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 ).

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.

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 saves x and y positional information to an onboard microSD card. The RAT also provides a programmable multi-function input/output pin that can be used for controlling other equipment, transmitting tracking information in real time, or receiving data from other devices. Finally, the RAT includes a real-time clock (RTC) for accurate time stamping of data files. Real-time image processing averts the need to save video, greatly reducing storage, data handling, and communication requirements. To demonstrate the capabilities of the RAT, we performed three validation studies: (1) a 4-d experiment measuring circadian activity patterns; (2) logging of mouse positional information alongside status information from a pellet dispensing device; and (3) control of an optogenetic stimulation system for a real-time place preference (RTPP) brain stimulation reinforcement study. Our design files, build instructions, and code for the RAT implementation are open source and freely available online to facilitate dissemination and further development of the RAT.

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.

Rodent Activity Detector (RAD), an Open Source Device for Measuring Activity in Rodent Home Cages.

Physical activity is a critical behavioral variable in many research studies and is, therefore, important to quantify. However, existing methods for measuring physical activity have limitations which include high expense, specialized caging or equipment, and high computational overhead. To address these limitations, we present an open-source, cost-effective, device for measuring rodent activity. Our device is battery powered and designed to be placed in vivarium home cages to enable high-throughput, long-term operation with minimal investigator intervention. The primary aim of this study was to assess the feasibility of using passive infrared (PIR) sensors and microcontroller-based dataloggers in a rodent home cages to collect physical activity records. To this end, we developed an open-source PIR based data-logging device called the rodent activity detector (RAD). We publish the design files and code so others can readily build the RAD in their own labs. To demonstrate its utility, we used the RAD to collect physical activity data from 40 individually housed mice for up to 10 weeks. This dataset demonstrates the ability of the RAD to (1) operate in a high-throughput installation, (2) detect high-fat diet (HFD)-induced changes in physical activity, and (3) quantify circadian rhythms in individual animals. We further validated the data output of the RAD with simultaneous video tracking of mice in multiple caging configurations, to determine the features of physical activity that it detects. The RAD is easy to build, economical, and fits in vivarium caging. The scalability of such devices will enable high-throughput studies of physical activity in research studies.

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.

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.

Integration of digital signal processing technologies with pulsed electron paramagnetic resonance imaging.

The integration of modern data acquisition and digital signal processing (DSP) technologies with Fourier transform electron paramagnetic resonance (FT-EPR) imaging at radiofrequencies (RF) is described. The FT-EPR system operates at a Larmor frequency (L(f)) of 300MHz to facilitate in vivo studies. This relatively low frequency L(f), in conjunction with our approximately 10MHz signal bandwidth, enables the use of direct free induction decay time-locked subsampling (TLSS). This particular technique provides advantages by eliminating the traditional analog intermediate frequency downconversion stage along with the corresponding noise sources. TLSS also results in manageable sample rates that facilitate the design of DSP-based data acquisition and image processing platforms. More specifically, we utilize a high-speed field programmable gate array (FPGA) and a DSP processor to perform advanced real-time signal and image processing. The migration to a DSP-based configuration offers the benefits of improved EPR system performance, as well as increased adaptability to various EPR system configurations (i.e., software configurable systems instead of hardware reconfigurations). The required modifications to the FT-EPR system design are described, with focus on the addition of DSP technologies including the application-specific hardware, software, and firmware developed for the FPGA and DSP processor. The first results of using real-time DSP technologies in conjunction with direct detection bandpass sampling to implement EPR imaging at RF frequencies are presented.

Direct detection and time-locked subsampling applied to pulsed electron paramagnetic resonance imaging.

The application of direct time-locked subsampling (TLSS) to Fourier transform electron paramagnetic resonance (FT-EPR) spectroscopy at radio frequencies (rf) is described. With conventional FT-EPR spectroscopy, the high Larmor frequencies (L(f)) often necessitate the use of intermediate frequency (IF) stages to down convert the received free induction decay (FID) signal to a frequency that can be acquired with common data acquisition technology. However, our research focuses on in vivo studies, and consequently utilizes a FT-EPR system with a L(f) of 300 MHz. This relatively low frequency L(f), in conjunction with the advent of bandpass sampling analog-to-digital conversion and signal processing technologies, has enabled us to omit the IF stage in our FT-EPR system. With this in mind, TLSS techniques have been developed to directly sample the 300 MHz FID signal at a sampling rate of 80 MHz providing a signal bandwidth of 20 MHz. The required modifications to the data acquisition and processing system specific to this application are described. Custom software developed to control the EPR system setup, acquire the signals, and post process the data, is outlined. Data was acquired applying both coherent averaging and stochastic excitation sequences. The results of these experiments demonstrate digital down conversion of the 300 MHz FID signal to quadrature baseband. Direct FID TLSS eliminates many noise sources common in EPR systems employing traditional analog receiver techniques, such as the IF mixer stage in single channel systems, and the quadrature baseband mixer stage in dual channel systems.

