An open-source system to integrate Calcium-imaging, Pupillometry, and Locomotion-estimated Tracking (CaPuLeT) in head-fixed mice
Designed by members of the Pégard and Rodríguez-Romaguera Labs at the University of North Carolina at Chapel Hill, CaPuLeT allows researchers to capture locomotion, pupil dynamics, and body kinematics simultaneously. Our system relies on CMOS cameras, an infrared camera placed at eye-level to capture pupil size and an additional camera to record body kinematics. Our system is capable of capturing locomotion, or the mouse's movement across time, as our wheel system is attached to a mounted rotary encoder. For more information, refer to Ortiz-Juza et al, 2024 [1].
The CaPuLeT MATLAB graphical user interface (GUI) allows users to trigger and manage the cameras and LEDs driven by an Arduino microcontroller with a custom shield. Within CaPuLeT, users can track locomotion, pupil-size, and in vivo calcium imaging data in real-time.
Behavioral and Imaging Data Collected:
- IR Camera → Pupil Dynamics (Dilation and Constriction)
- Additional Camera → Body Kinematics (Body and Tail Movement)
- Rotary Encoder → Locomotion (Speed and Acceleration)
- Multi-Photon Microscope → Neuronal Activity (Intracellular Calcium Concentration)
The head-fixation wheel system is driven and controlled by an Arduino Mega 2560 microcontroller board, with a custom printed circuit board (PCB) Arduino shield with built-in resistance, interactive feedback LEDs, real-time clock compatibility, and screw hole terminals. The printed circuit board shield expands the capabilities of the microcontroller board and allows for greater stability than a conventional solderless circuit board. The Arduino shield allows users to interface with each of the components of the CaPuLeT GUI.
The following component list details the manufacturer, part number, and quantity of each item required. This version of the component list contains a color coded key which provides an additional layer of support when building the system.
| Part Number | Distributor | Part Name | Distributor Part Number | Quantity Needed | Unit Price | Total Price |
|---|---|---|---|---|---|---|
| 1 | McMaster | 5 mm Linear Motion Shaft | 6112K37 | 1 | $10.98 | $10.98 |
| 2 | McMaster | Clear Impact-Resistant Polycarbonate Film | 85585K15 | 1 | $8.35 | $8.35 |
| 3 | McMaster | Female Threaded Round Standoff | 93330A541 | 10 | $2.13 | $21.30 |
| 4 | McMaster | Socket Head Screw M3 x 0.5 mm Thread, 6 mm Long | 91290A111 | 1 (Pack of 100) | $10.91 | $10.91 |
| 5 | Polulu | Aluminum Mounting Hub for 5mm Shaft | 1203 | 1 (Pack of 2) | $18.90 | $18.90 |
| 6 | Polulu | Machine Screw: #4-40, 1/2? Length, Phillips | 1962 | 1 (Pack of 25) | $1.39 | $1.39 |
| 7 | Thorlabs | 0.75 Inch Post | TR075 | 1 | $5.38 | $5.38 |
| 8 | Thorlabs | 2 Inch Post | TR2 | 2 | $5.90 | $11.80 |
| 9 | Thorlabs | 3 Inch Post | TR3 | 2 | $6.15 | $12.30 |
| 10 | Thorlabs | 4 Inch Post, 5 Pack | TR4-P5 | 1 (Pack of 5) | $30.00 | $30.00 |
| 11 | Thorlabs | 6 Inch Post | TR6 | 2 | $8.09 | $16.18 |
| 12 | Thorlabs | 8 Inch Post | TR8 | 2 | $9.69 | $19.38 |
| 13 | Thorlabs | 10 Inch Post | TR10 | 2 | $10.76 | $21.52 |
| 14 | Thorlabs | 2 Inch Post Holder | PH2 | 1 | $8.95 | $8.95 |
