Development of Optrodes and Instrumentation

for Wireless Optogenetic Application

H. E. Oshiro

1,2 a

, R. A. P. Andrade

2 b

, J. N. S. Junior

1 c

, M. Luppe

1 d

E. Colombari

3 e

, M. C. Dias

4 f

and J. P. Carmo

1 g

Group of Metamaterials Microwaves and Optics (GMeta), Department of Electrical Engineering (SEL), University of São

Paulo (USP), Avenida Trabalhador São-Carlense, Nr. 400, São Carlos 13566-590, SP, Brazil

brain4care, Avenida Bruno Ruggiero Filho, 971 - Parque Santa Felícia Jardim, São Carlos, SP, 13562-420, Brazil

Department of Physiology and Pathology, Faculty of Odonthology, São Paulo State University (UNESP), Rua Humaitá,

Nr. 1680, Araraquara 14801-385, SP, Brazil

Faculty of Medicine, University of Porto, Alameda Hernani Monteiro, piso 8, Unidade Cuidados Neurocriticos,

4200-319, Porto, Portugal

Keywords: Optrode, Optogenetics, Biopotentials.

Abstract: Optogenetics combines optical and genetic techniques to control and monitor neuronal activities. Recent

efforts focus on developing portable and wireless electronics for optical activation and biopotential

acquisition. These advancements aim to offer greater mobility and freedom for studying animals, contrasting

with the large equipment commonly found in laboratories for laser activation, signal amplification, and data

acquisition. This study presents the development of a wireless optrode system for optogenetics, integrating

optical stimulation and biopotential acquisition in a compact, portable format.

1 INTRODUCTION

Genetic engineering techniques are used to allow

neuronal populations to produce light-sensitive

proteins. These proteins can be stimulated or

inhibited by specific light patterns and wavelengths.

Deisseroth et al. (2006) defined optogenetics as a

technology that “combines genetic targeting of

specific neurons or proteins with optical technology

for imaging or control of the targets within intact,

living neural circuits”. To validate neuronal responses

to light stimulation, electrodes and techniques such as

patch-clamp electrophysiology can be employed,

allowing precise measurements of intracellular and

synaptic activity evoked by optical stimuli, as

demonstrated by Boyden et al. (2005).

https://orcid.org/0000-0003-0370-4700

https://orcid.org/0000-0002-7248-4636

https://orcid.org/0000-0002-1975-2267

https://orcid.org/0000-0001-7419-2154

https://orcid.org/0000-0002-1395-4036

https://orcid.org/0000-0003-0340-9808

https://orcid.org/0000-0001-7955-7503

In the current context, optogenetics applications

are generally carried out in laboratories equipped with

large optical equipment and instruments for reading

signals. Compact systems are being developed to

address limitations such as the need for wiring and

dependence on multiple pieces of equipment (light

emission controllers, acquisition hardware and

independent implantable parts), promoting greater

mobility in animal studies and allowing their free

behavior. The next chapters will address the

development of an optical stimulation system that

integrates with a biosignal acquisition system and

features an optrode module for use with an embedded

biosignal acquisition and optical stimulus control

system.

Oshiro, H. E., Andrade, R. A. P., S. Junior, J. N., Luppe, M., Colombari, E., Dias, M. C. and Carmo, J. P.

Development of Optrodes and Instrumentation for Wireless Optogenetic Application.

DOI: 10.5220/0013148100003911

Paper published under CC license (CC BY-NC-ND 4.0)

In Proceedings of the 18th International Joint Conference on Biomedical Engineering Systems and Technologies (BIOSTEC 2025) - Volume 1, pages 1073-1079

ISBN: 978-989-758-731-3; ISSN: 2184-4305

1073

2 OPSINS

Opsins are essential tools in optogenetics, allowing

neuronal circuits to be stimulated or inhibited by

light. “Opsins are membrane-bound proteins that are

activated by light, which results in activation

(depolarization), inhibition (hyperpolarization), or

modulation of the intracellular signaling cascade”

(Guru et al., 2015). Opsins function as ion channels

or pumps that are activated by specific wavelengths

of light. “Upon activation by light, these channels and

pumps respond by opening or closing, which

conducts the flow of ions into or out of the cell”

(Addgene, n.d.). Three commonly used opsins

include:

(1) Channelrhodopsins: channel-type opsins

that allow rapid depolarization of neurons through

direct stimulation of ion channels when exposed to

light (Addgene, n.d.). Channelrhodopsin-2 (CHR2) is

activated by light with wavelengths in the range of

400nm to 500nm (Dufour et al., 2015).

