Building a Simple and Versatile Illumination System for Optogenetic Experiments

Summary

This protocol describes how to perform optogenetic experiments for controlling gene expression with red and far-red light using PhyB and PIF3. Included are step-by-step instructions for building a simple and flexible illumination system, which enables the control of gene expression or other optogenetics with a computer.

Abstract

Controlling biological processes using light has increased the accuracy and speed with which researchers can manipulate many biological processes. Optical control allows for an unprecedented ability to dissect function and holds the potential for enabling novel genetic therapies. However, optogenetic experiments require adequate light sources with spatial, temporal, or intensity control, often a bottleneck for researchers. Here we detail how to build a low-cost and versatile LED illumination system that is easily customizable for different available optogenetic tools. This system is configurable for manual or computer control with adjustable LED intensity. We provide an illustrated step-by-step guide for building the circuit, making it computer-controlled, and constructing the LEDs. To facilitate the assembly of this device, we also discuss some basic soldering techniques and explain the circuitry used to control the LEDs. Using our open-source user interface, users can automate precise timing and pulsing of light on a personal computer (PC) or an inexpensive tablet. This automation makes the system useful for experiments that use LEDs to control genes, signaling pathways, and other cellular activities that span large time scales. For this protocol, no prior expertise in electronics is required to build all the parts needed or to use the illumination system to perform optogenetic experiments.

Introduction

Optogenetic tools are becoming ubiquitous and new technology is constantly being developed to optically control biological processes such as gene expression, cell signaling, and many more^1,2,3. The ability to control cellular processes with light allows for fast kinetics, tight spatial control, and dose-dependent regulation that can be controlled by light intensity and exposure time. To use these tools, a device to control these parameters is necessary. We have recently developed a genetically encoded PhyB-PIF3 mammalian gene switch that reversibly activates and deactivates genes using red/far-red light, respectively⁴. This system was tested in several mammalian cell lines and enabled the unparalleled induction of gene expression even with very small amounts of light, including pulses of light. Researchers wishing to use the PhyB switch and similar tools^5,6 frequently request information on methods to control illumination intensity and duration. Therefore, we developed this protocol with step-by-step instructions to enable broader adoption of these tools for optogenetics.

Before the widespread use of LEDs, broadband light sources with filters were used to study light-responsive proteins such as phytochromes⁷. Recently, some LED illumination systems have been published along with optogenetic tools^8,9,10,11,12, but those protocols can require significant expertise in electronics/software, require specialized equipment (e.g., 3D printers, laser cutting machines, or photomasks), or do not provide the step-by-step instructions some researchers would need to deploy for their research needs. While independent control of individual wells in a multiwell plate can be useful, it is often unnecessary when researchers only need to compare several different samples in light and dark or red light versus far-red light. Also, many existing commercial systems are expensive, with limited customization capability. However, the LEDs described in this protocol are cost-effective, bright, and can be mounted in many ways; therefore, they can be used to illuminate several different types of samples. With the protocol and the software provided, LEDs ranging from ultraviolet (UV) to NIR can be used and controlled with software to perform optogenetic experiments using UVR8^13,14, Dronpa^15,16, LOV domains^17,18, Step Function Opsins^19,20, CRY2^21,22, PhyB^4,23,24,25, bacterial phytochromes^26,27,28,29 and other light-responsive systems^30,31,32.

This protocol constitutes a tutorial for the assembly of the circuits and other hardware needed to control different parameters for light stimulation as well as the molecular/cellular tools to run an optogenetic experiment. In addition, we report plasmids optimized from Kyriakakis et al.⁴ that are smaller and more stable for cloning. Through this protocol, biologists without expertise in electronics and optics can build illumination systems that are flexible and robust. In a step-by-step fashion, we show how to build LED systems, removing the technical bottleneck for the broader adoption of optogenetic tools. This system can easily be used in most cell culture incubators, even if they do not contain wire ports. For example, we have kept the LED system in a humidified CO₂ incubator continuously for more than 6 months with no decrease in performance. We also explain how to connect the LED system to a computer and interface it with open-source software we provide on GitHub (https://github.com/BreakLiquid/LED-Control-User-Interfaces). Building a system using this protocol provides researchers the basic knowledge to debug potential issues, replace parts, and improve/extend functionalities.

System overview

Building the illumination system involves (1) building the electronic circuit, (2) building the peripherals (power supply cord, power switch, etc.), (3) building the LEDs, (4) assembling all of these components, and (5) installing the software to control the LEDs with a user interface (Figure 1A). Once completed, the illumination system can control up to four LEDs independently with a user interface (Figure 1B). The user interface enables each LED to pulse at specified time intervals and shut off after a specified time. There is also a start-delay to begin illumination programs at a specified time. Potentiometers (POTs) regulate each LED’s intensity independently or can be used for manual LED control without a computer. The wires to the LEDs can be any custom length, allowing them to be easily placed in an incubator or lab space. Due to these LEDs’ high power, they can be used to illuminate a large area with a single LED from a distance.

LED driver description

To power and control the LEDs' intensity, this protocol will go through steps to build an “LED driver”. Each LED has a range of voltages in which it operates (Figure 1C). During operation, the regulator's output voltage, which controls the light intensity, can be tuned by a potentiometer. The POT varies the resistance, adjusting the output voltage/brightness. Tuning with a 1kΩ (1 kilo-ohm) POT gives what we call the “high-voltage circuit” and has a range of 1.35 V to 2.9 V. Since 2.9 V is too high for operating the lower voltage LEDs (Figure 1C), we show a single modification (Resistor 3 or “R3” Supplementary Figure 1A) that limits the range to match the low voltage LEDs. R3 serves to decrease the maximum voltage applied to the LEDs to 1.85 V (assembly detailed in Supplementary Figure 8) when in parallel with the potentiometer. By using voltage to control brightness instead of current, the system is more flexible for LEDs with different operating voltages. Figure 1C contains a list of the high and low voltage LEDs to guide the optimal circuit selection. This design keeps the minimum voltage low enough so that the LED is completely off when the potentiometer is off and does not allow the voltage to go above the typical operating voltage of the LED. For PhyB optogenetics, we use deep red and far-red LEDs, which use the low-voltage circuit.

