Tutorials

Part 1. Overview of Wireless Optogenetic Probe Systems

The wireless optogenetic device presented here features a self-contained, head-mounted design that can be easily adapted and utilized for a variety of neuroscience studies, eliminating the need for costly and complex tools or equipment. The wireless optogenetic probe system consists of the following components:

1) MicroLED probe: This probe is designed to deliver light precisely to the target neural circuit within the brain.

2) Wireless control module: The wireless module enables untethered and programmable control of optogenetic stimulation in freely behaving animals.

3) Rechargeable Lithium Polymer (LiPo) battery: The lightweight LiPo battery (0.5 g) powers the wireless control module

By incorporating these components, researchers can seamlessly implement optogenetic stimulation in their studies, enhancing their ability to investigate neural circuits and behavior in freely moving animals.

The exemplary wireless optogenetic probe system introduced in the tutorial has bilateral probe configuration and provides the following key capabilities and features:

(Note that various probe configurations are available here.)

• Selective simultaneous control of multiple devices (up to maximum 7 devices)

• Selective simultaneous control of 2 microLED channels with different optical wavelengths (e.g., 470 nm (blue) and 589 nm (orange); combination with other wavelengths is also possible)

*Note: The number of controllable microLEDs can be selected based on two factors: (i) the number of probe shafts (e.g., 1 for unilateral design, 2 for bilateral design) and (ii) the number of microLED channels (e.g., 1 for single channel, 2 for dual channel). For example, a bilateral microLED probe with a dual channel allows control of a total of 4 microLEDs, controlling two microLEDs in each probe. Gerber files for different probe configurations are available for download from the above link as desired.

• Adjustable optical intensity of optogenetic stimulation in 3 levels:

(i) Lv.1: ~21 mW/mm2 @ 470 nm-wavelength; ~30 mW/mm2 @ 589 nm-wavelength 

(ii) Lv.2: ~61 mW/mm2 @ 470 nm-wavelength; ~82 mW/mm2 @ 589 nm-wavelength 

(iii) Lv.3: ~97 mW/mm2 @ 470 nm-wavelength; ~129 mW/mm2 @ 589 nm-wavelength

• Adjustable operation frequency of optogenetic stimulation with 5 options (i.e., (i) 5 Hz, (ii) 10 Hz, (iii) 20 Hz, and (iv) 40 Hz with fixed pulse width of 10 ms and (v) Continuous operation) 

• Bluetooth Low Energy (BLE) control through a custom-designed smartphone app (Available for Android)

• Device dimension:

  • Probe: i) body part: 8(w) X 15.5(l) X 2(t) mm3;

        ii) shaft part: 0.73(w) X 7.2(l) X 0.13(t) mm3

• Head-mounted body: 10 X 12 X 5 mm3 

• Weight: 0.9 g (with microLED probe)

The effectiveness and reliability of this wireless optogenetic device have been confirmed through extensive animal experiments conducted in partnership with neuroscientists from Washington University in St. Louis (McCall Lab), University of Washington (Bruchas Lab), KAIST (Lee Lab), and Yonsei University. Additionally, the wireless optogenetic devices have been distributed to other researchers in the field of neuroscience to assist their own research endeavors. The success of this wireless device and the significant findings derived from its usage have been published in several esteemed journals, including the followings:

• Jeon et al., “Adolescent parvalbumin expression in the left orbitofrontal cortex shapes sociability in female mice”, Journal of Neuroscience 43, 1555-1571 (2023) → Link

• Parker et al., “Customizable, wireless and implantable neural probe design and fabrication via 3D printing”, Nature Protocols 18, 3-21 (2023) → Link

• Qazi et al., “Scalable and modular wireless-network infrastructure for large-scale behavioural neuroscience”, Nature Biomedical Engineering 6, 771-786 (2022) → Link

• Kim et al., “Soft subdermal implant capable of wireless battery charging and programmable controls for applications in optogenetics”, Nature Communications 12, 535 (2021) → Link

• Byun et al., “Mechanically transformative electronics, sensors, and implantable devices”, Science Advances 5, eaay0418 (2019) → Link

• Qazi et al., “Wireless optofluidic brain probes for chronic neuropharmacology and photostimulation”, Nature Biomedical Engineering 3, 655-669 (2019) → Link

The tutorial presented in the following sections will guide you through the process of creating and implementing a wireless optogenetic probe system. It will demonstrate the method for constructing both the microLED probe and the wireless control module, as well as instruct you on how to upload the firmware to the wireless module and utilize our custom-designed smartphone app.