| 15 | Thorlabs | 3 Inch Post Holder | PH3 | 4 | $9.62 | $38.48 |
| 16 | Thorlabs | 6 Inch Post Holder | PH6 | 1 | $14.70 | $14.70 |
| 17 | Thorlabs | Right Angle End Clamp | RA180 | 4 | $12.34 | $49.36 |
| 18 | Thorlabs | Rotating Clamp | SWC | 1 | $26.91 | $26.91 |
| 19 | Thorlabs | Right Angle Post Clamp | RA90TR | 1 | $17.82 | $17.82 |
| 20 | Thorlabs | Mini Optical Post | MS3R | 1 | $9.09 | $9.09 |
| 21 | Thorlabs | Aluminum Breadboard 8" x 10" x 1/2", 1/4"-20 Taps | MB810 | 1 | $131.60 | $131.60 |
| 22 | Thorlabs | Aluminum Breadboard 12" x 18" x 1/2", 1/4"-20 Taps | MB1218 | 1 | $214.65 | $214.65 |
| 23 | Thorlabs | Breadboard Lifting Handles, Reinforced Polymer | BBH1 | 2 (Pack of 2) | $17.01 | $17.01 |
| 24 | Thorlabs | 4-40 Stainless Steel Cap Screw, 3/8" Long | SH4S038 | 1 (Pack of 50) | $9.31 | $9.31 |
| 25 | Thorlabs | Spring-Loaded 3/16" Hex-Locking Thumbscrew, 1/4"-20 Thread | TS25H | 2 | $2.87 | $5.74 |
| 26 | Thorlabs | 1/4"-20 Stainless Steel Setscrew, 5/8" Long (Adaptor) | SS25S0625 | 1 (Pack of 25) | $6.97 | $6.97 |
| 27 | Thorlabs | 1/4" Washer, M6 Compatible, Stainless Steel | W25S050 | 1 (Pack of 100) | $10.20 | $10.20 |
| 28 | Thorlabs | 1/4"-20 Stainless Steel Nut | N25S0440 | 1 (Pack of 50) | $6.43 | $6.43 |
| 29 | Omron | Rotary Encoder - 1024 P/R (Quadrature) | E6B2-CWZ3E (E69-C06B) | 1 | $48.50 | $48.50 |
| 30 | DB Unlimited | Speaker 80hm 5 w top port 104db | 2104-SP500208-3-ND | 1 | $6.79 | $6.79 |
| 31 | SullinsCorp | 2.54mm Pitch Single Row Pin Header Strip | PREC040SAAN-RC | 3 | $1.53 | $1.53 |
| 32 | Ardest | 2N2222 NPN TO-92 Power Transistors | 2N2222 | 6 | $1.44 | $1.44 |
| 33 | Boca Bearings | 5 mm Steel Ball Bearing | SMR115-ZZC #5 LD | 2 | $10.58 | $21.16 |
| 34 | Xenocam | 9-22mm 1/3" F1.4 M12-mount lens with IR filter | XC0922LENS | 1 | $19.99 | $19.99 |
| 35 | Monoprice | USB 2.0 Type-A Male to Mini-B 5pin Male Cable (Monoprice) | B002KL8J8W | 1 | $4.78 | $4.78 |
| 36 | Computar | 0.33-Inch Varifocal lens 2.8-12mm F1.3 Auto Iris (Computar) | B002HRE2ZS / TG4Z2813FCS-IR | 1(Used) | $19.99 | $19.99 |
| 37 | Chanzon | 3 mm UV light, 395-400nm (Chanzon) | 100F3T-YT-WH-PU | 1 (Pack of 100) | $6.99 | $6.99 |
| 38 | Adafruit | 5 mm super-bright IR LED, 940nm (Adafruit) | ADA388 / B00ULB0U44 | 1 (Pack of 25) | $10.98 | $10.98 |
| 39 | Fermerry | 24 AWG 50 feet Electrical Wire (Fermerry) | B089CX3FVV | 1 | $26.99 | $26.99 |
| 40 | Arduino | Arduino Mega 2560 Rev3 Microcontroller Board (Arduino) | A000067 / B0046AMGW0 | 1 | $48.99 | $48.99 |
| 41 | Arduino | USB 2.0 Cable Type A/B (Arduino) | M000006 / B013EOQUAW | 1 | $7.00 | $7.00 |
| 42 | Tnisesm | PCB Mount Screw Terminal Block Connectors (Tnisesm) | CATN-T03B | 1 | $8.99 | $8.99 |
| 43 | Adeep | Real Time Clock Module DS3231 AT24C32 IIC (Adeept) | 2pcsDS3231 / B097PMNMQZ | 1 (Pack of 2) | $7.99 | $7.99 |
| 44 | LiCB | 3V Lithium Battery (Junpower) | CR2032 / B07LGL9LQ1 | 1 (Pack of 10) | $4.99 | $4.99 |
| 45 | ELECFUN | Heat Shrink Tubing (Electfun) | RSG-KIT350 | 1 | $14.99 | $14.99 |
| 46 | Yancorp | Black Adhesive contact paper (Yancorp) | B082CPSVPW | 1 | $6.98 | $6.98 |
| 47 | Weld-On | IPS Weld-On 3 Acrylic Plastic Cement (Weld-On) | ?TLLPN-21847771 | 1 | $28.55 | $28.55 |
