FluOpti

FluOpti is an open-hardware device designed to study how bacterial gene networks respond to light stimuli through optogenetic control. The system integrates multichannel fluorescence detection, bright-field tracking of bacterial growth, and temperature regulation using an ITO glass heater. FluOpti supports independent programming and control of blue LEDs for exciting fluorescent reporters, white LEDs for bright-field growth measurements, and red/green LEDs for optogenetic control of the CcaS/CcaR system (green induces, red represses). The device combines and extends prior advances in automated microscopy, optogenetics, and open hardware for biology, including:

1. Fluopi: https://journals.plos.org/plosone/article?id=10.1371/journal.pone.0187163
2. Liquid-media control of the CcaS/R system in E. coli: https://pubmed.ncbi.nlm.nih.gov/24608181/
3. Temperature control using ITO glass (Tim Rudge and Kevin Simpson, unpublished).

Optical System Description

1. The fluorescence microscopy path uses 470 nm LEDs with a diffuser and a blue acrylic filter for excitation, plus an orange acrylic long-pass filter (cut-on >510 nm) that transmits green, yellow, and red emission.
2. Cellular state reporting relies on a green fluorescent protein (sfGFP) and a red fluorescent protein (mBeRFP, long Stokes shift with blue excitation), enabling simultaneous excitation of both reporters and two clearly separable signals without moving filter wheels.
3. The optogenetic module is based on the CcaS/R system developed by Jeff Tabor (Ong 2018), which is activated by green light and deactivated by red light. A ring of red and green LEDs provides optogenetic control.
4. Bright-field illumination for growth measurements uses white LEDs around the plate, enabling colony-growth quantification even for non-fluorescent strains.

The design also includes sensors to calibrate the system and monitor variables such as light intensity and temperature.

Hardware Specifications

1. Blue LEDs for sample illumination: Super Bright 5mm Blue
   • Peak wavelength: 470 nm
   • Forward current: 30 mA @ 3.3 V
   • Peak forward current: 100 mA
   • Maximum forward voltage: 3.8 V
2. Green LEDs for gene activation: Kingbright WP7083ZGD/G
   • Peak wavelength: 520 nm
   • Typical forward current: 20 mA @ 3.2 V
   • Peak forward current: 100 mA
   • Maximum forward voltage: 4.0 V
3. Red LEDs for gene deactivation: Deep-Red 5mm LED
   • Peak wavelength: 660 nm
   • Typical forward current: 20 mA @ 2.2 V
   • Peak forward current: 100 mA
   • Maximum forward voltage: 2.8 V
4. White LEDs are provided by a commercial LED strip, typically operated at:
   • Forward current: 20 mA @ 12 V

5. LED intensity is modulated by PWM control, chosen for its straightforward implementation.
6. A power-distribution stage supplies all submodules, the LEDs, and the Raspberry Pi.
7. The system can read sensors to measure and calibrate light intensity. Because control is provided by a Raspberry Pi, either digital sensors or an external ADC with signal conditioning is required. The light sensor should be low cost, easy to source, and have a flat response for the red and green LEDs. One option is the TEMT6000 phototransistor.
8. Temperature sensing uses an NTC thermistor (e.g., Adafruit #372).
9. Heating is resistive and powered from 9 V to 12 V. Current is PWM-controlled through a transistor. A possible heater is the Film Heating Panel.

Additional Design Considerations

Modularity is the primary design criterion to provide an adaptable and scalable solution. A PWM-generation module, controlled over an I2C serial bus, produces 16 PWM channels that drive LED driver modules. Drivers tailor the PWM signals to the electrical needs of the different LED circuits. Each driver can host one or multiple channels, and some PWM control lines are available to interface with existing driver boards if needed.

The power-distribution system generates all voltages required by the different circuit blocks from a single input supply, improving adaptability by delivering the necessary rails internally.

The board also includes a 4-channel ADC controlled via I2C to read up to four analog sensors. Because the Raspberry Pi lacks an ADC, this stage broadens compatibility with diverse analog sensors. Signal-conditioning stages are configured to match each specific sensor.

Summary of Proposed Solution

1. Adaptability: the entire board is controlled through a single I2C bus (3 pins), regardless of the number of LEDs or sensors.
2. Integration: the layout of PWM control channels accommodates both existing solutions and modules from different vendors.
3. Scalability: I2C enables chaining multiple boards from a single Raspberry Pi (or other microcontroller/processor). Two boards in series provide up to 32 LED control channels and 8 analog sensor channels.

Module Details

ADC

The chosen ADC is the Texas Instruments ADS1115. Adafruit offers a self-contained module for integrating this ADC with the Raspberry Pi, along with documentation and supporting libraries.

Key specifications:

• 4 single-ended channels
• 16-bit resolution
• Supply voltage: 2 V to 5 V
• I2C interface
• Internal reference

Signal Conditioning

The sensors used for measurements vary their electrical properties, typically producing a voltage change. A voltage divider connected to each sensor captures the measurement voltage. Proper acquisition requires conditioning so sensor outputs remain within the ADC and Raspberry Pi input limits while maximizing usable dynamic range.

Because the exact sensors may change, a generic conditioning circuit was implemented with amplification and offset stages. Solder jumpers (S1, S2, S3) allow configurations such as buffer-only, gain-only, offset-only, or gain plus offset. Resistor values can be tuned to set the appropriate gain and offset for each sensor.

PWM Generator

PWM generation is handled by the PCA9685 integrated circuit. Adafruit provides a Raspberry Pi-compatible module with documentation and libraries.

Key specifications:

• 16 PWM-dimmable channels
• 12-bit resolution
• Supply voltage: 2.3 V to 5.5 V
• I2C interface

Low-Current Driver

Low-current channels (e.g., red and green LED arrays) use the ULN2803, an array of eight Darlington transistors that allows control of up to eight channels.

Key specifications:

• 8-channel array with common supply
• Maximum current per channel: 500 mA (higher if channels are paralleled)

High-Current Driver

High-current channels use the IRF740 MOSFET, with one transistor per channel. For currents above 1 A, heatsinks or thermal pads are recommended to dissipate heat effectively.

Key specifications:

• Supports high switching frequencies
• Low operating losses
• Up to 10 A continuous control (up to 40 A pulsed)
• Simple implementation

Power Distribution

The electronics operate from a 12 V DC input, with DC-DC converters generating the required rails for analog and digital circuits, LED arrays, and the heater. The following converters are used:

• 5 V: LM2596R-5.0 — step-down switching regulator, 150 kHz switching, 3 A max output.
• 3.3 V: AP2112K-3.3 — linear LDO, 600 mA max output.
• 9 V: LM2696SX-ADJ — adjustable step-down switching regulator, 150 kHz switching, 3 A max output.
• 16 V / 20 V / 24 V: XL6008E1 — adjustable step-up switching regulator, 400 kHz switching, 3 A max output.

Power Supply

Because the circuit operates from DC, a switched-mode 12 V supply is used.

• Input voltage: 100–120 VAC @ 60 Hz or 200–240 VAC @ 50 Hz
• Output voltage: 12 V
• Maximum output current: 10 A
• Maximum output power: 120 W

Funding

This project is funded by the ANID Millennium Science Initiative Program (ICN17_022) and the Fondo de Desarrollo Cientifico y Tecnologico (FONDECYT Regular 1211218 and FONDECYT Regular 1241452, directed by Fernan Federici).