LED computer control system description

The LED illumination system can be used for constant illumination without a computer or microcontroller. However, for pulsing programs and for controlling individual LED timing, a microcontroller must be installed. To use a microcontroller to control the LEDs, a transistor is required to connect the microcontroller to the circuit. This transistor senses voltage from the microcontroller and switches from being conductive or insulating. To control the “on” and “off”, we use what is called an “NPN switching type transistor” (2N2222) as a controllable shunt across R2 (Supplementary Figure 1A). When the voltage from the microcontroller is applied to the transistor base, the transistor becomes conductive and makes the LED voltage low, turning the LED off. Thus, the LED and transistor on and off states are directly controlled by the microcontroller, which is controlled by the software installed on the PC.

For making the illumination system, the following steps are required: Build the electrical circuit; build the power supply, manual power switch, POTs, and microcontroller connection; build the LEDs; accommodate a black box to fit the illumination system; connect all of the wiring and devices; install the LED control software, stimulate the cells with light; measure gene expression using a dual luciferase assay.

Protocol

1. Build the electrical circuit

NOTE: The protocol for building a single circuit for an available LED is described here. Instructions to expand this up to four LEDs are included in the supplementary info.

1. Turn on the smoke absorber and the soldering iron. Add water to the wiping sponge, have the solder at hand.
   CAUTION: Make sure to take safety precautions to remove smoke and prevent burns.
2. Begin soldering circuit components to the printed circuit board (PCB board) in the order shown in the supplementary panels.
   NOTE: Use a small amount of solder on the soldering iron tip to heat the metal of the component and the PCB board first and melt additional solder directly onto the components; flux can help a lot.
3. Solder jumper wires and components (Supplementary Figure 2 and Supplementary Figure 3).
   1. For the jumper wires (the insulated wiring that connects two points on the circuit board), use two pieces of orange [7.6 mm (0.3 inch)] and yellow [12 mm (0.4 inch)] wires from the jumper kit.
   2. Clip the PCB board onto the “helping hands” and insert the jumper wires in the following pinholes, bend the terminals 45 degrees and add flux (Figure 2, Supplementary Figure 2 and Supplementary Figure 3): a1 and a3 → ground (-) (orange), a7 → power supply (+) #7 (yellow), d2 → d6 (yellow).
   3. Solder and then trim the back of the wires.
   4. Insert LM317T voltage regulator in the following pinholes, bend the pins, and add flux (Figure 2 and Supplementary Figure 4): Adj → e5, V_out → e6, V_in → e7.
   5. Solder the left and right terminals first, trim them, then solder and trim the middle terminal.
   6. To set the low voltage range of the circuit, insert an 820 Ω resistor all the way down into pinholes, solder and trim c2 → c5 (Figure 2 and Supplementary Figure 5).
   7. To enable LED control by the microcontroller, insert the transistor into b3–b5 (Figure 2 and Supplementary Figure 6): Collector → b3, Base → b4, Emitter → b5.
      NOTE: Be aware of the orientation of the transistor to insert correctly; check the specs to find the Collector, Base, and Emitter designation.
4. Solder the wire-to-wire connectors for the POT, LED, microcontroller, and power source.
   NOTE: Pay attention to the color of the wires of the wire-to-wire connectors and whether using a female or male wire-to-wire connector.
   1. Determine whether a “low voltage” circuit or “high voltage” circuit is required for the desired LED (Figure 1C).
      NOTE: If the LED is on the “low voltage” list, a resistor in parallel with the POT is required.
   2. For the “low voltage” or “high voltage” circuit, place the wire from a female wire-to-wire connector through hole a5 (Supplementary Figure 7). Do not solder into place yet if making the low voltage circuit.
      NOTE: Twist the bare wire ends so that the small wire hairs don’t flake out. If the wire seems too thick to push through the pinhole without fraying, cut 2–6 strands and then twist them back together (Supplementary Figure 7B–D).
   3. If making the “high voltage” circuit, skip to step 1.4.5. If making the “low voltage” circuit, push a 560Ω resistor through the same hole (a5) and solder with the wire-to-wire connector lead.
   4. Connect the other end of the resistor to the ground (Supplementary Figure 7G).
   5. Insert the other end of the female wire-to-wire connector soldered into a5 hole connecting it to the ground and solder it (Supplementary Figure 8A,B).
   6. For the microcontroller connection, insert one end of a male wire-to-wire connector into hole a4 and the other into a hole connected to the ground (Supplementary Figure 9A–C).
   7. For the LED connection, insert one end of a female wire-to-wire connector into hole a2 and the other end into a hole connected to the ground (Supplementary Figure 9D,E).