(2) Halorhodopsins (NpHR): opsins that pump

chloride ions into the neuronal membrane, causing

cellular hyperpolarization and inhibiting neuron

activation. NpHR is activated by light with

wavelengths in the range of 550nm to 620nm

(Dufour et al., 2015).

(3) Archaerhodopsins: light-controlled opsins

that pump protons out of the neuronal membrane,

causing cellular hyperpolarization and inhibiting

neuron activation. ArchT is activated by light with

wavelengths in the range of 500nm to 600nm

(Dufour et al., 2015).

In this way, an optrode can be equipped with

different light sources to suit the use in conjunction

with the different opsins used for different studies and

purposes.

3 SYSTEM ARCHITECTURE

The optrode developed in this project was designed

for use in a wireless system for optogenetic

stimulation and signal recording. The system is

compact, battery-operated, and integrates a

microcontroller with wireless communication, along

with circuits such as an analog-to-digital converter,

auxiliary sensors, and a power management system.

The acquisition system connects to the optrode via a

board-to-board connector.

The structure of the acquisition system is

represented in Figure 1.

3.1 Optrode Module

The optrode module connects to the acquisition

board, receiving the necessary power, the

communication buses and delivering the analog

signals to the ADC (analog to digital converter) in the

other board. Its architecture is shown in Figure 2.

The board incorporates a signal filtering stage to

ensure adequate visualization of the read signals, a

temperature sensor and auxiliaries to monitor the

activity of the study animal, in addition to means of

controlling the light pattern. As optrode module

design requirements, the following items have been

listed:

(1) It must have a means of delivering light;

(2) It must have an interface with electrodes;

(3) It must provide rapid integration and

configuration with the acquisition/control system;

(4) It must provide means for optical control of

excitation patterns;

(5) It must provide the necessary energy for the

components;

(6) It must provide an adequate analog

interface;

(7) It may include additional sensors.

3.1.1 Implantable Interface

To stimulate and measure the activity of a test animal,

it is necessary to perform optical delivery and reading

of the biopotentials of interest. Signal acquisition is

generally achieved through electrodes. As the optrode

module provides signal delivery to the acquisition

board's ADC, it is possible to adapt the module to

capture signals from other sensors, such as pressure

and temperature sensors, just by modifying the

interface with the test animal and without interfering

with the main acquisition and control module.

Light delivery can be done by optical fiber, or, as

adopted in this project, by small SMD LEDs.

4 DEVELOPMENT

4.1 Light Emitter

To stimulate or inhibit neurons expressing opsins,

light at the appropriate frequency and intensity must

reach the target. Each type of opsin responds to a

specific wavelength of light, as mentioned

previously. For ChR2 photostimulation,

Chen et al. (2016) mentions pulse widths between

1ms and 15ms as typical requirements, pulse

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Figure 1: Optrode module architecture.

Figure 2: System architecture for optogenetics.

frequency between 1Hz and 50Hz, and an optical

power at the fiber tip of at least 32µW. A minimum

irradiance of 1mW/mm² when delivering light to

neurons via an optrode (Freitas et al., 2021).

There are three common types of light sources

used for excitation in optogenetics: arc lamps, lasers,

and LEDs. The work by Lin (2012) mentions the

following characteristics for each of the light sources:

(1) Arc lamps: provides a continuous spectrum

of wavelengths in the visible range. However,

lighting control is done via a shutter, limiting the

stimulation frequency.

(2) Lasers: provides high-intensity light that

can be coupled to an optical fiber. It usually involves

a high equipment cost and wavelengths are limited

(3) LEDs (light-emitting diodes): physically

smaller than other options and can be mounted

directly on the brain. Due to its size and current, it can

generate heat. Its intensity is typically weaker

compared to the other options.

