MouseVUER: video based open-source system for laboratory mouse home-cage monitoring.

Video monitoring of mice in the home-cage reveals behavior profiles without the disruptions caused by specialized test setups and makes it possible to quantify changes in behavior patterns continually over long time frames. Several commercial home-cage monitoring systems are available with varying costs and capabilities; however there are currently no open-source systems for home-cage monitoring. We present an open-source system for top-down video monitoring of research mice in a slightly modified home-cage. The system is designed for integration with Allentown NexGen ventilated racks and allows unobstructed view of up to three mice, but can also be operated outside the rack. The system has an easy to duplicate and assemble home-cage design along with a video acquisition solution. The system utilizes a depth video camera, and we demonstrate the robustness of depth video for home-cage mice monitoring. For researchers without access to Allentown NexGen ventilated racks, we provide designs and assembly instructions for a standalone non-ventilated rack solution that holds three systems for more compact and efficient housing. We make all the design files, along with detailed assembly and installation instructions, available on the project webpage ( https://github.com/NIH-CIT-OIR-SPIS/MouseVUER ).

Optogenetic Signaling Activation in Zebrafish Embryos.

Signaling pathways orchestrate fundamental biological processes, including development, regeneration, homeostasis, and disease. Methods to experimentally manipulate signaling are required to understand how signaling is interpreted in these wide-ranging contexts. Molecular optogenetic tools can provide reversible, tunable manipulations of signaling pathway activity with a high degree of spatiotemporal control and have been applied in vitro, ex vivo, and in vivo. These tools couple light-responsive protein domains, such as the blue light homodimerizing light-oxygen-voltage sensing (LOV) domain, with signaling effectors to confer light-dependent experimental control over signaling. This protocol provides practical guidelines for using the LOV-based bone morphogenetic protein (BMP) and Nodal signaling activators bOpto-BMP and bOpto-Nodal in the optically accessible early zebrafish embryo. It describes two control experiments: A quick phenotype assay to determine appropriate experimental conditions, and an immunofluorescence assay to directly assess signaling. Together, these control experiments can help establish a pipeline for using optogenetic tools in early zebrafish embryos. These strategies provide a powerful platform to investigate the roles of signaling in development, health, and physiology.

Whole Animal Feeding FLat (WAFFL): a complete and comprehensive validation of a novel, high-throughput fly experimentation system.

Non-mammalian model organisms have been essential for our understanding of the mechanisms that control development, disease, and physiology, but they are underutilized in pharmacological and toxicological phenotypic screening assays due to their low throughput in comparison with cell-based screens. To increase the utility of using Drosophila melanogaster in screening, we designed the Whole Animal Feeding FLat (WAFFL), a novel, flexible, and complete system for feeding, monitoring, and assaying flies in a high-throughput format. Our 3D printed system is compatible with inexpensive and readily available, commercial 96-well plate consumables and equipment. Experimenters can change the diet at will during the experiment and video record for behavior analysis, enabling precise dosing, measurement of feeding, and analysis of behavior in a 96-well plate format.

Neural mechanism of spatio-chromatic opponency in the Drosophila amacrine neurons.

Visual animals detect spatial variations of light intensity and wavelength composition. Opponent coding is a common strategy for reducing information redundancy. Neurons equipped with both spatial and spectral opponency have been identified in vertebrates but not yet in insects. The Drosophila amacrine neuron Dm8 was recently reported to show color opponency. Here, we demonstrate Dm8 exhibits spatio-chromatic opponency. Antagonistic convergence of the direct input from the UV-sensing R7s and indirect input from the broadband receptors R1-R6 through Tm3 and Mi1 is sufficient to confer Dm8's UV/Vis (ultraviolet/visible light) opponency. Using high resolution monochromatic stimuli, we show the pale and yellow subtypes of Dm8s, inheriting retinal mosaic characteristics, have distinct spectral tuning properties. Using 2D white-noise stimulus and reverse correlation analysis, we found that the UV receptive field (RF) of Dm8 has a center-inhibition/surround-excitation structure. In the absence of UV-sensing R7 inputs, the polarity of the RF is inverted owing to the excitatory input from the broadband photoreceptors R1-R6. Using a new synGRASP method based on endogenous neurotransmitter receptors, we show that neighboring Dm8s form mutual inhibitory connections mediated by the glutamate-gated chloride channel GluClα, which is essential for both Dm8's spatial opponency and animals' phototactic behavior. Our study shows spatio-chromatic opponency could arise in the early visual stage, suggesting a common information processing strategy in both invertebrates and vertebrates.