2. Build power supply, manual power switch, POTs, and microcontroller connection

1. Build the power supply.
   1. Solder an orange [7.6 mm (0.3 inch)] jumper from a29 to the ground (Supplementary Figure 10).
   2. Solder a female wire-to-wire connector from a30 to the power supply (+) (Supplementary Figure 11A–C).
   3. Solder a male wire-to-wire connector from c29 to c30 (Supplementary Figure 11D–F).
   4. Cut the connector off a power supply cord, expose the wires, and strip them (Supplementary Figure 12A–C).
   5. Add flux to the wires prior to soldering using a flux pen (Supplementary Figure 3G).
   6. Place a 3.18 mm (1/8 inch) shrink tube around a male wire-to-wire connector and a thicker piece 4.76 mm (3/16 inch) over the power supply wire (Supplementary Figure 12D).
   7. Twist the wires from the power supply and the male wire-to-wire connector together and solder (Supplementary Figure 12E, 13A,B).
   8. Place the smaller diameter shrink tube 3.18 mm (1/8 inch) over the connections and shrink them with a heat gun (Supplementary Figure 13C,D).
   9. Place a larger diameter shrink tube 4.76 mm (3/16 inch) over the smaller shrink tube 3.18 mm (1/8 inch) and heat again (Supplementary Figure 13E,F).
2. Build the manual power switch.
   1. Place the shrink tube 3.18 mm (1/8 inch) over the wires for the switch (Supplementary Figure 14A).
   2. Twist and solder the wires of a male wire-to-wire connector (Supplementary Figure 14B,C).
   3. Place the shrink tube 3.18 mm (1/8 inch) over soldered sections and shrink with a heat gun (Supplementary Figure 14D,E).
3. Connect the male wire-to-wire connector to the POT.
   1. Loop the wire-to-wire connector's black wire around the middle terminal of the POT (Supplementary Figure 15B).
   2. Twist the wire that is looped tightly around the terminal and solder it (Supplementary Figure 15C).
      NOTE: Small precision pliers can assist in making a tight twist.
   3. Repeat with the red wire connection to the terminal, as in Supplementary Figure 15D.
   4. Use pliers to break the metal tab near the red arrow (Supplementary Figure 15E,F).
4. Build the microcontroller connection (only necessary for computer-controlled LEDs).
   1. If making a LED driver for more than one LED, cut off the black wires from all but one female wire-to-wire connector (Supplementary Figure 16A).
   2. Crimp the ends of the wire-to-wire connectors, as shown (Supplementary Figure 16B–D).
   3. Push the crimped ends through the rectangular connector (Supplementary Figure 16E).

3. Build the LEDs

1. Strip the wire ends (~5 mm) and apply flux using a flux pen as in Supplementary Figure 3G.
   NOTE: To efficiently solder the wires onto the LED base, flux must be added to the contacts on the LED base and the wires.
2. Tin the wire by heating the wire from below and adding solder from the top (Supplementary Figure 17B).
3. Use the flux pen to place flux onto the surface contact of the LED base (Supplementary Figure 17C).
4. Place a generous amount of solder onto a large soldering tip (~4–5 mm) (Supplementary Figure 17D), use it to heat the LED base at the contact (Supplementary Figure 17E). After a few seconds, drag the solder across the contact (Supplementary Figure 17F). Repeat steps 3.3–3.4 on the other contact (Supplementary Figure 17G).
   CAUTION: The LED base can get very hot during soldering. Place the LED base on a surface that will not melt or burn.
5. Clip the black wire onto the contact “C+” (the cathode) using the hair clips (Supplementary Figure 18A).
6. Place a generous amount of solder onto the large soldering tip (Supplementary Figure 18B) and press it down on the wire until the solder on the LED base melts (Supplementary Figure 18C). Hold down the wire (Supplementary Figure 18D) and remove the soldering iron while holding the wire in place (Supplementary Figure 18E).
7. Place a small amount of solder paste onto the pads for the LED connections (Supplementary Figure 19A,B) and place the LED over the pads using forceps (Supplementary Figure 19C).
   NOTE: If the placement is a little off, it is okay; it will go into place once the solder paste melts.
8. Hold the red wire on the “A+” (anode) and clip it with a hair clip (Supplementary Figure 20A–C).
9. Place a generous amount of solder onto the large soldering tip (Supplementary Figure 20D) and press it down onto the wire until the solder on the LED base and the solder paste under the LED melts (Supplementary Figure 20E).
   NOTE: After the solder paste melts, the color becomes silver (Supplementary Figure 20H,I).
10. Choose the length of the wire needed for the desired set up. Strip the LED wires, and a male wire-to-wire connector (Supplementary Figure 21A) then add flux as in Supplementary Figure 3G.
11. Place the shrink tube over the wires. Use a 3.18 mm (1/8 inch) shrink tube over the wire-to-wire connectors and a 4.76 mm (3/16 inch) shrink tube over the wire (Supplementary Figure 21B).
12. Clip the wire-to-wire connector with a “helping hands” and twist the connector end with the wire (Supplementary Figure 21C) and solder them. Repeat with the other wire (Supplementary Figure 21D,E).
13. Place the 3.18 mm (1/8 inch) shrink tubes over the solder and shrink (Supplementary Figure 21F–G).
14. Place the 4.76 mm (3/16 inch) shrink tube over the 3.18 mm (1/8 inch) shrink tube and shrink (Supplementary Figure 21H–I).
15. Clip the LED wires the “helping hands” with tape under it (Supplementary Figure 22A).
16. Mix epoxy according to the manufacturer’s instructions and spread over the top of the soldered LED (Supplementary Figure 22B). Leave overnight to cure.
17. If mounting using a touch fastener, cut a small piece of the touch fastener (Supplementary Figure 23A) and press it against the back of the LED for 30 s.
18. Use a high-speed rotary tool to make a notch on the lid of a black box (Supplementary Figure 23C–E).
19. Build a mounting for a single LED through a privacy film.
    1. Using the spade drill bit, drill a 1.75 cm (11/16 inch) hole through the top of a black box where the LED will be placed (Supplementary Figure 24A).
    2. Using a high-speed rotary tool, make a notch on one side of the hole to make room for the LED wire, as shown in Supplementary Figure 24A.
    3. Cut a piece of privacy film (25–30 mm) and tape onto the inside of the black box covering the hole that the LED will illuminate through (Supplementary Figure 24A).
    4. Place the LED outside the black box on top of the hole with privacy film and tape in place with electrical tape (Supplementary Figure 24B–E).