Part 2. Components Procurement

In order to construct the wireless optogenetic device, it is essential to fabricate the microLED probe and wireless control module, as well as prepare the firmware for the wireless module. These processes involve the utilization of diverse components, including printed circuit boards (PCBs) and electronic components. This section serves as an introductory guide to acquiring these essential elements for the device construction.

A.  MicroLED probe

1. Probe substrate 

• PCB manufacturing company can produce a probe substrate built on PCB by providing Gerber files. The company introduced in Component List #1 is available for online ordering from any location across the globe.

• We have provided Gerber files for several distinct probe designs customized for various target applications. You can access the Gerber files here. The photo of the fabricated PCB using one of the provided Gerber files can be seen in Figure #1.

• When placing an order with the PCB manufacturing company, please ensure to include the following specifications:

(i) Thickness of the flexible PCB: 0.13 mm

(ii) Thickness of the finished copper: 35 μm

Including these details will ensure that the PCBs are manufactured with optimal mechanical characteristics for implantation in brain tissue.

• Our optogenetic probe system is designed to facilitate optogenetic experiments using microLEDs of various wavelengths. 

• The microLEDs compatible with the provided probe designs are listed in Component List #1, along with their respective vendors.

2. Electrical connector

• The provided optogenetic probes are designed to be connected with the wireless control modules via 3-pin connectors.

• The probe requires a female-type connector.

• Examples of available products are listed in Component List #1 and Figure #1. When purchasing microLEDs, it is recommended to determine the desired quantity and wavelength beforehand.

• Alternative connectors can be used as long as they meet the following conditions:

(i) The number of pins should be 3.

(ii) The pitch of the pad should be 1.27 mm.

Figure #1: The materials required to fabricate a microLED probe.

B.  Wireless control module

3. Flexible PCB (FPCB) for the wireless control module

• We have uploaded the Gerber file here for the PCB for the wireless control module.

• Using the attached Gerber file, you can also order a stencil mask from the PCB manufacturing company. The stencil mask is a tool used to easily apply solder paste to the electrodes of FPCB. For detailed instructions on its usage, refer to Figure #19 in Part 4.

• Figure #2 illustrates the layout of components on the top layer of the FPCB, providing visual representation of the positioning details mentioned in Component List #2. All the items listed in Component List #2 are displayed in Figure #3.

4. Bluetooth Low Energy System-on-Chip (BLE SoC)

• This chip is a microcontroller with BLE communication capability.

• It is a third-party chip from Taiyo Yuden, based on Nordic Semiconductor’s nRF52832.

5. Voltage regulator

• This component is used to supply a constant voltage (3.3 V) to the circuit, regardless of the voltage variations over time in the battery.

6. Capacitors

• There are three types of capacitors used in the circuit, which are listed in Component List #2.

• In addition to the models mentioned in the table, capacitors with the same capacitance and size can also be used.

7. Indicator LEDs

• The purpose of the indicator LEDs is to indicate the status of the wireless control module.

• The indicator LEDs used in this device design are listed in Component List #2. As long as the LED size is 1005 metric (i.e., 1.0 mm X 0.5 mm) and the turn-on voltage is lower than 2.4 V, it is acceptable to use different models.

8. Resistors

• Component List #2 presents three types of resistors and their models, which are used in the circuit

• The models listed in the table can be replaced with resistors with the same resistance and size.

9. Electrical connector

• The wireless control module utilizes two types of male pin connectors.

• The 3-pin male connector is used for connection with the microLED probe. Alternative models can be used other than the product introduced in Component List #2, as far as they satisfy the following conditions: (i) number of pins = 3, (ii) pitch of pins = 1.27 mm, and (iii) height of pins = 1 mm

• The 2-pin male connector works for connection with the battery. It is acceptable to use different models of the connector as long as they meet identical conditions and specifications.

10. LiPo Battery

• The 2-pin female connector is attached to the battery, and it is listed in Component List #2.

• The 12 mAh battery listed in Component List #2 can continuously turn on one microLED channel (i.e., 2 microLEDs) for the following duration: ~130 min for optical intensity Lv.1 (~21 mW/mm2 for 470 nm microLED), ~80 min for optical intensity Lv.2 (~61 mW/mm2 for 470 nm microLED), and ~65 min for optical intensity Lv.3 (~97 mW/mm2 for 470 nm microLED).