In this project, the use of LED was chosen to meet

the need to obtain a compact and low energy

consumption system in a wearable device. To

interface the optrode module in delivering light to the

test animal, an arrow-shaped printed circuit board

with a width of 0.7mm and a pointed end was

manufactured. Figure 3 shows a photograph of a

prototype of the implantable interface, which has only

two tracks and pads for soldering the LED, near the

pointed end, and for soldering wires to connect to the

optrode module, at the other end. As it has two

terminals, it is also possible to use the same board

design as an electrode to read potentials. The total

length is 20.7mm and 0.5mm thickness.

Development of Optrodes and Instrumentation for Wireless Optogenetic Application

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Figure 3: Photograph of an optrode module prototype with

Dialight 598-8091-107F LED.

4.2 LED Driver Circuit and Other

Components

The TLC5940 integrated circuit from Texas

Instruments was chosen to be used to control the

LEDs. Key characteristics that influenced this choice

include:

(1) Capability to control up to 16 LED

channels;

(2) Programmable and individual current control

for each channel in 64 levels;

(3) 4096-level individual PWM control;

(4) Compact size, 5.00mm×5.00mm in VQFN

version;

(5) Serial communication up to 30MHz.

The integrated circuit's communication protocol

does not follow a recognized standard, however it is

possible to use SPI with some modifications.

Additionally, a WLCSP−4 package EEPROM

measuring 0.77mm×0.77mm was included in the

design for quick recognition and configuration by the

acquisition board. To validate the project, a 20 mm x

20 mm printed circuit board was manufactured to

interconnect the aforementioned components. The

following were also included:

(1) Solder pads for 2 LEDs;

(2) Solder pads for 2 signal reading pairs, which

can be connected to an electrode, temperature sensor,

or both;

(3) Solder pads for resistors and capacitors on the

analog signal tracks for RC filter implementation;

(4) 40-pin connector, on the back of the board, for

connection to the acquisition module;

(5) Electrical test points to facilitate testing.

The optrode module, together with the interface

board is shown in Figure 4, while Figure 5 shows the

optrode module coupled to the acquisition board.

Figure 4: Optrode module and interface board.

Figure 5: Optrode module connected to the main acquisition

module.

5 BENCH TESTS

The optical power of commercial LED models and

the performance of optical pattern execution for

optogenetics applications were evaluated under

bench test conditions

5.1 Optical Power Test of LEDs

A search for LED models was conducted, and five

models were selected and tested. For the selection,

only models with an emitted wavelength close to 473

nm— the region in which CHR2 is maximally

activated—were considered.

The current of each LED was adjusted to close to

its nominal value and close to its maximum value by

varying a potentiometer to adjust the current while it

was measured by a Fluke 287 multimeter, and the

optical power was recorded using the Thorlabs

PMD100D Optical Power Meter setled to 473nm

wavelength in an environment with a dark chamber.

contacts

LED

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5.2 Optical Pattern Execution Tests for

Optogenetics

Figure 6 illustrates a structure that was created to

describe the pulses to be executed. The parameters

that can be configured when defining stimulation

pulses are listed below:

(1) Amplitude: The amplitude of the optical pulse

will be given by the current control, being directly

related to the light intensity

(2) Delay: Delay in starting activation;

(3) Width: Length of activation time;

(4) Interval: Time interval between activations;

(5) Pulse Number: Number of train pulses;

(6) Repeat Interval: Time interval between

repetitions;

(7) Repeat Number: Number of repetitions.

Figure 6: Description of a pulse pattern.

The optrode module received commands to

reproduce the following pulse pattern:

(1) Delay: 500ms,

(2) Width: 2ms,

(3) Interval: 500µs,

(4) Pulse Number: 5,

(5) Repetition interval: 2s,

(6) Number of repetition: 3.

To check the optical pulses, a circuit with a

photodiode and a resistor in series was assembled, so

that the reverse current generated in the photodiode

when sensitized by the pulse pattern of the optrode

module generates a variation in the resistor voltage.

Figure 7 illustrates the schematic of this circuit. The

voltage across the resistor is monitored on an

oscilloscope, and the shape of the generated wave is

compared to the pulse pattern performed.

Figure 7: Schematic of the optical sensor circuit for testing

pulse patterns.