| 48 | CENTROPOWER | 12V 5A DC Power Cable Male Female Connector (Centro Power) | DCCORD-MF | 1 (Pack of 10) | $9.19 | $9.19 |
| 49 | CENTROPOWER | 12V DC Power Connector Barrel Adaptor (Centro Power) | B07C61434H | 1 (Pack of 10) | $7.99 | $7.99 |
| 50 | ALITOVE | 12V Red LED Flexible Strip Ribbon Light (Alitrove, 16.4 feet) | AL5RWPBK12V | 1 | $13.99 | $13.99 |
| 51 | Mifasopower | Power Strip Surge Protector 10Ft (Mifaso Power) | MFS311T | 1 | $19.99 | $19.99 |
| 52 | Sumato Stuff | Anti Vibration Pads (Functional Necessities) | SSHI00003 | 1 (Pack of 4) | $14.99 | $14.99 |
| 53 | Focusound | 1" x 12" x 12" Sound Absorption Wall Panels (Xin & Log) | FS0302A | 1 (Pack of 24) | $17.99 | $17.99 |
| 54 | TANTTI | 4 inch Zip Ties with 18 lb Tensile Strength | 604477847283 | 1 (Pack of 200) | $3.99 | $3.99 |
| 55 | 3M | 3M Scotch Super 33+ Electrical Tape - 3/4 in x 52 ft | MMM06133 | 1 | $4.98 | $4.98 |
| 56 | Amazon | Hirose 4 Pin Female HR10A-7P-4S to Flying Leads Power Cable (Alvin’s Cables) | HR10A-7P-4S(73) | 1 | $25.99 | $25.99 |
| 57 | Custom Machined Parts | Rotary Encoder Adaptor (Aluminum 304) | CNC Machining (eMachineShop) | 6 (Bulk Purchase) | $28.43 | $170.62 |
| 58 | Custom Machined Parts | Head Plate Holder (Aluminum 304) | CNC Machining (eMachineShop) | 12 (Bulk Purchase) | $30.20 | $362.44 |
| 59 | Custom Machined Parts | Head Plate (Top Piece) (Aluminum 304) | CNC Machining (eMachineShop) | 6 (Bulk Purchase) | $39.14 | $234.85 |
| 60 | Custom Machined Parts | Head Plate (Bottom Piece) (Aluminum 304) | CNC Machining (eMachineShop) | 6 (Bulk Purchase) | $51.01 | $306.04 |
| 61 | Custom Machined Parts | Head rings (Aluminum 6061-T6) | CNC Machining (eMachineShop) | 100 (Bulk Purchase) | $5.16 | $258.22 |
| 62 | Custom Machined Parts | Rotary Wheel (1/8 Thick Clear Cast Acrylic) | Laser Cutting (Ponoko) | 2 | $61.06 | $61.06 |
| 63 | Custom Machined Parts | Rotary Wheel Curved Surface (Clear Cast Acrylic) | Laser Cutting / Manual Cutting | 1 | $0.00 | $0.00 |
| 64 | Custom Machined Parts | Rotary Wheel Curved Surface (Black Adhesive Contact Paper) | Laser Cutting / Manual Cutting | 1 | $0.00 | $0.00 |
| 65 | Custom Machined Parts | Waste Management Ramp (Resin) | 3-D Printing (Craftcloud3d) | 1 | $57.76 | $57.76 |
| 66 | Custom Machined Parts | Arduino Mega 2560 Rev3 Protective Case (Resin) | 3-D Printing (Craftcloud3d) | 1 | $16.06 | $16.06 |
| 67 | Custom Machined Parts | Arduino Mega 2560 Rev3 Shield SMT PCB | PCB Manufacturing (JLC PCB) | 1 | X | X |
| 68 | FLIR | Firefly S Camera | FFY-U3-16S2M-S | 1 | $234.00 | $234.00 |
| 69 | FLIR | Tripod Adapter for Firefly S Camera | ACC-01-0017 | 1 | $32.10 | $32.10 |
| 70 | FLIR | USB 3.1 Locking Cable | ACC-01-2300 | 1 | $24.60 | $24.60 |
| 71 | FLIR | GPIO cable w/ 6 pins | ACC-01-3015 | 1 | $9.60 | $9.60 |
| 72 | The Imaging Source | USB 2.0 CMOS Monochrome Camera | DMK-22BUC03 | 1 | $234.00 | $234.00 |
| 73 | SYSRACKS | 15U 35" Depth Under Desk Sound Proof Server Cabinet | SP15.900 | 1 | $1,122.00 | $1,122.00 |
This document outlines the design of each of the custom pieces and the file types found within this repository that can be used to replicate the system. More details regarding the specifications of the custom parts can be found here. The following spreadsheet outlines the total cost of the system.