• A battery with a larger capacity can be applied to maintain optogenetic stimulation for a longer duration. However, it is recommended to decide on a battery model by considering their size and weight as needed:

(i) 12 mAh LiPo battery: 9 X 10 X 3 mm3, 0.5 g

(ii) 30 mAh LiPo battery: 10 X 12 X 4 mm3, 1.0 g

Figure #2: The top layer of the flexible PCB for the wireless control module illustrating the positions of each circuit component.

Figure #3: The circuit components that are necessary for construction of the wireless control module.

C.  Firmware upload

11. J-Link Lite

• J-Link is a circuit that facilitates communication between a PC and a BLE SoC.

• Multiple versions are available, including Lite, and each version uses a different connector.

• In this tutorial, information is provided specifically for J-Link Lite.

• When purchasing J-Link Lite from Segger, it comes conveniently packaged with a mini-USB cable and an insulation displacement contact (IDC) cable. These accessories (Figure #4) are included to ensure a seamless and hassle-free setup and connection process.

12. Mini-USB cable

• A cable with a mini pin on one end and a USB pin on the other end is used for connection between J-Link and PC. This cable is listed in Component List #3.

• Any other cables can be used without any issues if the pin types are the same.

13. IDC cable

• IDC cable is used to connect the J-Link and the custom board. It is listed in Component List #3.

14. Custom board for direct programming of BLE SoC

• The electrodes on the BLE SoC are very small, making it challenging to connect wires directly.

• To address this, a custom board was designed to connect BLE SoC to the IDC cable for firmware upload.

• The Gerber file for this board is available here.

• When ordering the PCB, simply choose the standard PCB option (FR-4 material), and there are no other specific considerations to be aware of.

15. BLE SoC

• The BLE SoC used to upload the firmware is the same BLE SoC used for the development of the wireless control module.

• It is essential to complete uploading the firmware (bootloader) to the BLE SoC through a wired connection prior to soldering it onto the wireless control module PCB.

This section describes the method for constructing an implantable optogenetic probe based on flexible printed circuit boards (PCBs). The process involves soldering the microLEDs and electrical connectors onto the flexible PCBs and utilizing a 3D-printed supporter to protect the probe.

A.  Soldering microLED on the probe substrate built using flexible PCB

1. Secure the probe substrate PCB well on a glass slide using Kapton tape to ensure it does not move during soldering. Avoid using heat-sensitive tapes (e.g., Scotch tape) since soldering requires high temperatures. As the complete process of microLED soldering is demonstrated in Video #1, it will be convenient to follow and replicate the procedure by simultaneously referring to both the video and the text explanation.

2. Apply a small amount of solder paste to electrodes (anode: +, cathode: -) at the tip of the probe using a thin needle.

3. Place microLEDs onto the electrodes at the tip of the probe substrate PCB using a tweezer (Figure #5). Ensure that the front plane of the microLEDs is facing upwards and both electrodes (i.e., cathode and anode) are positioned correctly (Figure #6).

Figure #5: Schematic diagram illustrating the positions of the microLED on the probe tip. (Note that only one microLED can be                      soldered per probe depending on your need.)

Figure #6: Front (left) and back (right) side views of the 470 nm- and 589 nm-wavelength microLEDs. The backside electrodes                       of microLED are gold in color, and they are the area in contact with the electrodes of the probe. 

4. Apply heat to the solder paste using a soldering iron (temperature ~180 °C). Gently touch the soldering iron to the interconnects, allowing the heat to transfer to the solder paste and facilitating soldering (Video #1). Observe the solder paste as it solidifies, ensuring that the microLED remains securely in place. Exercise caution to avoid applying excessive temperature or pressure with the soldering iron, as this could potentially damage the device. 

5. Verify if the light turns on by applying voltage to both electrodes of the microLED. It is convenient to apply voltage to the large pad on the probe, which is used for attaching the pin connector. The recommended voltage range is 2.7–3.0 V. You can refer to Video #1 for a demonstration of this process.

6. Soldering can be accomplished using alternative methods such as a reflow oven or a hot air gun, in addition to a soldering iron. Regardless of the soldering technique employed, Steps 1-3 remain unchanged, and only Step 4 requires modification as follows:

1) When using a reflow oven: Place the flexible PCB mounted with microLEDs using soldering paste into the reflow oven and execute the solder paste reflow process as per the recipe below (Figure #7):

a) Preheat at 90 °C for 90 s.

b) Heat at 130 °C for 90 s.

c) Solder with a peak temperature at 170 °C for 60 s.

d) Cool down for more than 15 min until the temperature drops to the room temperature.