Digital video recorder for Raspberry PI cameras with multi-camera synchronous acquisition.

Video acquisition and analysis have become integral parts of scientific research. Two major components of a video acquisition system are the choice of camera and the acquisition software. A vast variety of cameras are available on the market. Turnkey multi-camera synchronous acquisition software, however, is not as widely available. For prototyping applications, the Raspberry Pi (RPi) has been widely utilized due to many factors, including cost. There are implementations for video acquisition and preview from a single RPi camera, including one implementation released by the RPi organization itself. However, there are no multi-camera acquisition solutions for the RPi. This paper presents an open-source digital video recorder (DVR) system for the popular RPi camera. The DVR is simple to setup and use for acquisition with a single camera or multiple cameras. In the case of multiple cameras, the acquisition is synchronized between cameras. The DVR comes with a graphical user interface (GUI) to allow previewing the camera streams, setting recording parameters, and associating "names" to cameras. The acquisition code as well as the DVR GUI are written in Python. The open-source software also includes a GUI for playback of recorded video. The versatility of the DVR is demonstrated with a life science research application involving high-throughput monitoring of fruit-flies.

Spatial control of oxygen delivery to three-dimensional cultures alters cancer cell growth and gene expression.

Commonly used monolayer cancer cell cultures fail to provide a physiologically relevant environment in terms of oxygen delivery. Here, we describe a three-dimensional (3D) bioreactor system where cancer cells are grown in Matrigel in modified six-well plates. Oxygen is delivered to the cultures through a polydimethylsiloxane (PDMS) membrane at the bottom of the wells, with microfabricated PDMS pillars to control oxygen delivery. The plates receive 3% oxygen from below and 0% oxygen at the top surface of the media, providing a gradient of 3-0% oxygen. We compared growth and transcriptional profiles for cancer cells grown in Matrigel in the bioreactor, 3D cultures grown in 21% oxygen, and cells grown in a standard hypoxia chamber at 3% oxygen. Additionally, we compared gene expression of conventional two-dimensional monolayer culture and 3D Matrigel culture in 21% oxygen. We conclude that controlled oxygen delivery may provide a more physiologically relevant 3D system.

Three-Dimensional Pose Estimation for Laboratory Mouse From Monocular Images.

Video-based activity and behavior analysis of mice has garnered wide attention in biomedical research. Animal facilities hold large numbers of mice housed in "home-cages" densely stored within ventilated racks. Automated analysis of mice activity in their home-cages can provide a new set of sensitive measures for detecting abnormalities and time-resolved deviation from the baseline behavior. Large-scale monitoring in animal facilities requires minimal footprint hardware that integrates seamlessly with the ventilated racks. The compactness of hardware imposes the use of fisheye lenses positioned in close proximity to the cage. In this paper, we propose a systematic approach to accurately estimate the 3D pose of the mouse from single-monocular fisheye-distorted images. Our approach employs a novel adaptation of a structured forest algorithm. We benchmark our algorithm against existing methods. We demonstrate the utility of the pose estimates in predicting mouse behavior in a continuous video.

Enhanced Sample Handling for Analytical Ultracentrifugation with 3D-Printed Centerpieces.

The centerpiece of the sample cell assembly in analytical ultracentrifugation holds the sample solution between windows, sealed against high vacuum, and is shaped such that macromolecular migration in centrifugal fields exceeding 200 000g can proceed undisturbed by walls or convection while concentration profiles are imaged with optical detection systems aligned perpendicular to the plane of rotation. We have recently shown that 3D printing using various materials allows inexpensive and rapid manufacturing of centerpieces. In the present work, we expand this endeavor to examine the accuracy of the measured sedimentation process, as well as short-term durability of the centerpieces. We find that 3D-printed centerpieces can be used many times and can provide data equivalent in quality to commonly used commercial epoxy resin centerpieces. Furthermore, 3D printing enables novel designs adapted to particular experimental objectives because they offer unique opportunities, for example, to create well-defined curved surfaces, narrow channels, and embossed features. We present examples of centerpiece designs exploiting these capabilities for improved AUC experiments. This includes narrow sector centerpieces that substantially reduce the required sample volume while maintaining the standard optical path length; thin centerpieces with integrated window holders to provide very short optical pathlengths that reduce optical aberrations at high macromolecular concentrations; long-column centerpieces that increase the observable distance of macromolecular migration for higher-precision sedimentation coefficients; and three-sector centerpieces that allow doubling the number of samples in a single run while reducing the sample volumes. We find each of these designs allows unimpeded macromolecular sedimentation and can provide high-quality sedimentation data.

