Need in synthetic biology. Optogenetic circuits require precise, programmable illumination inside a CO₂ incubator without heating cells, leaking light, or disturbing other cultures. Commercial solutions are expensive, bulky, or hard to customize.

Our solution. SPARKbox is a low-cost, light-tight, single-plate illumination insert for standard CO₂ incubators. It delivers programmable pulses at defined wavelengths, maintains gas exchange via a “light-maze” vent (no light leaks), removes heat with an aluminum sink, and is controlled by a Raspberry Pi web app. It was designed with user feedback, validated in real cell experiments, and fully documented for easy reproduction.

What it enables. Reliable, reproducible optogenetic stimulation (violet/blue/green/NIR), parallel dark controls in the same incubator, and rapid iteration for light-controlled gene circuits.

In optogenetic research, photosensitive proteins allow precise, non-invasive regulation of gene expression or signaling pathways. However, to activate these proteins reliably in the lab, researchers need illumination equipment that delivers specific wavelengths under controlled and reproducible conditions.

Commercial light systems are often costly, rigid, or incompatible with incubator environments. Therefore, we designed SPARKbox, a low-cost, programmable, and modular illumination device tailored for mammalian optogenetics.

SPARKbox was built to meet the following requirements:

• Emit light of defined wavelengths (violet, blue, green, and near-infrared);
• Completely block light leakage to protect control samples;
• Maintain ventilation and stable CO₂ exchange for normal cell survival;
• Provide adjustable timing and intensity for flexible experimental control.

After several rounds of design, user feedback, and testing, our final SPARKbox meets all these goals and functions successfully inside standard CO₂ incubators. It offers an accessible, easy-to-use, and reproducible platform for any lab performing light-controlled biological experiments.

To operate the system:

1. Place the SPARKbox inside the incubator.
2. Connect the power cable and regulate voltage.
3. Open the web interface via Raspberry Pi.
4. Input illumination parameters (time, cycles).
5. Click Submit to start the program, the LEDs automatically turn on/off according to schedule.

Our first-generation illumination system, built upon the 2021 NUDT-CHINA design, was intended to provide blue-light exposure for cell experiments.

The setup included:

• A Raspberry Pi single-board computer for program control;
• A relay module for LED switching;
• A custom LED matrix with 24 LEDs (6 per plate region);
• A heat sink for cooling;
• A semi-transparent acrylic cover.

The Raspberry Pi executed a simple Python script to regulate light cycles (ON/OFF duration). Users could adjust light duration and interval through an external screen, mouse, and keyboard (Figure 1). Detailed code is on our software page.

Fig 1 Construction of the preliminary illumination system showing open structure and heat sink

While functional, this early system had several limitations:

• Only one wavelength (blue light);
• Significant light leakage, requiring aluminum foil-wrapped control groups;
• Bulky design that occupied an entire incubator shelf;
• Frequent rewiring needed to change light colors or adjust layouts.

These issues made long-term or multi-condition experiments cumbersome and limited reproducibility.

Problem Identification

To identify improvement needs, we consulted Dr. Shaowei Zhang, a synthetic biologist from the National University of Defense Technology (NUDT), who uses optogenetic systems regularly.

He noted two key issues:

1. Light leakage can compromise other samples in the incubator, limiting the throughput of the experiment.
2. Aluminum foil wrapping around control plates can restrict gas exchange and harm cell viability. (Figure 2)

Fig 2 The preliminary illumination device cannot work simultaneously with two wavelengths.
(A) Heat sink equipped with two light LEDs. (B) The light generated by green LEDs affects the cells placed on the red LED sets.

Dr. Zhang recommended developing a compact, sealed illumination box that can house a single cell culture plate and maintain gas circulation without light leakage (Figure 3).

Fig 3 Interview with Dr. Shaowei Zhang and design of the ventilation part

Design Solution

Inspired by his advice, we designed a closed, modular illumination box, the SPARKbox. Its key innovative feature is a “light-maze” ventilation system, which allows air and CO₂ exchange while preventing photons from escaping. This design leverages the difference between straight-line light propagation and omnidirectional gas diffusion.

To verify feasibility, we modeled the design using SolidWorks 2020, adding:

• A dual-layer light baffle (the light maze) on top and bottom for airflow;
• A A square heat-dissipation opening beneath the LED matrix;
• A positioning frame ensuring precise alignment between LEDs and culture wells;
• An outer casing made of opaque, 3D-printed panels.

The final SPARKbox measures 183 mm × 115 mm × 163 mm, allowing over 16 units to fit in a single incubator, supporting large-scale parallel experiments. (Figure 4)

Practice and Results

Assembly and Testing

SPARKbox components were 3D-printed and assembled manually (assembly gap tolerance: 0.2 mm). Minor seams were sealed with black tape to ensure perfect light-blocking. The modular structure simplifies LED replacement, using Wago Amphenol connectors for quick swaps of LED colors. When assembled , the box was tested for: Light containment, Gas permeability, and Cell viability. (Figure 5)

Fig 5 Physical components and assembled SPARKbox device.