Here is a list of the manufacturers we purchased materials from:
- McMaster-Carr
- Pololu
- Thorlabs
- SparkFun
- Digikey
- Amazon
- FLIR
- The Imaging Source
- SYSRACKS
Here is a list of companies you can purchase custom parts from, if not fabricated in-house:
- eMachineShop (CNC Machining)
- Ponoko (Laser Cutting)
- Craftcloud3d (3-D Printing)
- JBL PCB (PCB Printing / Soldering)
A fully functioning system requires users to build the following elements from the materials purchased:
- Locomotion Wheel
- Headplate Fixation System
- IR / UV LEDs
Additionally, the following commercial materials need to modified to be used within our system:
- Rotary Encoder
- GPIO Cable
- Hirose Cable
- Overhead Speaker
- Ambient Noise Fan
- Red LED Strips
This following guide provides a step-by-step walkthrough for building and modifying each of the required components. This document includes diagrams and explanations for assembling each of the final components of the build.
Following this installation guide will ensure that your system will be able to run the set of MATLAB scripts and Arduino IDE sketches we have provided. Our system is run primarily on MATLAB's interface. While other software is used to first initialize components, such as Arduino IDE for the rotary encoder and Spinnaker SDK for the cameras, the subsequent input signals and data acquisition steps are performed exclusively with MATLAB.
Required Software and Applications:
Our MATLAB scripts are based on MATLAB R2022a, a previous version of the software application. Running our scripts on a more recent version of the application will result in a number of errors. After downloading MATLAB R2022a, you will need to download a number of toolboxes and add-ons.
Base Toolboxes:
- Data Acquisition Toolbox
- Image Acquisition Toolbox
- Image Processing Toolbox
- Statistics and Machine Learning Toolbox
Additional Toolbox Support Packages:
- Data Acquisition Toolbox Support Package for National Instruments NI-DAQmx Devices
- Image Acquisition Toolbox Support Package for GenICam Interface
- Image Acquisition Toolbox Support Package OS Generic Video Interface
- Image Acquisition Toolbox Support Package for Point Grey Hardware
- Image Acquisition Toolbox Support Support Package for GigE Vision Hardware
- MATLAB Support Package for Arduino Hardware
Finally, users will need to download the following drivers to generate a connection between their computer system and an external hardware device. In our case, we need to download two drivers to interface with the additional camera used in our system.
Driver Requirements:
- USB3 Driver
- DMK 22BUC03 Driver
- Spinnaker SDK USB3 Vision Cameras Driver
- IC Matlab Plugin for Matlab (Version 3.4.0.58)
Library Requirements:
Further information and requests for resources should be directed to and will be fulfilled by the lead contact and corresponding author, Jose Rodriguez-Romaguera ([email protected]). Example pupillometric and locomotion data is available upon request to the technical contact and corresponding author, Nicolas C. Pégard ([email protected]).
If you replicate this system in your own labs or use parts of our designs and code, we kindly ask that you please cite Ortiz-Juza et al, 2024 [1].
[1] Ortiz-Juza, M. M., Tormes-Vaquerano, J., Hegel, S. M., Curtis, V. R., Alghorazi, R. A., Miller, N. W., McTaggart, E. M., Pégard, N. C., & Rodriguez-Romaguera, J. (2024). Protocol for an open-source system to integrate calcium imaging, pupillometry, and locomotion-estimated tracking in head-fixed mice. STAR Protocols, 5(4), 103331. https://doi.org/10.1016/j.xpro.2024.103331