Towards reduction of SAR in scaling up in vivo pulsed EPR imaging to larger objects.

An excessive RF power requirement is one of the main obstacles in the clinical translation of EPR imaging. The radio frequency (RF) pulses used in EPR imaging to excite electron spins must be very short to match their fast relaxation. With traditional pulse schemes and ninety degree flip angles, this can lead to either unsafe specific absorption rate (SAR) levels or unfeasibly long repetition times. In spectroscopy experiments, it has been shown that stochastic excitation and correlation detection can reduce the power while maintaining sensitivity but have yet to be applied to imaging experiments. Stochastic excitation is implemented using a pseudo-random phase modulation of the input stimulus. Using a crossed coil resonator assembly comprised of an outer saddle coil and an inner surface coil, it was possible to obtain a minimum isolation of ∼50 dB across a 12 MHz bandwidth. An incident peak RF power of 5 mW was used to excite the system. The low background signal obtained from this resonator allowed us to generate images with 32 dB (>1000:1) signal-to-noise ratio (SNR) while exciting with a traditional pulse sequence in a phantom containing the solid paramagnetic probe NMP-TCNQ (N-methyl pyridinium tetracyanoquinodimethane). Using two different stochastic excitation schemes, we were able to achieve a greater than 4-fold increase in SNR at the same peak power and number of averages, compared to single pulse excitation. This procedure allowed imaging at significantly lower RF power levels than used in conventional EPR imaging system configurations. Similar techniques may enable clinical applications for EPR imaging by facilitating the use of larger RF coils while maintaining a safe SAR level.

Measuring Risky Driving Behavior Using an mHealth Smartphone App: Development and Evaluation of gForce.

BACKGROUND: Naturalistic driving studies, designed to objectively assess driving behavior and outcomes, are conducted by equipping vehicles with dedicated instrumentation (eg, accelerometers, gyroscopes, Global Positioning System, and cameras) that provide continuous recording of acceleration, location, videos, and still images for eventual retrieval and analyses. However, this research is limited by several factors: the cost of equipment installation; management and storage of the large amounts of data collected; and data reduction, coding, and analyses. Modern smartphone technology includes accelerometers built into phones, and the vast, global proliferation of smartphones could provide a possible low-cost alternative for assessing kinematic risky driving. OBJECTIVE: We evaluated an in-house developed iPhone app (gForce) for detecting elevated g-force events by comparing the iPhone linear acceleration measurements with corresponding acceleration measurements obtained with both a custom Android app and the in-vehicle miniDAS data acquisition system (DAS; Virginia Tech Transportation Institute). METHODS: The iPhone and Android devices were dashboard-mounted in a vehicle equipped with the DAS instrumentation. The experimental protocol consisted of driving maneuvers on a test track, such as cornering, braking, and turning that were performed at different acceleration levels (ie, mild, moderate, or hard). The iPhone gForce app recorded linear acceleration (ie, gravity-corrected). The Android app recorded gravity-corrected and uncorrected acceleration measurements, and the DAS device recorded gravity-uncorrected acceleration measurements. Lateral and longitudinal acceleration measures were compared. RESULTS: The correlation coefficients between the iPhone and DAS acceleration measurements were slightly lower compared to the correlation coefficients between the Android and DAS, possibly due to the gravity correction on the iPhone. Averaging the correlation coefficients for all maneuvers, the longitudinal and lateral acceleration measurements between iPhone and DAS were rlng=0.71 and rlat=0.83, respectively, while the corresponding acceleration measurements between Android and DAS were rlng=0.95 and rlat=0.97. The correlation coefficients between lateral accelerations on all three devices were higher than with the corresponding longitudinal accelerations for most maneuvers. CONCLUSIONS: The gForce iPhone app reliably assessed elevated g-force events compared to the DAS. Collectively, the gForce app and iPhone platform have the potential to serve as feature-rich, inexpensive, scalable, and open-source tool for assessment of kinematic risky driving events, with potential for research and feedback forms of intervention.

Physiological Recording in the MRI Environment (PRiME): MRI-Compatible Hemodynamic Recording System.

