Article Figures & Data
Figures
- Figure 1.
Camera software setup. A, Insert a clean micro-SD card into a computer. B, Download the latest release of the Show-me webcam image file from its official GitHub page. C, Install the Show-me webcam image file on the micro-SD card using the official Raspberry Pi Imager. Image source: https://github.com/raspberrypilearning/components.
- Figure 2.
Camera hardware setup. A, Essential components required to build a Pi USB Cam including the main Raspberry Pi board, a USB to USB-C cable, a micro-SD card, the camera module, and a camera ribbon flex cable (listed from left to right). B, Remove the attached IR LEDs from the main camera board by unscrewing the four screws outlined in red. C, Connect the main camera board to the Raspberry Pi 4B board via a camera ribbon flex cable. See Extended Data Figure 2-1 for a labeled diagram of the Pi motherboard. D, Insert the prepared micro-SD card (see Fig. 1 for software setup) into the Raspberry Pi board. E, Power up the Pi USB Cam by connecting it to a computer using the USB cable. F, House the camera in custom 3D-printed case and mount tools for protection and installation in behavioral testing environment.
- Figure 3.
Adjusting camera settings. A, Manually enable IR sensitivity by covering the photoresistor with nontranslucent tape (1) or enable IR correction by unplugging the connector on the back of the camera (2). B, Use of our recommended low-light custom settings (right) eliminates slightly purple hue obtained using the camera’s default settings (left) when recording in total darkness. C, In contrast, the same low-light settings produce an unwanted yellow hue (right) under bright light conditions when default settings (left) produce a more appropriate image. D, To adjust camera settings, connect the Pi USB Cam containing the micro-SD card into a host computer. E, Identify the COM port for the Pi USB Cam in Windows Device Manager. F, Connect to the Pi USB Cam using the open-source software PuTTY. G, Enter the command shown in (1) to access all adjustable camera setting (2). Settings we recommend adjusting for low-light recordings are highlighted in red. H, Adjust the focus by unscrewing the small screw that secures the lens in place (arrow) and twisting the lens in either direction until your desired image quality is achieved. *Recommended settings for each lighting condition.
- Figure 4.
Wiring IR LEDs independent from Pi USB Cam. A, Unwanted flare artifacts are apparent when IR LED boards remain attached to and powered directly by the camera board and the camera is mounted over a reflective surface. Detaching the IR LEDs from the camera board allows for flexible placement in a configuration that avoids these flares as well as shadows cast by other equipment such as what is observed when the LEDs are mounted overhead (B) versus away from the reflective surface (C). D, Custom-made jumper wires (1–3) necessary to wire the IR LED boards in parallel and power them via a pair of 3v3 power and ground pins on the Pi board. See Extended Data Figures 4-1 and 4-2 for additional wiring instructions. E, Fully connected IR LED unit and Pi board. The locations of 3v3 power pins (highlighted in pink) and ground pins (highlighted in in black) on the 40-pin header are illustrated in the inset. F, Heat sinks should be installed on the back of the LED boards (1) to prevent overheating after housing them in the custom 3D-printed case (2). G, CAD rendering of the IR LED housing and mount parts (1) and a close-up photograph of a fully assembled and mounted IR LED unit (2).
- Figure 5.
Set up for multicamera recordings. A, The following steps should be done in OBS Studio from a host computer connected to one or more Pi USB Cams. B, Verify in the Windows Device Manager that no more than three cameras are connected to each USB controller. C, Main OBS Studio interface. Menu, Sources, and Controls panels are highlighted. Users can generate a “Profile” from the drop-down menu (D1, D2) and use the “Controls” panel to access various recording settings (D3–D7). Image source: https://github.com/obsproject/obs-studio/blob/master/UI/forms/images/obs_256x256.png.
- Figure 6.
Recommended configuration for separate recordings from multiple video sources. For each Pi USB Cam, create a “Scene Collection” from the drop-down menu (A1, A2) and add the camera as a video source using the “Sources” panel (A3–A7). B, Launch additional instances of OBS Studio to record from multiple camera sources. See Extended Data Figure 6-1 for a detailed explanation of OBS Studio configurations.