Figure #7: Photograph showing the placement of the PCB with soldering paste and microLED on it into the reflow oven for                            soldering.

2) When using a hot air gun: Direct the airflow from the hot air gun towards the components at a temperature of approximately 215 °C to melt the solder paste (Figure #8). Ensure that the airflow is gentle enough to prevent components from being dislodged or blown away.

Figure #8: Demonstration of soldering the microLED to the PCB using a hot air gun.

B.  Parylene C coating on the microLED probe for waterproofing

7. Place microLED-attached probes on a glass slide for Parylene C encapsulation for waterproofing and biocompatibility as shown in Figure #9. Cover the connector electrode pads of the probe with Kapton tape to prevent Parylene C deposition in that area. To ensure comprehensive coating of the probe shank with Parylene C from all angles, suspend it in the air without making contact with the glass slide. Stack multiple glass slides to create a thickness of 2 mm or more, allowing ample space for the probe shank to float freely in the air during the Parylene C coating process. This setup ensures uniform coating of the probe shank from all directions.

Figure #9: Probes mounted on a glass slide with connector electrode pads protected by Kapton tape for Parylene C coating. 

8. Deposit 7 μm of Parylene C on the probe using a Parylene coater. If there is no Parylene coater available in your lab or institution, you can reach out to the companies listed in Vendor List #1 to get the necessary deposition service.

9. Remove the Kapton tape attached to the connector electrode pad of the probe after Parylene C coating (Figure #10).

Figure #10: Connector electrode pads exposed in the air without a Parylene C film after removing the Kapton tape.

10. Drop a water droplet on the top of the microLED and apply voltage to it through the connector pad to check for any abnormalities in the probe and its Parylene C encapsulation (Figure #11).

Figure #11: Water droplet on the microLED demonstrating the waterproof characteristics of the Parylene C coating.

C.  Assembly of probe and supporter 

11. Apply solder paste to the connector pad electrodes of the probe.

12. Refer to Figure #12 and Figure #13, align and solder a 3-pin female connector to the connector pad electrodes of the probe.

Figure #12: Photograph showing alignment of the 3-pin female connector with the connector pad electrodes of the probe.

Figure #13: Photograph showing the soldering process (top) and the resulting soldered connection (bottom) of the 3-pin                        female connector onto the probe.

13. Print a probe supporter using a 3D printer. The support structures are specifically designed to provide mechanical support for the probe, and you can download various .stl files with different shapes here. Figure #14 shows an example of a 3D-printed probe supporter. In the event that a 3D printer is not available in your lab, you can consider outsourcing the printing job to the companies listed in Vendor List #2 for 3D printing services. In this tutorial, we used a 3D printer called ‘B9 Core 530’, which uses Stereolithography (SLA), with ‘B9R-4-Yellow resin’ from B9Creations (Figure #15).

Figure #14: 3D-printed supporter for probe protection.

Figure #15: 3D printer (B9 Core 530), resin (B9R-4-Yellow), and other accessories.

14. Attach the probe to the 3D-printed supporter using double-sided tape (Figure #16).

Figure #16: Probe attached to the 3D-printed supporter by double-sided tape.

15. Apply 5-minute epoxy carefully to reinforce the physical connection between the probe and the supporter (Figure #17). Ensure that the amount of 5-minute epoxy is sufficient to prevent detachment of the connector during assembly/disassembly of the wireless module. Conversely, excessive application of 5-minute epoxy may obstruct the holes of the female connector, impeding the formation of an electrical connection with the wireless control module. For a visual demonstration of this procedure, you can refer to Video #2, which will provide helpful guidance.

Figure #17: The probe in its final state after applying epoxy to the side area of the 3-pin connector.

16. Immerse the Parylene C-coated shank of the probe in a saline solution for a period of 24 hours. Once this step is completed, proceed to connect the wireless module while the probe remains submerged in the saline solution. Verify the functionality of the microLED by operating the wireless module with a smartphone and confirming that the light turns on as intended. Please refer to Figure #18 for the waterproofing test setup.

This section provides step-by-step instructions for constructing a wireless control module and uploading the firmware onto it. The wireless control module can be created by soldering a Bluetooth Low Energy System-on-Chip (BLE SoC) to the bottom layer side of the flexible printed circuit board (PCB). On the top layer side, surface-mount device (SMD) components and connectors are attached using solder paste.