Hemodynamic recording during interventional cardiovascular procedures is essential for procedural guidance, monitoring patient status, and collection of diagnostic information. Recent advances have made interventions guided by magnetic resonance imaging (MRI) possible and attractive in certain clinical scenarios. However, in the MRI environment, electromagnetic interference (EMI) can cause severe distortions and artifacts in acquired hemodynamic waveforms. The primary aim of this paper was to develop and validate a system to minimize EMI on electrocardiogram (ECG) and invasive blood pressure (IBP) signals. A system was developed which incorporated commercial MRI compatible ECG leads and pressure transducers, custom electronics, user interface, and adaptive signal processing. Measurements were made on pediatric patients (N = 6) during MRI-guided catheterization. Real-time interactive scanning, which is known to produce significant EMI due to fast gradient switching and varying imaging plane orientations, was selected for testing. The effectiveness of the adaptive algorithms was determined by measuring the reduction of noise peaks, amplitude of noise peaks, and false QRS triggers. During real-time gradient-intensive imaging sequences, peak noise amplitude was reduced by 80% and false QRS triggers were reduced to a median of 0. There was no detectable interference on the IBP channels. A hemodynamic recording system front-end was successfully developed and deployed, which enabled high-fidelity recording of ECG and IBP during MRI scanning. The schematics and assembly instructions are publicly available to facilitate implementation at other institutions. Researchers and clinicians are provided a critical tool in investigating and implementing MRI guided interventional cardiovascular procedures.

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.

Rapid diagnosis of lymph node metastasis in breast cancer using a new fluorescent method with γ-glutamyl hydroxymethyl rhodamine green.

Sentinel lymph node biopsy is performed as a standard procedure in breast cancer surgery, and the development of quick and simple methods to detect metastatic lesions is in high demand. Here, we validated a new fluorescent method using γ-glutamyl hydroxymethyl rhodamine green to diagnose metastatic lymph nodes in breast cancer. One hundred and forty-nine lymph nodes from 38 breast cancer patients were evaluated in this study. Comparison of fluorescent and pathological images showed that this fluorescent method was successful for visualizing breast cancer cells in lymph nodes. This method had a sufficiently high sensitivity (97%), specificity (79%) and negative predictive value (99%) to render it useful for an intraoperative diagnosis of cancer. These preliminary findings suggest that this novel method is useful for distinguishing non-cancerous specimens from those in need of careful examination and could help save time and cost for surgeons and pathologists.

Rapid intraoperative visualization of breast lesions with γ-glutamyl hydroxymethyl rhodamine green.

We previously developed γ-glutamyl hydroxymethyl rhodamine green (gGlu-HMRG) as a tool to detect viable cancer cells, based on the fact that the enzyme γ-glutamyltranspeptidase (GGT) is overexpressed on membranes of various cancer cells, but is not expressed in normal tissue. Cleavage of the probe by GGT generates green fluorescence. Here, we examined the feasibility of clinical application of gGlu-HMRG during breast-conserving surgery. We found that fluorescence derived from cleavage of gGlu-HMRG allowed easy discrimination of breast tumors, even those smaller than 1 mm in size, from normal mammary gland tissues, with 92% sensitivity and 94% specificity, within only 5 min after application. We believe this rapid, low-cost method represents a breakthrough in intraoperative margin assessment during breast-conserving surgery.

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.

Multiple redundant medulla projection neurons mediate color vision in Drosophila.

The receptor mechanism for color vision has been extensively studied. In contrast, the circuit(s) that transform(s) photoreceptor signals into color percepts to guide behavior remain(s) poorly characterized. Using intersectional genetics to inactivate identified subsets of neurons, we have uncovered the first-order interneurons that are functionally required for hue discrimination in Drosophila. We developed a novel aversive operant conditioning assay for intensity-independent color discrimination (true color vision) in Drosophila. Single flying flies are magnetically tethered in an arena surrounded by blue and green LEDs (light-emitting diodes). The flies' optomotor response is used to determine the blue-green isoluminant intensity. Flies are then conditioned to discriminate between equiluminant blue or green stimuli. Wild-type flies are successfully trained in this paradigm when conditioned to avoid either blue or green. Functional color entrainment requires the function of the narrow-spectrum photoreceptors R8 and/or R7, and is within a limited range, intensity independent, suggesting that it is mediated by a color vision system. The medulla projection neurons, Tm5a/b/c and Tm20, receive direct inputs from R7 or R8 photoreceptors and indirect input from the broad-spectrum photoreceptors R1-R6 via the lamina neuron L3. Genetically inactivating these four classes of medulla projection neurons abolished color learning. However, inactivation of subsets of these neurons is insufficient to block color learning, suggesting that true color vision is mediated by multiple redundant pathways. We hypothesize that flies represent color along multiple axes at the first synapse in the fly visual system. The apparent redundancy in learned color discrimination sharply contrasts with innate ultraviolet (UV) spectral preference, which is dominated by a single pathway from the amacrine neuron Dm8 to the Tm5c projection neurons.