- Figure 8.
Comparison of different Pi USB Cam-compatible fisheye lenses. Pictures of a Pi USB Cam equipped with various M12 fisheye lenses (1), including 180° (A), 140° (B), 118° (C), 100° (D), 73° (E), 67° (F), 33° (G), and 26° (H). Corresponding snapshots of videos acquired from a centered position overhead of the arena at a distance of ∼20 cm from the rod floor are depicted for each lens (2). *Indicates the default lens that comes with the Arducam day-night vision camera. See accompanying extended figures for digital fisheye distortion correction (Extended Data Fig. 8-1), comparison of lenses at different object distances (Extended Data Fig. 8-2), and position tracking comparisons under distorted and low-distortion video acquisition settings (Extended Data Fig. 8-3).
- Figure 7.
Video quality comparison between Pi USB Cam and commercial webcam. Snapshots of videos acquired using Pi USB Cam (left column) and Logitech C930e webcam (right column) mounted overhead at a distance that accommodated the entire field of view (A). Recordings were made under (B) no light, (C) cue light illumination, (D) red light illumination, (E) white light illumination, and (F) white light illumination + cue lights.
- Figure 9.
Camera implementation for operant box monitoring. Use of custom 3D-printed components allows for versatile camera installation options including overhead (A, B) and side (C, D) mounted configurations. For each option, (1) depicts the CAD drawing of 3D-printed components. Close-up photographs of each configuration are provided in (2) with object distance indicated in (3). For overhead viewing, Pi USB Cam can be mounted on a commutator balance arm post (A) or directly on the roof panel (B). Similar post (C) and wall (D) mount options are available for side view configuration. Both overhead (E) and side view (F) configurations allow for full view of the operant arena. Video snapshots acquired under no-light conditions.
- Figure 10.
Camera implementation for home cage monitoring. Custom 3D-printed components can also be configured to accommodate home cage recordings in a variety of settings including mount to a ring stand (A–C) for recordings performed offsite (e.g., testing room) and on wire shelving (D–F) for recordings inside the vivarium. For each option (1) depicts the CAD drawing of 3D-printed components. Close up photographs of each configuration are provided in (2) with object distance indicated in (3). Pi USB Cam is easily mounted to a ring stand (A) to accommodate offsite recordings in the home cage shown here in both bright-light (B) and no-light (C) conditions. Note that in this configuration the IR LEDs are used in the default configuration such that they are attached to and powered directly by the main camera board. Using the custom 3D-printed G clamp, Pi USB Cam (and independent IR LEDs) can also be mounted directly to shelving (D) shown here in both bright-light (E) and no-light (F) conditions.
- Figure 11.
Camera configuration for large apparatus video recordings. Together with custom 3D-printed components, Pi USB Cam can be configured to record from large behavioral testing arenas like the conditioned place preference apparatus shown here. A1, CAD drawing of the 3D-printed components used in this configuration. A2, Close-up photograph of Pi USB Cam mounted overhead on a wire shelf. A3, The entire testing arena is visualized overhead with the camera mounted at a distance of ∼30 cm using a 100° HFOV fisheye lens. Video snapshots acquired with this configuration under bright-light (B) and no-light (C) conditions.
- Figure 12.
Pi USB Cam scalability is limited only by USB bandwidth and host computer specifications. A, Multisubject multisite recordings are easily achieved using the free and open-source video capture software OBS Studio depicted here with eight independent Pi USB Cams installed in eight separate operant boxes. B, Windows task manager shows the CPU usage of each instance of OBS Studio during multicamera recordings (n = 8) using the same video acquisition settings shown in Figure 5D and a host computer with the specifications depicted in C, including CPU, memory (1), and add-on USB controllers from a USB PCIe expansion card (2).