*IMPORTANT! Bootloader must be uploaded on the BLE SoC prior to soldering onto the wireless control module PCB. For this process, please refer to Part 5A. You can proceed with Parts 5B and C after constructing the wireless module in this Part 4.

A.  BLE SoC attachment

1. Place a flexible PCB designed for the wireless control module on a glass slide with the bottom layer side facing upwards. Secure both sides of the PCB using Kapton tape to ensure it remains in place during the assembly process.

2. Place a stencil mask over the surface of the flexible PCB, ensuring that each hole on the stencil mask aligns with every electrode on the PCB surface. Position the mask carefully and secure its position by attaching Kapton tape to the corners of the stencil mask. This ensures that the stencil mask remains in the correct position during subsequent steps.

3. Apply a solder paste onto the stencil mask and traverse the surface longitudinally using a razor blade. After that, detach a Kapton tape and remove a stencil mask (Figure #19). Make sure that all 28 electrodes on the bottom layer side are properly covered with the solder paste.

Figure #19:  Photographs showing the bottom layer side electrodes of the flexible PCB, with (left) and without a stencil mask                            (right) covered with solder paste. The PCB has a total of 28 electrodes. 

4. Attach a Kapton tape on the side of the BLE SoC that does not have an electrode. While holding the Kapton tape on both sides, lower it vertically from the top to position the BLE SoC correctly on the flexible PCB surface (Figure #20). The bottom overlay layer, indicated by a white line, marks the boundary of the BLE SoC position on the flexible PCB surface. This overlay layer will assist in identifying the appropriate placement while aligning it with a top view.

Figure #20: Photograph showing the attachment process of the BLE SoC on the bottom layer surface of the flexible PCB.

5. Apply pressure to the entire area of the Kapton tape to firmly secure the BLE SoC in place above the flexible PCB (Figure #21).   

Figure #21: Photograph of the flexible PCB after attaching the BLE SoC.

6. Place the glass slide containing the attached flexible PCB and BLE SoC into the reflow oven (Figure #22) and follow the specified recipe to reflow the solder paste:

a) Preheat at 90 °C for 90 s. 

b) Heat at 130 °C for 90 s. 

c) Solder with a peak temperature of 170 °C for 60 s. 

d) Allow for a cooling period of at least 15 min until the temperature drops to room temperature.

(If a reflow oven is not available, an alternative option is to use a hot air gun set to approximately 215 °C. Additionally, you can opt to utilize the services of chip bonding companies listed in Vendor List #3 for professional assistance if needed.)

Figure #22: Flexible PCB with the BLE SoC mounted on top, placed inside a reflow oven.

7. Once the temperature drops to room temperature (<30 °C), take out the glass slide from the reflow oven. Carefully remove each piece of Kapton tape from the assembly (Figure #23).

Figure #23: Photograph of the Flexible PCB with the attached BLE SoC after reflow soldering.

8. Test the electrical connection between the BLE SoC and the flexible PCB by applying a voltage (between 2.7–3.3 V) to the electrodes using a DC power supply. Make sure to connect the anode probe (+) to one of the output voltage electrodes (marked in red), and the cathode probe (-) to one of the GND electrodes (marked in blue) (Figure #24, left and middle). If a device named "BOOTLOADER" is detected through Bluetooth (Figure #24, right), it indicates that the BLE SoC is correctly attached to the flexible PCB and the electrical connection is functioning properly. 

Figure #24: Photograph illustrating the testing of the electrical connection between the BLE SoC and the electrodes on the                              flexible PCB.

B.  Surface-mount device (SMD) component attachment

9. Position the flexible PCB with the attached BLE SoC on the bottom layer side onto a glass slide, ensuring that the top layer side is facing upwards. Secure the flexible PCB in place by attaching Kapton tape to one side of the corner, taking care not to cover any electrodes with the tape. This will fix the flexible PCB securely to the glass slide while maintaining accessibility to the electrodes.

10. Apply solder paste to each electrode on the surface of the flexible PCB using a stencil mask (Figure #25), following the same method described in Step 3. Make sure that all 29 electrodes on the top layer side are properly covered with solder paste.

Figure #25: Top layer side electrodes of the flexible PCB, with a total of 29 electrodes, covered with solder paste.