6 EXPERIMENTAL RESULTS

AND DISCUSSION

6.1 Optical Power of Commercial

LEDs

In total, five LED models were tested. Each of them

has been tested close to its rated current and its

maximum current, when possible. Figure 8 shows the

LED approach to the sensor in the image on the left

and the measurement panel on the right. Table 1

summarizes the results obtained.

Figure 8: Optical power test: (a) sensitive component and

(b) power measurement.

The LED model DA2432, manufactured by Cree,

demonstrated high light power (18.7mW) despite its

small physical size. Furthermore, its maximum current

is 100mA. However, its compact dimensions (240 µm

by 320 µm) make soldering challenging and increase

the risk of losses. Additionally, obtaining more units

of this model was not possible, as it is an obsolescent

product. Cree's CLM3A-BKW-CUAVA453 model

exhibited similar optical power levels at a lower

current, but its volume is approximately 652 times

larger. Roithner's B5B-437-IX model delivered

slightly more than half the optical power of the

previous models and has the largest physical size

among them. Dialight's 597-3601-207F model

presented the lowest optical power even though it was

larger than the 598-8091-107F model from the same

brand, which, in turn, obtained 5.15mW of optical

power and the second smallest size among them.

Figure 9: Measurement of the performed pulse pattern.

Development of Optrodes and Instrumentation for Wireless Optogenetic Application

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Table 1: Measurements of LED power.

Supplier/Model

Dimension [mm]

Wavelength [nm]

Nominal current [mA]

Measured optical

power [mW]

Cree DA2432

0.240.320.14

470

5.6 @ 19.6mA

18.7 @ 99mA

Cree CLM3A-BKW-

CUAVA453

2.72.01.3

470

18.8 @ 19.6mA

10.4 @ 10.0mA

Dialight 598-8091-

107F

1.60.80.7

473

5.15 @ 19.6mA

Dialight 597-3601-

207F

3.52.81.9

465

1.7 @ 20mA

Roithner B5B-437-IX

5 (diameter)

468

8.3 @ 21mA

10.6 @ 30mA

Figure 10: Train of pulses captured by the oscilloscope.

Figure 11: Programmed delay time measured in logic analyser.

6.2 Execution of Optical Patterns for

Optogenetics

The photodiode was assembled in series with a

resistor, as shown in Figure 7. Figure 9 shows the

assembly of the photodiode and the approach of the

interface board to the LED performing the pulse

patterns. The LED used in the tests was the Dialight

598-8091-107F.

The voltage across the resistor was measured

using a Hantek 6022BL digital oscilloscope. The

captured pulse train is presented in Figure 10, where

the pulse width, inter-pulse interval, and repetition

times were analyzed.

The start delay (delay parameter) was defined as

the time interval between the last clock pulse of the

TLC5940 initial configuration transmission and the

beginning of the first pulse transmission. This

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measurement was performed using a Hantek 6022BL

logic analyzer in conjunction with Saleae Logic

1.2.40 software. The results of the configured and

measured parameters are summarized in Table 2.

Table 2: Configured values and measured values when

executing pulse patterns.

Parameter

Configured

Measured

Delay [µs]

500

497

Width [ms]

Interval [µs]

500

595

Pulse Number

Repeatition Interval [s]

1.98

Repeat Number

7 CONCLUSIONS

In bench tests, the optrode module successfully

reproduced the patterns required for optogenetics

experiments, demonstrating both adequate optical

power and temporal precision. These results support

the continuation of the project’s development. The

next steps include full integration and testing with the

acquisition electronics, enabling simultaneous

stimulation and signal acquisition. Subsequently, in

vivo tests will be necessary to validate the system's

performance in practical scenarios. Additionally,

developing alternative interfaces for light delivery

and signal capture, as well as exploring other

wavelengths, is of interest to expand the system's

applicability to diverse contexts.

ACKNOWLEDGEMENTS

This work was partially supported by the CNPq

(Conselho Nacional de Desenvolvimento Cientifico e

Tecnológico) through the project with the reference

CNPq 402752/2023-6. Professor João Paulo Carmo

was supported by a PQ scholarship with the reference

CNPq 305858/2023-8.

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