4. Accommodate a black box to fit the illumination system

1. For a four LED system, drill four 0.83 cm (21/64 inch) holes on the lid 3.81 cm (1.5 inch) apart where the potentiometers will be attached (Supplementary Figure 25).
2. Using a high-speed rotary tool, cut a 1.19 cm x 1.90 cm (0.47 inch x 0.75 inch) rectangular hole in the top-left corner (Supplementary Figure 25).
3. Using the spade drill bit, drill a 1.75 cm (11/16 inch) hole on the black box (Supplementary Figure 26).
4. File the holes and insert the grommet into the hole drilled in (Supplementary Figure 26).
5. For the computer-controlled LEDs, sandpaper the area where the microcontroller will be glued in a black box, as well as the bottom side of the microcontroller holder.
6. Snap the microcontroller onto the holder before securing the holder in the black box and then epoxy them in place (Supplementary Figure 27A).
7. Use sandpaper to sand the bottom of two clips and the area in a black box where the circuit will be placed and secure the clips inside the black box with the epoxy (Supplementary Figure 27A).
8. Secure the PCB board into clips (Supplementary Figure 27B).
9. Push the power switch through the square hole in the lid made in Supplementary Figure 25 and snap it into place (Supplementary Figure 28A).
10. Push the POTs through the holes on the lid, screw into place (Supplementary Figure 28A), and put the knob onto the POT (Supplementary Figure 28B).

5. Connect all of the wiring and devices

1. Label the wire-to-wire connectors (e.g., LED, POT, COM) (Supplementary Figure 29A).
2. Attach the crimped connectors made in step 2.4 (Supplementary Figure 16) to the male wire-to-wire connector between the two female connectors (POT and LED) (Supplementary Figures 7A and S37).
3. Connect the crimped ends into the microcontroller (Supplementary Figure 30).
4. Pull the USB cable through the grommet and plug it into the microcontroller.
5. Pull the wires for the LEDs through the grommet and connect to the female wire-to-wire connector on the left of the microcontroller connection (Supplementary Figures 9D and 38).
6. Pull the wire for the power supply through the grommet and connect it to the male wire-to-wire connector on the right side of the PCB board (Supplementary Figure 11D).
7. Connect the male wire-to-wire connector from the power switch to the female wire-to-wire connector on the right of the PCB board (Supplementary Figure 11A).
8. Connect the male wire-to-wire connectors from the POTs on the lid to the female wire-to-wire connectors on the PCB board (Supplementary Figures 8 and 36).
   NOTE: Do not turn on the circuit without the potentiometers connected.

6. Install the LED control software

NOTE: See the detailed Software Installation Instructions in the supplementary file on Github. https://github.com/BreakLiquid/LED-Control-User-Interfaces

1. Download and install the software for programming the microcontroller
2. Download and install the package manager.
3. Program the microcontroller.
4. Download and install the runtime engine.
5. Download the user interface.

7. Stimulate the cells with light

1. Transfect HEK293 cells.
   1. Plate HEK293 cells at 100k cells per well in a 24-well plate.
   2. Use the example table to calculate serum-free media, Polyethylenimine (PEI), and DNA volumes (Supplementary Figure 39) and transfect using the manufacturer’s protocol.
2. Stimulating cells with light.
   NOTE: Cells must be kept in the dark after transfection or handled using a light source that does not excite the optogenetic system.
   1. Decide what type of stimulation will be used on the cells (continuous light, pulsing intensity, etc.).
   2. With the POTs turned off (counterclockwise), turn on the LED power supply.
   3. Place a light meter inside the black box where the cells will be placed and place the lid with the LED over the meter. Adjust the light intensity as needed.
   4. If using the computer to control the LEDs, open the user interface software.
   5. Program the user interface (Figure 5A,B).
      1. On the top left panel, select the COM port for the microcontroller and click on Connect.
      2. Use the panels on the right to program each LED. For continuous light, select any time except for zero in the “Time On” and set the “Time Off” to zero.
      3. On the bottom right panel, program the main timing control.
         1. To delay illumination, select a start delay (HH:MM).
         2. To shut off all LEDs after a designated time, select a run time (HH:MM).
         3. Start the illumination program by clicking on the Run button (Figure 5A).

8. Measure gene expression using a dual luciferase assay

1. Prepare luciferase reagent by mixing 10 mL of luciferase buffer with luciferase reagent and aliquot in 1 mL tubes to be stored at -80 °C for up to 1 year.
2. Prepare lysis buffer 5x into 1x for 100 µL for N + 2 wells. e.g., for 30 samples, 30 x 20 µL of 5X lysis buffer, and 30 x 80 µL of MQ H₂O.
3. Prepare Renilla substrate solution: 20 µL of Renilla substrate for 1 mL of Renilla buffer (this amount is suitable for 10 assays).
4. Remove cells from the incubator, aspirate the media, add 100 µL of 1x lysis buffer per well and place it on a shaker at 100 RPM for 15 min.
5. Place in -20 °C for at least 1 h.
6. Add 100 µL of luciferase reagent per sample into a well of a white 96-well plate.
7. Set up the plate reader for luminescence. Using the luminometer module of the plate reader, set the integration for 1 s.
8. Add thawed lysates in wells below the luciferase reagent. Using a multichannel pipette, mix 20 µL of sample into the luciferase reagent and measure luminescence immediately.
9. After the readings plateau, add 100 µL of Renilla substrate solution and scan again.
10. Divide the Luciferase signal by the Renilla signal to account for transfection efficiency.
11. Compare luciferase signals normalized for transfection efficiency (e.g., compare the signal from red light illuminated and far-red light illuminated samples).

Discussion

The LED system described here has been used in our lab to optimize, characterize, and work with several optogenetic tools. In Kyriakakis et al.⁴, we tested many combinations of PhyB-PIF gene switches in parallel. We then used this system to test pulses of light at different frequencies to measure the gene switch kinetics and effective light intensity. This system was also used to optimize and characterize two optogenetic systems that use blue light for stimulation^5,6. Since only one LED needed to be bright enough to activate most optogenetic tools, buying a system with large numbers of LEDs over each well is not always necessary. This setup is inexpensive, reliable, easy to reconfigure, and requires no prior electrical expertise to follow the assembly protocol.

