mass of 29.7 kDa with a root-mean-square deviation (rmsd) of 0.0032 OD₄₈₉, and residuals as shown in the lower plot.</p

SCORHE:A novel and practical approach to video monitoring of laboratory mice housed in vivarium cage racks

The System for Continuous Observation of Rodents in Home-cage Environment (SCORHE) was developed to demonstrate the viability of compact and scalable designs for quantifying activity levels and behavior patterns for mice housed within a commercial ventilated cage rack. The SCORHE in-rack design provides day- and night-time monitoring with the consistency and convenience of the home-cage environment. The dual-video camera custom hardware design makes efficient use of space, does not require home-cage modification, and is animal-facility user-friendly. Given the system’s low cost and suitability for use in existing vivariums without modification to the animal husbandry procedures or housing setup, SCORHE opens up the potential for the wider use of automated video monitoring in animal facilities. SCORHE’s potential uses include day-to-day health monitoring, as well as advanced behavioral screening and ethology experiments, ranging from the assessment of the short- and long-term effects of experimental cancer treatments to the evaluation of mouse models. When used for phenotyping and animal model studies, SCORHE aims to eliminate the concerns often associated with many mouse-monitoring methods, such as circadian rhythm disruption, acclimation periods, lack of night-time measurements, and short monitoring periods. Custom software integrates two video streams to extract several mouse activity and behavior measures. Studies comparing the activity levels of ABCB5 knockout and HMGN1 overexpresser mice with their respective C57BL parental strains demonstrate SCORHE’s efficacy in characterizing the activity profiles for singly- and doubly-housed mice. Another study was conducted to demonstrate the ability of SCORHE to detect a change in activity resulting from administering a sedative