Tables
Product Vendor Catalog number Price Comment RASPBERRY PI BOARD: Raspberry Pi 4 Model B
(1/2/4/8GB)Anywhere n/a $35–75 Can be purchased as a kit with accessories at a higher cost CAMERA MODULE: Arducam Wide Angle Day-Night Vision for Raspberry Pi Camera
[FOV: 170° (D) × 140° (H)]UCTRONICS
https://bit.ly/39PKPH8B003507 $32.99 ACCESSORIES: MicroSD Card (16GB) PiShop.us
https://bit.ly/38Nc45c936 $7.95 Any microSD card with a minimum storage space of 64MB would work Raspberry Pi 4 Compatible Heavy-Duty Aluminum Alloy Case with Pre-installed Ready to Connect Fan Vilros
https://bit.ly/3GnbeIQVILP015 $14.99 Any case with camera ribbon cable and GPIO access would work;
Metal cases with preinstalled cooling fan would be preferred to prevent overheating of the Raspberry Pi boardStandard USB to USB-C Cable (6 ft) Vilros
https://bit.ly/3alpw0CVILP103 $4.99 Any USB-C type cable capable of data transferring Jumper Cable Pin Header Connector Housing Assortment Kit Amazon
https://amzn.to/3z3Z0muB077X8XV2J $12.98 For wiring the IR LED boards to use them independently of the camera board Ring Terminal, Non Insulated, 22–16 Wire Size, #4 Stud Size Amazon
https://amzn.to/3LKqUH9B005GDFMSG $11.23 For wiring the IR LED boards to use them independently of the camera board Red Black 2 Pin Wires 22 AWG (100 ft) Amazon
https://amzn.to/3wJhnvNB0793N3WZZ $18.99 For wiring the IR LED boards to use them independently of the camera board Heat Shrink Tubing Kit Jameco
https://bit.ly/3NxH5bX2095963 $17.95 For wiring the IR LED boards to use them independently of the camera board Crimping Tool Set Amazon
https://amzn.to/38Fy1mRB0045CUMLQ $66.74 For wiring the IR LED boards to use them independently of the camera board Wire Stripper Grainger
https://bit.ly/3MOaMps1XFZ6 $34.25 For wiring the IR LED boards to use them independently of the camera board 4-Port USB PCIe Card CDW
https://bit.ly/3aoX6Tz6409687 $114.99 For expanding the total USB bandwidth of the host computer during multicamera recording STL file Picture STL file Picture CAMERA CASE: IR LED CASE: Pi_Cam_Case_Top 
Pi_LED_Case_Top Pi_Cam_Case_Bottom_w_Rod Pi_LED_Case_Bottom_w_Mount Pi_Cam_w_LED_Case_Top IR LED MOUNT TOOLS: Pi_Cam_w_LED_Case_Bottom_w_Rod Pi_LED_Arm_5CM_M2F CAMERA MOUNT TOOLS: Pi_LED_Base Pi_Cam_Arm_7CM_M2F Pi_LED_Ring_180D Pi_Cam_Arm_7CM_M2M_90D ACCESSORIES: Pi_Cam_Arm_7CM_M2M_180D M2.5_Nut_Knob Pi_Cam_Ring_90D G-Clamp_Clamp Pi_Cam_Ring_180D G-Clamp_Screw Pi_Cam_Rod_Base G-Clamp_Press Comprehensive list of all 3D-printed components used in the current build. STL files and print instruction are accessible at https://www.thingiverse.com/gloverlab/designs.
Camera Lighting
conditionRecording
length (s)Video file
length (s)Target
FPSTrue
FPSTotal dropped
framesDropped frames
per minCommercial Red house light 300.56 ± 6.08 124.77 ± 2.52 20 8.30 ± 0.00 3515.00 ± 71.25 702.00 ± 0.00 Pi USB cam Red house light 300.60 ± 6.09 300.54 ± 6.09 20 20.00 ± 0.00 0.00 ± 0.00 0.00 ± 0.00 Commercial White house light 311.05 ± 4.39 179.57 ± 17.43 20 11.57 ± 1.16 2628.60 ± 377.60 505.60 ± 69.40 Pi USB cam White house light 311.02 ± 4.41 310.96 ± 4.40 20 20.00 ± 0.00 0.00 ± 0.00 0.00 ± 0.00 Five-minute videos (640 × 480 resolution, 20 FPS, avi format) were recorded from each camera via Synapse software for five trials under red and white house light illumination. Results are expressed as mean ± SEM.