In the supplementary Supplementary Figures 31–38, we describe how to incorporate up to four LEDs into the system. While this may limit some experiments requiring a large number of parallel conditions, more LEDs can be added by replacing the 9 Volt power supply used in this protocol with a higher wattage one. Similarly, several lower power LEDs can be connected in parallel to each circuit. In this latter arrangement, some LEDs will not be controlled individually, but this can be useful when many LEDs are required to cover a larger area. Once familiar with the electronics of this system, there are many ways to customize it. Additional strategies for customizing the system include placing the LED farther or closer to the sample and illuminating through filters/diffusers for homogenous illumination conditions or to prevent heating as in (Supplementary Figure 23) and Allen et al.⁵. Another notable feature of our LED design is that it is encapsulated in epoxy and has a touch fastener on the back; this allows the LED to be securely placed with ease virtually anywhere: in incubators, fish tanks, animal cages, walls, etc.

Many experiments that use optogenetics to control genes, signaling pathways, and other cellular activity often require pulsing, span large time scales, or need to be performed in an incubator, therefore requiring automatization or remote manipulation without a microscope. This LED system has been tested continuously for several months inside a humidified CO₂ incubator without any issues. Additionally, with reversible systems such as PhyB optogenetic systems, the experimenter may need to program-specific pulsing illumination schedules. In our previous work⁴, we used pulsing programs to test the reversibility dynamics of a PhyB-PIF3 switch in mammalian cells through the user interface. Using the methodology described in this manuscript, programming a pulsing protocol is easy, providing the flexibility and autonomy needed for many types of optogenetic experiments in a user-friendly manner.

The most critical steps in building this system include putting together the electrical circuit on the PCB board and connecting the components, which are detailed in section 1 and section 2. It is essential to carefully follow each step in these sections and double-check the pinhole numbers line-by-line before soldering each component. Section 2 explains how to set up the components that will be connected to the circuit. So that the components connect in the correct orientation, it is particularly important to ensure that the colors of the black and red wires on the wire-to-wire connectors match. Small oversights in these two sections will very likely affect the system’s functionality. Indeed, the first step in troubleshooting this method will be to check that the circuit was built correctly and that all the connections are in place. Secondly, checking the soldering quality for loose connections and the wires for flaring wirehairs that may be shorting the circuit is of particular importance. A third step would be to ensure that the LEDs are working correctly, which can be done using a power supply or a 1.5 V battery by clipping the LED's two terminals with alligator clips. Another potentially critical consideration is to prevent heating (when using the LEDs at high power) or diffusing the light for wider spread illumination. To address these considerations, the LEDs can be mounted outside of a black box with “privacy film” on the inside, as described in Supplementary Figure 23 and Allen et al.⁵. Because of the simplicity of this system, taking it apart to verify, modify, upgrade, or repair modular components is not difficult.

Another critical factor for inducible gene systems is to consider how much activation is required or how much leakiness is acceptable for the biological system being controlled. As shown in Figure 6, these can vary with the amount of reporter DNA. In addition, the transfection efficiency and, therefore, the copy number of reporter constructs in each cell will vary. It may be advantageous for some experiments to make a cell line with a fixed amount of reporter or PhyB gene-switch components and screen for clones with the desired range of induced expression, as is commonly done with drug inducible systems. Due to the size and instability of lentiviral plasmid pPK-230⁴, we also made non-lentiviral plasmid versions of the PhyB switch in the pcDNA backbone pPK-351 (Addgene #157921) and pPK-352 (Addgene #157922).

By building this LED illumination system following this protocol, users have all the components necessary to perform a wide array of optogenetics experiments in vitro and in vivo. Combined with the instructions for using PhyB-PIF3 in mammalian cells, this protocol will allow non-engineers and biologists to, flexibly and effectively, use PhyB-based optogenetics in a variety of contexts.