A hard-wired glutamatergic circuit pools and relays UV signals to mediate spectral preference in Drosophila.

Many visual animals have innate preferences for particular wavelengths of light, which can be modified by learning. Drosophila's preference for UV over visible light requires UV-sensing R7 photoreceptors and specific wide-field amacrine neurons called Dm8. Here we identify three types of medulla projection neurons downstream of R7 and Dm8 and show that selectively inactivating one of them (Tm5c) abolishes UV preference. Using a modified GRASP method to probe synaptic connections at the single-cell level, we reveal that each Dm8 neuron forms multiple synaptic contacts with Tm5c in the center of Dm8's dendritic field but sparse connections in the periphery. By single-cell transcript profiling and RNAi-mediated knockdown, we determine that Tm5c uses the kainate receptor Clumsy to receive excitatory glutamate input from Dm8. We conclude that R7s→Dm8→Tm5c form a hard-wired glutamatergic circuit that mediates UV preference by pooling ∼16 R7 signals for transfer to the lobula, a higher visual center.

Atlas based AAM and SVM model for fully automatic MRI prostate segmentation.

Automatic prostate segmentation in MR images is a challenging task due to inter-patient prostate shape and texture variability, and the lack of a clear prostate boundary. We propose a supervised learning framework that combines the atlas based AAM and SVM model to achieve a relatively high segmentation result of the prostate boundary. The performance of the segmentation is evaluated with cross validation on 40 MR image datasets, yielding an average segmentation accuracy near 90%.

Microfabricated polymeric vessel mimetics for 3-D cancer cell culture.

Modeling tumor growth in vitro is essential for cost-effective testing of hypotheses in preclinical cancer research. 3-D cell culture offers an improvement over monolayer culture for studying cellular processes in cancer biology because of the preservation of cell-cell and cell-ECM interactions. Oxygen transport poses a major barrier to mimicking in vivo environments and is not replicated in conventional cell culture systems. We hypothesized that we can better mimic the tumor microenvironment using a bioreactor system for controlling gas exchange in cancer cell cultures with silicone hydrogel synthetic vessels. Soft-lithography techniques were used to fabricate oxygen-permeable silicone hydrogel membranes containing arrays of micropillars. These membranes were inserted into a bioreactor and surrounded by basement membrane extract (BME) within which fluorescent ovarian cancer (OVCAR8) cells were cultured. Cell clusters oxygenated by synthetic vessels showed a ∼100µm drop-off to anoxia, consistent with in vivo studies of tumor nodules fed by the microvasculature. Oxygen transport in the bioreactor system was characterized by experimental testing with a dissolved oxygen probe and finite element modeling of convective flow. Our study demonstrates differing growth patterns associated with controlling gas distributions to better mimic in vivo conditions.

Performance characteristics of a positron projection imager for mouse whole-body imaging.

INTRODUCTION: We describe a prototype positron projection imager (PPI) for visualizing the whole-body biodistribution of positron-emitting compounds in mouse-size animals. The final version of the PPI will be integrated into the MONICA portable dual-gamma camera system to allow the user to interchangeably image either single photon or positron-emitting compounds in a shared software and hardware environment. METHODS: A mouse is placed in the mid-plane between two identical, opposed, pixelated LYSO arrays separated by 21.8-cm and in time coincidence. An image of the distribution of positron decays in the animal is formed on this mid-plane by coincidence events that fall within a small cone angle perpendicular to the two detectors and within a user-specified energy window. We measured the imaging performance of this device with phantoms and in tests performed in mice injected with various compounds labeled with positron-emitting isotopes. RESULTS: Representative performance measurements yielded the following results (energy window 250-650keV, cone angle 3.5°): resolution in the image mid-plane, 1.66-mm (FWHM), resolution ±1.5-cm above and below the image plane, 2.2-mm (FWHM), sensitivity: 0.237-cps/kBq (8.76-cps/µCi) (18)F (0.024% absolute). Energy resolution was 15.9% with a linear-count-rate operating range of 0-14.8MBq (0-400µCi) and a corrected sensitivity variation across the field-of-view of <3%. Whole-body distributions of [(18)F] FDG and [(18)F] fluoride were well visualized in mice of typical size. CONCLUSION: Performance measurements and field studies indicate that the PPI is well suited to whole-body positron projection imaging of mice. When integrated into the MONICA gamma camera system, the PPI may be particularly useful early in the drug development cycle where, like MONICA, basic whole-body biodistribution data can direct future development of the agent under study and where logistical factors (e.g., available imaging space, non-portability, and cost) may be limitations.