Camera Lighting condition Recording
length (s)Target
FPSTrue
FPSTotal duplicate
framesDuplicate frames
per minuteCommercial Red house light 301.29 ± 3.81 30 8.28 ± 0.01 6545.20 ± 83.92 1303.40 ± 0.24 Pi USB cam Red house light 301.18 ± 4.09 30 30.00 ± 0.00 0.00 ± 0.00 0.00 ± 0.00 Commercial White house light 300.37 ± 3.77 30 8.33 ± 0.02 6509.00 ± 79.33 1300.20 ± 1.20 Pi USB cam White house light 300.65 ± 3.47 30 30.00 ± 0.00 0.00 ± 0.00 0.00 ± 0.00 Five-minute videos (640 × 480 resolution, 30 FPS, mp4 format) were recorded from each camera via OBS Studio for five trials under red and white house light illumination. Results are expressed as mean ± SEM.
Movies
- Movie 1.
Comparison of Pi USB Cam and commercial webcam performance using specialized neural recording software for video acquisition.
- Movie 2.
Comparison of Pi USB Cam and commercial webcam performance using OBS Studio.
- Movie 3.
Example video showing versatile implementation of Pi USB Cam for behavioral monitoring.
- Movie 4.
Example video showing versatile implementation of Pi USB Cam for rodent home cage monitoring.
- Movie 5.
Example video showing position tracking performed on a video acquired with a fisheye lens before and after distortion correction, and a video obtained with a low-distortion lens.
- Movie 6.
Example video showing real-time closed-loop behavioral control by a commonly used video-tracking system combined with Pi USB Cam.
Extended Data Table 1-1
List of store-bought components including alternative options and accessories. Download Table 1-1, XLS file.
Extended Data Figure 2-1
Diagram of the Raspberry Pi 4B motherboard. Image source: https://github.com/raspberrypi/documentation/blob/develop/documentation/asciidoc/computers/os/using-gpio.adoc. Download Figure 2-1, TIF file.
Extended Data Figure 4-1
Step-by-step instructions for crimping ring terminal connectors. A, Gather all essential parts: (1) electrical wire AWG22, noninsulated ring terminal connector AWG22-16 #4 stud size, heat shrink tubing 1/8”, (2) Astro crimping tool with B-Jaw for noninsulated terminals, and wire stripper. B–D, Strip the wire to expose ∼4 mm of conducting wire, comparable to the length of the wire barrel of the connector. D, Cut one short segment of 1/8” heat shrinking tubing that is long enough to cover the entire wire barrel (≥6 mm) and put it on the wire before crimping. E, Crimp the connector to the bare wire using the first die (DIN 0.5–1.5 mm2) on the B-Jaw of the Astro crimping tool. F, Make sure to visually inspect and perform a gentle pull test to confirm the crimp is successful. G, Fully cover the entire wire barrel with heat shrink tubing and use a heat gun to shrink the tubing. Download Figure 4-1, TIF file.
Extended Data Table 1-2
Pi USB Cam expenses. Laboratories can choose equipment based on budget constraints. Tools and supplies are listed separately because they are only required if users intend to use the IR LEDs independently from the camera as shown in Figure 4 and/or if laboratories are not already stocked with these items. Note that the grand total (n = 8) for the upper bound includes the cost of one set of tools and supplies as well as the cost for eight sets of essential equipment. Our university rate for 3D-printing services of $30/kg of PLA plastic was used to calculate the upper bound cost of 3D-printed components, whereas the lower bound was calculated as the expense for only the purchase of plastic for those laboratories that already have a 3D printer. A complete camera (Fig. 2F) and IR LED (Fig. 4G) holder is estimated to require ∼130 g of PLA plastic based on our recommended print settings. Commercial Webcam = Logitech C930e Webcam; Industrial camera estimates based on quotes from June 2022 from three widely used behavioral neuroscience suppliers. Download Table 1-2, XLS file.