Materials

Name	Company	Catalog Number	Comments
18AWG 2pin RED Black wire	Amazon	15M-28AWG-2468	Inexpensive wire to connect LEDs to the power circuit. https://www.amazon.com/gp/product/B072KGYH1M/ref=oh_aui_detailpage_o05_s00?ie=UTF8&psc=1
1K Ohm potentiometer	Amazon	52161500	2 x 1K Ohm potentiometer potential + 2 x black control Knob. https://www.amazon.com/gp/product/B00XIWA2GO/ref=oh_aui_detailpage_o00_s00?ie=UTF8&psc=1
20 Gauge Silicone JST Connector	Amazon	SIM&NAT 5.9 inch 2 Pin Male Female JST RCY Plug Connectors	These are very common and there are many equivalents. https://www.amazon.com/gp/product/B071XN7C43/ref=oh_aui_detailpage_o00_s01?ie=UTF8&psc=1
22 AWG solid jumper wires	Amazon	WJW-60B-R	Jameco Valuepro WJW-60B-R Wire Jumper Kit 350 each 22 AWG, 14 Lengths 10 Colors 25 Of Each Length. https://www.amazon.com/Jameco-Valuepro-WJW-60B-R-Jumper-Lengths/dp/B01KHWEB3W/ref=sr_1_5?s=industrial&ie=UTF8&qid=1519261370&sr=1-5&keywords=solid+wire+breadboard&dpID=51UopZhPJeL&preST=_SX342_QL70_&dpSrc=srch
560 ohm 1/2watt 1% tolerance	Amazon	a14051600ux0301	Uxcell a14051600ux0301 60 Piece Axial Lead 1% Tolerance Colored Ring Metal Film Resistor Resistance, 560 Ohm 1/2W. https://www.amazon.com/a14051600ux0301-Tolerance-Colored-Resistor-Resistance/dp/B016ZU2DGC/ref=pd_day0_328_9?_encoding=UTF8&pd_rd_i=B016ZU2DGC&pd_rd_r=XTM6KHQ3NT8DHWB1QWZN&pd_rd_w=txGNx&pd_rd_wg=ELyii&psc=1&refRID=XTM6KHQ3NT8DHWB1QWZN
820 ohm 1/2watt 1% tolerance	Amazon	TTL-A-8035-50Ea	Set of 50Ea Metal Film Resistor 820 Ohm 1% 1/2W (0.5W). https://www.amazon.com/50Ea-Metal-Film-Resistor-0-5W/dp/B00VGU2SS0/ref=sr_1_14?s=industrial&ie=UTF8&qid=1518045187&sr=1-14&keywords=1%2F2W+820+Ohm+resistor
A Male to B Male Cable (10 Feet)	Amazon	Part# 30-001-10B	The cable that comes with the Arduino doesn't fit well in the box. https://www.amazon.com/gp/product/B001MSU1HG/ref=oh_aui_detailpage_o07_s00?ie=UTF8&psc=1
Ardiuino UNO equivilent	Amazon	Elegoo EL-CB-001	UNO R3 Board ATmega328P ATMEGA16U2 with USB Cable for Arduino. https://www.amazon.com/gp/product/B01EWOE0UU/ref=oh_aui_detailpage_o03_s00?ie=UTF8&psc=1
Arduino holder	Digikey	X000018	Fits very snug. https://www.digikey.com/product-detail/en/arduino/X000018/1050-1150-ND/8135632
Black boxes for circuits and light chambers	Amazon	1591ESBK	Hammond 1591ESBK ABS Project Box Black. https://www.amazon.com/gp/product/B0002BSRIO/ref=oh_aui_detailpage_o07_s00?ie=UTF8&psc=1
Blue LED	Digikey	LXML-PB01-0040	LED LUXEON REBEL BLUE SMD. Uses "Saber 20 mm Star base" https://www.digikey.com/product-detail/en/lumileds/LXML-PB01-0040/1416-1029-1-ND/3961134
Cable ties	Amazon	sd027	Tarvol Nylon Zip Ties (Pack of 100) 8 Inch with Self Locking Cable Ties (White). https://www.amazon.com/Tarvol-Nylon-Locking-Cable-White/dp/B01MRD0JRR/ref=sr_1_7?s=hi&ie=UTF8&qid=1519261882&sr=1-7&keywords=Cable+ties&dpID=51zUNmuUjyL&preST=_SY300_QL70_&dpSrc=srch
Command Fridge Clips	Amazon	17210CLR	Clips for holding circuit board inside of the black box. Command strips can also be used. https://www.amazon.com/gp/product/B0084M69YM/ref=oh_aui_detailpage_o00_s01?ie=UTF8&psc=1
Cyan LED	Digikey	LXML-PE01-0070	LED LUXEON REBEL CYAN SMD. Uses "Saber 20 mm Star base" https://www.digikey.com/products/en?keywords=1416-1031-1-nd
Electrical tape - 3M Scotch #35 Electrical Tape Value Pack	Amazon	03429NA	Scotch 700 Electrical Tape, 03429NA, 3/4 in x 66 ft. https://www.amazon.com/Scotch-Electrical-Tape-4-Inch-66-Foot/dp/B001ULCB1O/ref=psdc_256161011_t1_B001B19FDK
Farred LED 720nm	Luxeon Star LEDs	LXML-PF01	Far Red (720nm) LUXEON Rebel LED. Uses "Saber 20 mm Star base" https://www.luxeonstar.com/lxml-pf01-far-red-luxeon-rebel-led-260mW
Farred LED 740nm	Ushio	EDC740D-1100-S5	Uses "STAR XP 3535" base https://www.ushio-optosemi.com/jp/products/led/power/pdfs/edc/EDC740D-1100-S5.pdf
Farred LED 780nm	Ushio	EDC780D-1100	Uses "STAR XP 3535" base http://www.ushio-optosemi.com/jp/products/led/power/pdfs/edc/EDC780D-1100.pdf
Farred LED 810nm	Ushio	EDC810D-1100	Uses "STAR XP 3535" base http://www.ushio-optosemi.com/jp/products/led/power/pdfs/edc/EDC810D-1100.pdf
Farred LED 850nm	Ushio	EDC850D-1100	Uses "STAR XP 3535" base http://www.ushio-optosemi.com/jp/products/led/power/pdfs/edc/EDC850D-1100.pdf
Grommets	Amazon	Pico 6120D	These are very common and there are many equivalents. https://www.amazon.com/Pico-6120D-Vinyl-Grommets-Package/dp/B0002ZG47G