Stochastic excitation and Hadamard correlation spectroscopy with bandwidth extension in RF FT-EPR.

The application of correlation spectroscopy employing stochastic excitation and the Hadamard transform to time-domain Fourier transform electron paramagnetic resonance (FT-EPR) spectroscopy in the radiofrequency (RF) band is described. An existing, time-domain FT-EPR spectrometer system with a Larmor frequency (L(f)) of 300 MHz was used to develop this technique by incorporating a pseudo-random pulse sequence generator to output the maximum length binary sequence (MLBS, 10- and 11-bit). Software developed to control the EPR system setup, acquire the signals, and post process the data, is outlined. The software incorporates the Hadamard transform algorithm to perform the required cross-correlation of the acquired signal and the MLBS after stochastic excitation. To accommodate the EPR signals, bandwidth extension was accomplished by sampling at a rate many times faster than the RF pulse repetition rate, and subsequent digital signal processing of the data. The results of these experiments showed that there was a decrease in the total acquisition time, and an improved free induction decay (FID) signal-to-noise (S/N) ratio compared to the conventional coherent averaging approach. These techniques have the potential to reduce the RF pulse power to the levels used in continuous wave (CW) EPR while retaining the advantage of time-domain EPR methods. These methods have the potential to facilitate the progression to in vivo FT-EPR imaging of larger volumes.

High frequency of BRAF mutations in nevi.

To evaluate the timing of mutations in BRAF (v-raf murine sarcoma viral oncogene homolog B1) during melanocytic neoplasia, we carried out mutation analysis on microdissected melanoma and nevi samples. We observed mutations resulting in the V599E amino-acid substitution in 41 of 60 (68%) melanoma metastases, 4 of 5 (80%) primary melanomas and, unexpectedly, in 63 of 77 (82%) nevi. These data suggest that mutational activation of the RAS/RAF/MAPK pathway in nevi is a critical step in the initiation of melanocytic neoplasia but alone is insufficient for melanoma tumorigenesis.

Anionic Halomolybdate(III) Chemistry. Tetrahydrofuran Loss from [MoX(3)Y(THF)(2)](-) (X, Y = Cl, Br, I), Preparation and Properties of [Mo(3)X(12)](3)(-) (X = Br, I), and Crystal Structure of the Edge-Sharing Trioctahedral [PPh(4)](3)[Mo(3)I(12)].

By interaction of MoX(3)(THF)(3) with [Cat]X in THF, the salts [Cat][MoX(4)(THF)(2)] have been synthesized [X = I, Cat = PPh(4), NBu(4), NPr(4), (Ph(3)P)(2)N; X = Br, Cat = NBu(4), PPh(4) (Ph(3)P)(2)N]. Mixed-halide species [MoX(3)Y(THF)(2)](-) (X, Y = Cl, Br, I) have also been generated in solution and investigated by (1)H-NMR. When the tetraiodo, tetrabromo, and mixed bromoiodo salts are dissolved in CH(2)Cl(2), clean loss of all coordinated THF is observed by (1)H-NMR. On the other hand, [MoCl(4)(THF)(2)](-) loses only 1.5 THF/Mo. The salts [Cat](3)[Mo(3)X(12)] (X = Br, I) have been isolated from [Cat][MoX(4)(THF)(2)] or by running the reaction between MoX(3)(THF)(3) and [Cat]X directly in CH(2)Cl(2). The crystal structure of [PPh(4)](3)[Mo(3)I(12)] exhibits a linear face-sharing trioctahedron for the trianion: triclinic, space group P&onemacr;; a = 11.385(2), b = 12.697(3), c = 16.849(2) Å; alpha = 76.65(2), beta = 71.967(12), gamma = 84.56(2) degrees; Z = 1; 431 parameters and 3957 data with I > 2sigma(I). The metal-metal distance is 3.258(2) Å. Structural and magnetic data are consistent with the presence of a metal-metal sigma bond order of (1)/(2) and with the remaining 7 electrons being located in 7 substantially nonbonding orbitals. The ground state of the molecule is predicted to be subject to a Jahn-Teller distortion, which is experimentally apparent from the nature of the thermal ellipsoid of the central Mo atom. The [Mo(3)X(12)](3)(-) ions reacts with phosphines (PMe(3), dppe) to form products of lower nuclearity by rupture of the bridging Mo-X bonds.