 

Experimental Validation

To evaluate biological compatibility, we compared HEK293T cells cultured inside and outside SPARKbox for 48 hours. Results showed no significant difference in survival rate or morphology, confirming sufficient ventilation. More importantly, we observed no significant light leakage in the incubator when activating a 740 nm LED in the SPARKbox. (Figure 6)

Fig 6 There is no significant influence to cells in SPARKbox

(A) Images of cells cultured inside and outside SPARKbox for 48h

(B) No significant difference in survival rate of cells

(C) Working SPARKbox can avoid light leakage

Also, SPARKbox allows efficient opto-activation of optoSPARK system, resulting in efficient green- and NIR-light-inducible protein secretion in HEK-293T cells. (Figure 7)

Fig 7 Through experiments using SPARKbox, Signal-controlled protein dissociation enables green- and near-infrared light-inducible secretion in mammalian cells.

We invited Prof. Jiawei Shao (Zhejiang University), an expert in mammalian optogenetics, to evaluate our device. (Figure 8)

He provided several valuable suggestions:

• Use low-power and increase the number of LEDs (1–2 mW each) to eliminate edge effects from high-power (3 W) emitters;
• Integrate the controller (Raspberry Pi and power supply) into a single compact module;
• Develop PCB-based LED arrays for higher spatial precision and scalability.

In response, our next design iteration will focus on:

• New illumination boards for uniform light intensity;
• Miniaturized control units for multiple-box operation;
• Raspberry Pi–controlled power regulation, reducing cost and complexit

Fig 8 Consultation with Prof. Jiawei Shao and future roadmap for SPARKbox optimization.

Cost and Accessibility

SPARKbox is fully open-source, affordable, and easy to reproduce.

All 3D models (STL/STEP), wiring diagrams, and Python control scripts are available on our GitLab and Software pages.

Materials	Quantity	Price ($)	Total Price ($)
LED	6	0.67	4.02
Raspberry Pi 4B	1	45	45
Screen	1	1.4	1.4
Aluminum heat sink (127×85×15mm)	1	5.76	5.76
Relay module	1	1.14	1.14
Raspberry Pi 15W USB-C Power Supply	1	6.74	6.74
3D print	1	77.22	77.22
Acrylic board (100×142mm)	1	1.05	1.05
Fire-resistant wire	10m	0.74	0.74
Wago amphenol connector	3	0.37	1.11
Dupont thread	2	0.02	0.04
Keyboard and mouse	1	3.62	3.62
Adjustable power supply	1	2.92	2.92
Thermal conductive silicone	1	1.39	1.39
Total	152.15

Note: 3D printing can be replaced by opaque acrylic panels to further reduce cost.

User Manual

Fig 9 Steps in manual.

1. Connect Raspberry Pi to screen, keyboard and mouse, and complete the preparation of Raspberry Pi (if you are a new hand, there are official tutorials: https://www.raspberrypi.com/documentation/computers/getting-started.html), remembering connect the Raspberry to the network;
2. Connect Raspberry Pi to relay module via Dupont thread; (Figure 9A)
3. Ensure you have installed Git, if not, please enter: https://git-scm.com/downloads;
4. Glued LEDs on aluminum heat sink via thermal conductive silicone, while every LED is corresponding to 4 wells; (Figure 9B)
5. Weld LEDs with fire-resistant wire (series connection), and the positive and negative electrodes should connect one wire respectively;
6. Connect these two places to one Wago amphenol connector:
   • Positive electrode of the LEDs and the positive pole of the adjustable power supply;
   • Negative electrode of the LEDs and normally opened terminal of the relay of relay module;
   • The common terminal and the negative pole of the adjustable power supply; (Figure 9C)
7. Assemble these 3D print models, and the second groove on the side plates is prepared for acrylic board. Remember place the heat sink to the bottom of the box and pull wires out of the hole;
8. Insert the dam-board, and seal the gap on places where two boards are joined (Commonly occurring places are noted in the picture); (Figure 9D)
9. Place your cell culture plate on the acrylic board and cover the lid;
10. Get and run our codes:
    • Power the Raspberry Pi and enter the command line page; (Figure 9E) Use “cd” command to enter a directory you want to save the program: input “cd file_location“ to get into the location named ”file_location”. Then input “git clone https://gitlab.igem.org/2025/software-tools/nudt-china.git” to install our program into Raspberry Pi from Gitlab. The file app.py is saved in directory “SPARKbox”, so input “cd nudt-china/SPARKbox” the next time, and finally input “python3 app.py” to run the script.

    • Directly get into our Gitlab, install the codes, and run the file app.py in directory SPARKbox.

    git clone https://gitlab.igem.org/2025/software-tools/nudt-china.git

    cd nudt-china/SPARKbox

    python3 app.py

11. Enter the popped-up website address, and input parameters you want;
12. Press “Submit” button and the hardware will start running.