Hair/Alligator Clips	Amazon		1-3/4 Inch (45 Mm)- Hair Clips Single Prong Metal Alligator Clips Hairbow Accessory -Silver,50 Pcs. https://www.amazon.com/gp/product/B00K09T3L8/ref=oh_aui_detailpage_o00_s00?ie=UTF8&psc=1
LED base	Luxeon Star LEDs	LXB-RS20A	Saber 20 mm Star Blank Aluminum MCPCB Base For Rebel LEDs https://www.luxeonstar.com/saber-20mm-star-blank-mcpcb-base-for-a-rebel-leds
LED PCB fopr Ushio LEDs	Adura LED solutions	STAR XP 3535 Package LED	Fits many other LEDs by Ushio http://aduraled.com/product/pcb/1901-star-xp-3535-package-led
Loctite Epoxy Clear Multi-Purpose, 0.85-Fluid Ounce Syringe	Amazon	1943587	Loctite Epoxy Clear Multi-Purpose, 0.85-Fluid Ounce Syringe. https://www.amazon.com/Loctite-Multi-Purpose-0-85-Fluid-Syringe-1943587/dp/B011INNBN0/ref=psdc_256243011_t4_B0044FBB8C
NTE Heat Shrink 2:1 Assorted Colors and Sizes 160 PCS	Amazon	B000FIDTYG	These are very common and there are many equivalents. https://www.amazon.com/NTE-Heat-Shrink-Assorted-Colors/dp/B000FIDTYG/ref=sr_1_1?s=hi&ie=UTF8&qid=1519261282&sr=1-1&keywords=nte+shrink&dpID=41L5l7LCfiL&preST=_SX342_QL70_&dpSrc=srch
Picture Hanging Velcro Strips	Amazon	PH204-16NA	With these you can hang the LEDs in many places. https://www.amazon.com/Command-Picture-Hanging-16-Pairs-PH204-16ES/dp/B073XS3CHV/ref=pd_sim_60_5?_encoding=UTF8&pd_rd_i=B073XS3CHV&pd_rd_r=112KGQJNRRYD0RAT598H&pd_rd_w=3n34Q&pd_rd_wg=sRvec&psc=1&refRID=112KGQJNRRYD0RAT598H
Power supply	Amazon	tb013	Any other 9V 1.5Z AC/DC converter will do becuase we cut the end off anyway. https://www.amazon.com/gp/product/B06Y1LF8T5/ref=oh_aui_detailpage_o07_s01?ie=UTF8&psc=1
Power switch Rocker Switch	Amazon	SIXQJZML	These are very common and there are many equivalents. https://www.amazon.ca/COOLOOdirect-Solder-Rocker-Switch-Toggle/dp/B071Y7SMVQ/ref=sr_1_31?_encoding=UTF8&c=ts&dchild=1&keywords=Boat+Rocker+Switches&qid=1594434474&s=sports&sr=1-31&ts_id=2438617011
Rectangular Connectors - for crimped wires	Digikey	2183-1905-ND	6 Rectangular Connectors - Housings Black 0.100" (2.54mm) https://www.digikey.com/product-detail/en/pololu-corporation/1905/2183-1905-ND/10450382?utm_adgroup=Rectangular%20Connectors%20-%20Housings&utm_source=google&utm_medium=cpc&utm_campaign=Shopping_Product_Connectors%2C%20Interconnects_NEW&utm_term=&utm_content=Rectangular%20Connectors%20-%20Housings&gclid=Cj0KCQjwvIT5BRCqARIsAAwwD-QmETT-ko07ote5VQgodKvWU0uDG8GYN7Vj-6WVBBOWdSgPaPd9azAaAhVLEALw_wcB
Red LED	Digikey	LXM3-PD01	LED LUXEON REBEL DEEP RED SMD. Uses "Saber 20 mm Star base" https://www.digikey.com/products/en?keywords=1416-1701-1-nd
Sandpaper	Amazon	B002NEV6GS	3M Wetordry Sandpaper, 03022, 800 Grit, 3 2/3 inch x 9 inch. https://www.amazon.com/3M-03022-Imperial-Wetordry-Sandpaper/dp/B002NEV6GS/ref=sr_1_1?dchild=1&keywords=3M+Imperial+Wetordry+3-2%2F3+in.+x+9+in.+800+Grit+Sandpaper+Sheets+%2810+Sheets-Pack%29&qid=1594435012&sr=8-1
Solder for soldering wires and circuit components	Amazon	Mudder Lead Free Solder Wire Sn99 Ag0.3 Cu0.7 with Rosin Core for Electrical Soldering 0.22lbs (0.6 mm)	These are very common and there are many equivalents. https://www.amazon.com/Mudder-Solder-Electrical-Soldering-0-22lbs/dp/B01B61TWGY
Solder-able Breadboard for building the circuit	Amazon	GK1007	Gikfun Solder-able Breadboard Gold Plated Finish Proto Board PCB Diy Kit for Arduino (Pack of 5PCS) GK1007. https://www.amazon.com/gp/product/B071R3BFNL/ref=oh_aui_detailpage_o04_s00?ie=UTF8&psc=1
Spade drill bit	Amazon	Irwin 88811	These are very common and there are many equivalents. https://www.amazon.com/Speedbor%C2%AE-Blue-GrooveTM-Standard-Length-Woodboring/dp/B017S9JXB2/ref=sr_1_10?ie=UTF8&qid=1519516560&sr=8-10&keywords=11%2F16+spade+bit
Transistor	Newark	2N2222A	Can buy from many places. http://www.newark.com/nte-electronics/2n2222a/bipolar-transistor-npn-40v-to/dp/10M4197
Voltage regulator	Newark	LM317T	Equivilent to NTE956. https://www.newark.com/stmicroelectronics/lm317t/adjustable-linear-regulator-1/dp/89K0685?gclid=CjwKCAiAu9vwBRAEEiwAzvjq-1rmUi6lvOIFFt-BxttHXvmAeUoni4NM0BW-BtM_LMliSqxA9Xq4KxoCfikQAvD_BwE&mckv=sQqHZDDRz_dc|pcrid|219869297712|plid||kword|lm317t|match|p|slid||product||pgrid|35966450488|ptaid|kwd-541160713|&s_kwcid=AL!8472!3!219869297712!p!!g!!lm317t&CMP=KNC-GUSA-SKU-MDC
Windows 10 tablet	Amazon	B08BYTT79Y	Any Windows 10 PC will do. https://www.amazon.com/gp/product/B08BYTT79Y/ref=ppx_yo_dt_b_asin_title_o01_s00?ie=UTF8&psc=1
Cell Culture Reagents
Human Embryonic Kidney 293 cells HEK293	ATCC	ATCC CRL-1573	Common Cell line. https://www.atcc.org/products/all/CRL-1573.aspx