11. Place the SMD components on the electrodes covered with solder paste using a tweezer (Figure #26). Watching Video #3 will provide a visual demonstration of this process. There are a total of 13 SMD components to be attached, which are listed below: 

a) 1 μF Capacitor (0603 metric) - Quantity: 1

b) 3.3 V Voltage regulator - Quantity: 1

c) 0.1 μF Capacitor (0603 metric) - Quantity: 2

d) 4.7 μF Capacitor (0603 metric) - Quantity: 1

e) 624 nm LED (1005 metric) - Quantity: 1

f) 573 nm LED (1005 metric) - Quantity: 1

g) 6.04 kΩ Resistor (0603 metric) - Quantity: 2

h) 150 Ω Resistor (0603 metric) - Quantity: 2

i) 750 Ω Resistor (0603 metric) - Quantity: 2

Please refer to Component List #2 and Figure #2 in Part 2B for the positions of SMD components (i.e., Positions A-I in Figure #2) on the top layer surface of the flexible PCB. Also, please refer to the image below for the correct electrode polarity of the voltage regulator (b) and the indicator LEDs (e and f).

Figure #26: SMD components (a total of 13 components) mounted on the electrodes covered with solder paste.

12. Put the glass slide, which contains the flexible PCB mounted with the SMD components, in the reflow oven and follow the same process as described in Step 6.

13. Once the temperature drops to room temperature (<30 °C), carefully remove the glass slide from the reflow oven and detach all the Kapton tape.

14. To test the electrical connection of the SMD components, apply a voltage (between 3.0-4.2 V) to the electrodes using a DC power supply. Connect the anode probe (+) to the positive (red) electrode and the cathode probe (-) to the negative (GND; blue) electrode among the pair of battery electrodes (Figure #27, left and middle). If a device named "BOOTLOADER" is detected through Bluetooth (Figure #27, right), it indicates that the BLE SoC is successfully attached to the flexible PCB and the electrical connection is functioning properly.

Figure #27: Photograph showing the testing of the electrical connection between the SMD components and the flexible PCB                           electrodes.

C.  Connector attachment

15. After applying solder paste on the connector electrodes, place 3-pin male and 2-pin male connectors on them (Figure #28). Please refer to Component List #2 and Figure #2 in Part 2B for the positions of male components (i.e., Positions J and K in Figure #2) on the top layer surface of the flexible PCB.

Figure #28: Photograph of the flexible PCB with 3-pin and 2-pin male connectors mounted on electrodes with solder paste                               applied.

16. While gently applying pressure to the connector using a tweezer, solder the paste using a soldering iron (Figure #29). It is recommended to set the soldering iron temperature to a range of 180–220 °C.

This section provides instructions for uploading firmware to a Bluetooth Low Energy System-on-Chip (BLE SoC) within a wireless control module. If the BLE SoC has no existing programs, the bootloader should be uploaded first through a wired connection with a PC. Once the bootloader is successfully installed, you can wirelessly upload new firmware using a smartphone. If you want to make modifications to the firmware, you will need to switch back to the bootloader state.

A.  Upload bootloader using PC 

1. Install ‘SEGGER Embedded Studio (SES)’ on your PC. Make sure to install ARM version of SES. In SES, access the 'Package manager' through the 'Tools' menu at the top of the interface and download the 'CMSIS-CORE Support Package' and 'NRF CPU Support Package'.

2. Download the ‘nRF connect app’ from the app store on the smartphone. Refer to Figure #30 and Software List #1 for a list of required software and apps to be installed. 

Figure #30: Screenshots of the required software for uploading firmware to the BLE SoC.

3. Connect the J-Link to the PC using a Mini-USB cable. Use an IDC cable to establish a connection between the custom board and the J-Link. Supply a voltage of ~3 V to the custom board using a DC power supply. Finally, place the BLE SoC onto the custom board, ensuring proper alignment (Figure #31).

Figure #31: Hardware setup for uploading the bootloader to the BLE SoC.

4. Open the file ‘secure_bootloader_ble_s132_pca10040.emProject’ on your PC using SES. You can find the file here.

5. Click on ‘Target’ at the top and select ‘Download secure_bootloader_ble_s132_pca10040.emProject’ from the list. Repeat this process until the log shows ‘Download successful’. If you encounter any errors, they may be caused by misalignment between the BLE SoC and the custom board (Figure #32).

Figure #32: Screenshots showing the installation of the bootloader on the BLE SoC from SEGGER Embedded Studio.

B.  Upload firmware wirelessly 

6. Save the firmware in .zip format with names such as “Device01.zip”, “Device02.zip”, and so on (e.g., “Device03.zip”, “Device04.zip”, “Device05.zip”, “Device06.zip”, and “Device07.zip”), which will be uploaded to the wireless control module. You can save these files in the internal memory of your smartphone or in cloud storage.