Extended Data Figure 4-2
Step-by-step instructions for crimp pin terminal connectors. A, Gather all essential parts: (1) electrical wire AWG22, male or female pin terminal connector, plastic housing, (2) Astro crimping tool with H-Jaw for open barrel terminals, and wire stripper. B, C, Strip the wire to expose ∼2.5 mm of conducting wire. D, Insert the stripped wire into the pin terminal connector while making sure that the bare wire falls within the wire barrel of the connector and the wire insulation is inside the insulation barrel. Gently bend the insulation barrel around the wire insulation to prevent connector or wire from moving out of place during crimping. E, Crimp the connector to the wire using the middle die (DIN 0.5 mm2) on the H-Jaw of the Astro crimping tool. Make sure that the open barrel is facing towards the “nest” of the crimping die and both wire barrel and insulation barrel are positioned in the appropriate spots of the crimping die. F, Visually inspect and perform a gentle pull test to confirm the crimp is successful. G, Insert the crimped connector into a plastic housing. Visually inspection and gentle pull test should be performed to make sure that the connector is securely housed in order to avoid issues with mating pins. Download Figure 4-2, TIF file.
Extended Data Figure 6-1
Schematics showing several options to preview and/or record from multiple video sources. A, One scene with multiple video sources operating within one instance of OBS Studio recorded into a single video file. B, Multiple scenes each with their own video source operating within one instance of OBS Studio. Recording produces video from a single scene/video source. C, Multiple scene collections each with their own video source running in independent instances of OBS Studio produce independent video files. Image source: https://github.com/obsproject/obs-studio/blob/master/UI/forms/images/obs_256x256.png. Download Figure 6-1, TIF file.
Extended Data Figure 8-1
Digital correction of fisheye distortion. A, Download the OBS ShaderFilter plugin from its GitHub page and install on a Windows PC equipped with OBS Studio. B, Download the “fisheye.shader” text file located in its GitHub repository. In the main OBS interface, add a prerecorded video as a “Media source” for offline fisheye correction or a camera as a “Video Capture Device” for real-time fisheye correction (C1, C2). Enable the ShaderFilter plugin and fisheye correction for each video source (C3–C7). Download Figure 8-1, TIF file.
Extended Data Figure 8-2
Comparison of Pi USB Cam-compatible fisheye lenses in terms of object distance and fisheye correction. Pictures showing the object distance of a Pi USB Cam (1) equipped with various M12 fisheye lenses including 180° (A), 140° (B), 118° (C), 100° (D), and 73° (E) for overhead viewing in a standard operant box. Snapshots of raw videos acquired from a centered position at the corresponding object distances showing comparable captured fields (2), and the same videos after the fisheye image distortion was digitally corrected using the OBS ShaderFilter plugin (3). Power settings used are indicated on each image. *Indicates the default lens that comes with the Arducam day-night vision camera. Download Figure 8-2, TIF file.
Extended Data Figure 8-3
Effects of fisheye distortion on position tracking and locomotor measures. Snapshots of ANY-maze position tracking (1) performed on a video acquired with a 100° HFOV fisheye lens without distortion correction (A), after digital distortion correction (B), and from a video acquired in tandem using a 70° HFOV low-distortion lens (C). Representative center-point tracking plots (2) and heatmaps (3) from the same video show similar results. D, Effects of fisheye distortion and correction on position tracking accuracy were demonstrated by comparing various locomotor activity measures, including the total distance travelled (1) and average speed (2) during the entire test duration, as well as the total distance travelled (3, 4), average speed (5, 6), and time spent (7, 8) in either side of the testing apparatus. Download Figure 8-3, TIF file.
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