Fetal Bovine Serum	ThermoFisher	26140079	These are very common and there are many equivalents. https://www.thermofisher.com/order/catalog/product/26140079#/26140079
Dulbecco’s Modified Eagle Medium High Glucose	ThermoFisher	11965−092	These are very common and there are many equivalents. https://www.thermofisher.com/order/catalog/product/11965118?SID=srch-srp-11965118#/11965118?SID=srch-srp-11965118
10,000 units/mL of penicillin and 10,000 µg/mL of streptomycin	ThermoFisher	15140122	These are very common and there are many equivalents. https://www.thermofisher.com/order/catalog/product/15140122?SID=srch-srp-15140122#/15140122?SID=srch-srp-15140122
White Corning 96-Well Solid Black or White Polystyrene Microplates	ThermoFisher	07-200-589	White plates are preferred. Do not use clear plates. https://www.fishersci.com/shop/products/costar-96-well-black-white-solid-plates-8/p-152852
PEI MAX - Transfection Grade Linear Polyethylenimine Hydrochloride (MW 40,000)	PolySciences	24765-1	Can be replaced with another transfection reagent. https://www.polysciences.com/default/catalog-products/life-sciences/transfection-reagents/polyethylenimine-max-mw40000-high-potency-linear-pei/
Name of Equipment
Diagonal Cutting Plier (110mm)	Amazon	Proskit 1PK-037S	These are very common and there are many equivalents. https://www.amazon.com/iExcell-Diagonal-Cutting-Nippers-Chrome-Vanadium/dp/B076XYVS6Y/ref=sr_1_11?dchild=1&keywords=diagonal+cutting+pliers&qid=1594436230&sr=8-11
Dremil 3000 with cutting tool and grinder	Amazon	Dremel 3000	Dremel 3000-2/28 Variable Speed Rotary Tool Kit- 1 Attachments & 28 Accessories- Grinder, Sander, Polisher, Router, and Engraver. https://www.amazon.com/Dremel-3000-2-28-Attachments-Accessories/dp/B005JRJE7Y/ref=sr_1_3?dchild=1&keywords=Dremel+200-1%2F15+Two-Speed+Rotary+Tool+Kit&qid=1594436404&s=hi&sr=1-3
Dremil cutting and grinding tool	Amazon	Dremel 200-1/15	Any similar Dremil will work. https://www.amazon.com/Dremel-200-1-Two-Speed-Rotary-Tool/dp/B002BAHF8W/ref=sr_1_1?s=hi&ie=UTF8&qid=1519268058&sr=1-1&keywords=dremel+200&dpID=41h9ZucnTYL&preST=_SY300_QL70_&dpSrc=srch
Dremil grinding tip	Amazon	Dremel 84922	Silicon Carbide Grinding Stone. https://www.amazon.com/Dremel-84922-Silicon-Carbide-Grinding/dp/B00004UDKD/ref=sr_1_fkmr0_1?s=hi&ie=UTF8&qid=1519268585&sr=1-1-fkmr0&keywords=dremel+tip+84922
EDSYN The Original Deluxe SOLDAPULLT	Amazon	DS017	For removing solder/mistakes. https://www.amazon.com/EDSYN-The-Original-Deluxe-SOLDAPULLT/dp/B006GOKVKI
Helping Hand with Magnifying Glass	Amazon	SE MZ101B	These are very common and there are many equivalents. https://www.amazon.com/SE-MZ101B-Helping-Magnifying-Glass/dp/B000RB38X8/ref=sr_1_4?s=hi&ie=UTF8&qid=1519268108&sr=1-4&keywords=Helping+hands&dpID=31GEhMw7WvL&preST=_SX300_QL70_&dpSrc=srch
Pointed Nose Micro Pliers	Amazon	Hakko CHP PN-20-M	Steel Super Specialty Pointed Nose Micro Pliers with Smooth Jaws, 1.0mm Nose. https://www.amazon.com/Hakko-PN-20-M-Specialty-Pointed-Pliers/dp/B00FZPGUBI/ref=sr_1_1?s=hi&ie=UTF8&qid=1519268153&sr=1-1&keywords=Hakko+CHP+PN-20-M+Steel+Super+Specialty+Pointed+Nose+Micro+Pliers+with+Smooth+Jaws%2C+1.0mm+Nose&dpID=3109XRgwn3L&preST=_SX342_QL70_&dpSrc=srch
Small screw drivers	Amazon	Wiha 26197	These are very common and there are many equivalents. https://www.amazon.com/26197-Precision-Slotted-Phillips-Screwdrivers/dp/B01L46TEN2/ref=sr_1_1?s=hi&ie=UTF8&qid=1519268018&sr=1-1&keywords=Wiha+precision+set
Soldering iron	Amazon	Yihua 939D+ Digital Soldering Station	These are very common and there are many equivalents. https://www.amazon.com/Professional-Digital-Soldering-Station-Switch/dp/B07YSCBZ4F/ref=psdc_13837391_t1_B07RVMZNYR
TraceTech No-Clean Flux Pen	Amazon	2507-N	Tech Spray 2507-N No-Clean Flux Dispensing Pen, 11.5 mL. https://www.amazon.com/Tech-Spray-2507-N-No-Clean-Dispensing/dp/B00DDF2FYS/ref=sr_1_1?dchild=1&keywords=2507-N&qid=1595469618&sr=8-1
Weller WSA350 120v Bench Top Smoke Absorber	Amazon	WSA350	For soldering safety. https://www.amazon.com/Weller-WSA350-Bench-Smoke-Absorber/dp/B000EM74SK
Wire strippers	Amazon	CSP-30-7	These are very common and there are many equivalents. https://www.amazon.com/Hakko-CSP-30-7-Stripper-Maximum-Capacity/dp/B00FZPHY7M/ref=psdc_553398_t5_B00FZPHMUG
IWISS IWS-3220M Micro Connector Pin Crimping Tool 0.03-0.52mm² 32-20AWG	Amazon	IWS-3220M	These are very common and there are many equivalents. https://www.amazon.com/gp/product/B078WPT5M1/ref=ppx_yo_dt_b_search_asin_title?ie=UTF8&psc=1