7. Launch the ‘nRF Connect’ app on your smartphone, and follow Figure #33. Scan for available devices and connect to the one labeled as ‘BOOTLOADER’. Once connected, the screen will switch and a ‘CONNECTED’ message will be displayed.

8. Tap on the ‘DFU’ button located at the top of the screen and choose ‘Distribution packet (ZIP)’ as the file type.

9. Browse to the location where the firmware is stored and select the corresponding .zip file. This will initiate the immediate upload of the firmware to the BLE SoC.

10. Once the upload is completed, the device and smartphone will be disconnected. The ‘BOOTLOADER’ will no longer be visible for scanning.

Figure #33: Procedure for wirelessly uploading firmware using the nRF Connect app.

C.  Return to bootloader 

11. The device containing the firmware will be identified as ‘Device01”. Find the specific device that requires switching to the bootloader and establish a connection with the smartphone.

12. Once connected, tap on ‘Unknown Service’ to expand it and reveal its contents.

13. Within the expanded tab, click on the upward arrow icon next to ‘Unknown Characteristic’. This will open a command delivery screen.

14. Switch the command type to ‘TEXT’, enter the character ‘z’ as the command, and then press ‘SEND’.

15. The device will enter the bootloader state, and the connection will be terminated. To upload new firmware, follow the previously described process. For a more detailed demonstration, please refer to Figure #34 and Video #4, which provides step-by-step instructions using this ‘nRF Connect’ app.

This section provides an overview of the smartphone control app, including its installation process and instructions on how to use it for controlling a wireless control module.

A.  App installation 

1. Download the APK file of the app here and move it to your smartphone.

2. Run the APK file to begin the installation process (Figure #35).

Figure #35: Procedure for installing the smartphone app by executing the APK file.

3. Grant all the permissions requested during the installation process. When prompted for the app location permission after installation, select "Allow all the time" (Figure #36).

Figure #36: Procedure for granting permissions requested during app installation.

B.  App usage instructions

4. Open the smartphone control app (Figure #37(i)).

5. The app has the ability to scan for nearby BLE devices. To start scanning, locate and tap the black square button in the upper right corner of the app interface (Figure #37(ii)).

6. The scanned BLE devices will be listed. To establish a connection with the target wireless module (device name: Device01, Device02, etc.), perform a long-press on the desired device and then tap 'CONNECT' at the top of the screen to establish a connection with your smartphone. Once successfully connected, the device block will change its color to blue (Figure #37(iii)). It is possible to establish connections with multiple devices.

7. Once connected to the wireless control module, a screen will appear, allowing you to control the wireless module (Figure #37(iv)). Here, you can selectively adjust the intensity and frequency of the microLEDs on the optogenetic probe. When selected, all command buttons will appear as dark gray.

LED icons are accompanied by status indicator. ‘NULL’ indicates the initial state where no commands have been sent yet, ‘Lv.( ) – ( )Hz’ shows the optical intensity level and operation frequency of the optogenetic stimulation, and ‘XX’ represents a disconnection between the BLE device and the app. 

8. The intensity of the microLEDs can be adjusted across three levels (i.e., Lv.1 (~21 mW/mm2 for 470 nm-wavelength stimulation), Lv.2 (~61 mW/mm2 for 470 nm-wavelength stimulation), and Lv.3 (~97 mW/mm2 for 470 nm-wavelength stimulation)). Choose your desired intensity level and tap 'SEND' (Figure #37(v)). Upon successful transmission of the command, the 'NULL' indicator next to the LED icons will change to reflect the selected intensity level.

9. The microLEDs can be set to blink at frequencies of 5 Hz, 10 Hz, 20 Hz, or 40 Hz or they can remain continuously on. Select your desired operation and tap ‘SEND' to activate microLEDs accordingly (Figure #37(vi)). The status indicators next to the LED icons will be updated accordingly. Both microLEDs can be turned on simultaneously or independently.

10. To turn off a specific microLED, press the button corresponding to its current operation state and then press 'SEND'. 

11. To turn off all the microLEDs at once, click 'LED Off' followed by 'SEND' sequentially (Figure #37(vii)).

12. If you want to change the intensity and frequency while the other microLEDs are turned on, follow the same process mentioned above.

13. To end the control of the module, tap 'DISCONNECT' (Figure #37(viii)).

All the components that make up a wireless optogenetic probe system, including the microLED probe, wireless control module, and LiPo battery, need to be assembled for use in neuroscience experiments. The wireless module and LiPo battery can be enclosed using Kapton tape, providing protection and secure placement. As for the microLED probe, it can be easily connected to the wireless module by plugging it in using 3-pin male/female connectors.

A.  Enclosure of wireless module and LiPo battery

1. Place a wireless module on top of a LiPo battery. Using a double-sided tape between them will secure the position of the wireless module and facilitate the next steps.   

2. Wrap the wireless module and LiPo battery horizontally and vertically, making 1-2 turns in each direction, using Kapton tape (Figure #38). Prior to wrapping, create 5 small holes in the Kapton tape that align with the pin positions of the 3-pin and 2-pin male connectors. This will make the process easier. For a more detailed demonstration, refer to Video #5.

Figure #38: Photograph of a wireless module enclosed with a LiPo battery.

B.  Assembly of wireless module and microLED probe

3. Prior to assembling the microLED probe, connect the 2-pin female connector of the LiPo battery to a 2-pin male connector of the wireless module (Figure #39). Make sure that the positive pin (connected to the red cable) and negative pin (connected to the black cable) of the 2-pin female connector are correctly connected to the inner pin and outer pin of the 2-pin male connector, respectively.

Figure #39: Photograph of a wireless module powered by a LiPo battery through assembly of female and male pin connectors.

4. Connect the 3-pin female connector of the microLED probe to the 3-pin male connector of the wireless module (Figure #40). Ensure that the probe is facing away from the 2-pin battery connector. Otherwise, the two channels (i.e., LED 1 and LED 2) of the microLED probe will be inverted.

The operation of a wireless optogenetic probe system needs to be tested thoroughly before conducting neuroscience experiments. This guide outlines the process of operating the device and highlights key points to be checked during the operation. The steps for operation are also demonstrated in Video #6.

A.  Testing device connection

1. Connect the 2-pin female connector of the LiPo battery to the 2-pin male connector of the wireless control module before assembling the microLED probe. In this process, ensure that the wireless module is powered properly by observing the red and green indicator LEDs. They should blink once the wireless module is powered.

: If any of the indicator LEDs do not blink, first check if the battery is sufficiently charged (>3.0 V). Once you have verified that the battery is adequately charged, you can proceed to the next step. The indicator LEDs do not play a significant role in the system, so if they are not blinking despite a charged battery, it is not a major concern.   

B.  Testing smartphone app connection

(See Part 6B for a detailed tutorial on using a smartphone app.)

2. Open the smartphone app and check if the device is detected through Bluetooth with the set name (e.g., Device01, Device02, etc.).

: If a device is not detected in the smartphone app, first check if Bluetooth is enabled and ensure that the app has been granted location access the smartphone setting. If the device is still not detected and none of the indicator LEDs are blinked during Step 1, it indicates a potential issue with the wireless module. In this case, it is necessary to inspect the wireless circuit for any possible faults.  

3. To control the desired wireless module(s), press and hold them for 2-3 seconds, then tap the 'CONNECT' button to establish a wireless connection.

C.  Testing microLED Operation

4. Once the device is connected, send the desired commands for operation (Figure #41). Verify if the status (e.g., Lv.1-OFF, Lv.3-Cont., etc.) is accurately updated in the space located on the left side of the command buttons on the screen.

: In rare cases, commands may not be sent properly. If this happens, deselect the command button, select it again, and tap the 'SEND' button. This should ensure that the command is sent successfully.

5. Simultaneously, verifiy the proper functional of both channels (i.e., LED 1 and LED 2) of the microLED probe. For this testing, there is no need to change the frequency (or duty cycle) of microLED operation, but it is important to change the optical intensity. We recommend sending the following 6 commands: (i) INT Lv.1 & LED 1 Cont., (ii) INT Lv.2 & LED 1 Cont., (iii) INT Lv.3 & LED 1 Cont., (iv) INT Lv.1 & LED 2 Cont., (v) INT Lv.2 & LED 2 Cont., (vi) INT Lv.3 & LED 2 Cont.

: If either LED 1 and LED 2 channel does not work, you need to determine whether the issue lies with the microLED probe or the wireless module. Supply a voltage (2.7-3.0 V) to the microLED probe using a DC power supply and operate both LED channels. If either LED 1 and LED 2 channel still does not work, this means that there is an issue with the microLED probe. However, if both LED channels work fine, it suggests that the wireless control module requires repair.