Optogenetic Implants

Hubin Zhao

ContentsIntroduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2Biomedical Applications . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4Design Requirements . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5

Miniaturized Size . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5Device Materials . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 6Fabrication Process . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 6Thermal Effect . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 6Power Consumption and Power Delivery . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 7Light Sources . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 7Neural Inhibition . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 7Intensity Programmability . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 8Spatial-Temporal Resolution . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 8Closed-Loop System . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 8Integrated and Intelligent Devices . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 8Diagnostic Function . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 9

Technology Developments . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 9Discrete Optogenetic Implants . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 9Integrated Optogenetic Implants . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 14

Design Example: HUBIN Optrode – A Microchip-Based Optogenetic Implant . . . . . . . . . . . . . . . 18Open-Loop HUBIN Optrode . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 18Closed-Loop HUBIN Optrode . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 22Exploration: Scalable Architecture of HUBIN Optrode . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 24

Conclusion . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 26References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 26

H. Zhao (*)Department of Medical Physics & Biomedical Engineering, University College London,London, UKe-mail: hubin.zhao@ucl.ac.uk

© Springer Science+Business Media, LLC, part of Springer Nature 2020M. Sawan (ed.), Handbook of Biochips,https://doi.org/10.1007/978-1-4614-6623-9_48-1

1

Abstract

Using optogenetics for neuromodulation demonstrates a high potential, and itmay become a useful tool to analyze complicated neural circuits and provide aneffective gene therapy for chronic brain illnesses. One of the key challenges is todevelop a miniaturized, intelligent, integrated, multi-site/multilayer, multimodaloptogenetic implant. This chapter first introduces the optogenetics and its typicalbiomedical applications. Then this chapter gives an overview of the recentadvance in the technology developments in optogenetic implants. Both discreteoptogenetic implants and integrated optogenetic implants are described. Particu-larly, a microchip-based integrated approach (HUBIN optrode) demonstrated apossibility toward the development of new-generation intelligent, integrated,miniaturized, multimodal optogenetic implants.

Keywords

Optogenetics · Implants · Microchip · Intelligent · Multimodal · NeuralStimulation · Neural Recording · Closed-loop

Introduction

Optogenetics, a combination of genetic and optical methods for neuromodulation,can enable individual neurons controlled by light. This technology thus can have thefeasibility to be a useful neuroscience tool to explore complicated neural circuits andpotentially provide effective gene therapies for chronic brain illnesses such asParkinson’s disease, blindness, and epilepsy (Zhao 2017).

The first exploration of using light for neural stimulation was performed at 1971,and a blue light source was adopted to successfully generate action potentials inAplysia ganglia. Subsequently, at 1999, Nobel laureate Francis Crick came up with adelicate assumption of optogenetics: a new optical method which could be used totrigger or silence specific types of neurons without any influence on other neuronpopulations (Zhao 2017; Fan and Li 2015). In the past 20 years, numerous attemptsof optogenetic tools have been made, but these tools are stem from exogenouscofactors or combining the expression of different types of proteins, which arechallenging to be applied into in vivo studies (Kravitz and Kreitzer 2011). In theyear 2003, Nagel et al. found an expression of a single photosensitive protein –ChR2, which can be transgenically expressed in individual nerve cells; with appro-priate illumination from light sources (~470 nm wavelength), the photosensitizedneurons can be activated. This new optogenetic method has been increasinglyadopted to control both in vitro and in vivo neuronal activities with relatively precisespatial-temporal resolution (Boyden et al. 2005; Nagel et al. 2005). While ChR2demonstrated great potential and suitability to activate neural activities, an increas-ing demand for a specific tool that can be used to silence nerve cells has beenidentified. Neuroscientists thus have made significant advancement on this topic, and

2 H. Zhao

in 2007 Natronomas pharaonis halorhodopsin (NpHR) was discovered from archae-bacterium Natronomas pharaonic (Zhang et al. 2007) that can be utilized to inhibitneural activities from individual neurons using yellow light (with a wavelength of~570 nm). The field of optogenetics has been fully established since then. Figure 1illustrates how ChR2 and NpHR can be used for optogenetic stimulation.

Compared to conventional electrical stimulation methods, optogenetic stimula-tion provides a cell-specific approach for neuromodulation. Moreover, both CHR2and NpHR require comparatively low optical intensity (1 and 7 mW/mm2) toactivate/inhibit neurons. And many neuroscience and biological studies have beenconducted to realize deep explorations of complies neural circuits and complexneural illness.

For wider and better applications of optogenetics, technology development ofnovel optogenetic implants using suitable engineering techniques is highlydemanded, so as to accurately and reliably deliver the light into the targeted areaof the brain with satisfactory spatial-temporal resolution. In the past 10 years,significant progress has been made toward a miniaturized, lightweight, integrated,multi-site optogenetic implant. To achieve an intelligent-oriental system, microchip-based optoelectronic implant demonstrates a new possibility for next-generationoptogenetic engineering tools.

NaCa

ClH

K

Na

c

b

a

Ca Cl

H

K

460nm light 570nm light

ChR2 HR

1s 2ms

Fig. 1 Diagram of usingChR2 and NpHR foroptogenetic stimulation (Luanet al. 2015). (a) The default“off” states of the ChR2 andNpHR ion channels. (b) Withthe stimulation of blue/yellowlight (460/570 nm), positiveand chloride ions pass throughthe neuron via ChR2 andNpHR ion channels,respectively. (c) While ChR2activates action potentials ofneurons, NpHR deactivatesneural activities. (This figureis reprinted with permission)

Optogenetic Implants 3

Based on the approaches of light delivery, all the optogenetic implants can becatalogued into two different categories: discrete optogenetic implants and inte-grated optogenetic implants. This chapter gives an overview and tutorial of thestate of the art of technology development in (both types of) optogenetic implants.Moreover, the design requirement of an “ideal” optogenetic implant is characterized.Furthermore, a design example of microchip-based optogenetic implant is alsointroduced. The following sections are organized as follows: section “BiomedicalApplications” describes some examples of typical applications of optogenetic tech-nologies; section “Design Requirements” illustrates a list of requirements toward an“ideal” new-generation intelligent optogenetic implant; section “Technology Devel-opment” introduces both types of discrete optogenetic implants and integratedoptogenetic implants, and the limitations in current designs are correspondinglyrecognized; section “Design Example: HUBIN Optrode – A Microchip-BasedOptogenetic Implant” demonstrates the trend toward the development ofnew-generation integrated, intelligent optogenetic implant; section “Conclusion”concludes the whole chapter.

Biomedical Applications

In recent 10 years, the applications of optogenetics into clinical studies have beenincreasingly progressed. An encouraging application is to apply optogenetic tech-nologies to analyze possible principles and treatment mechanisms of Parkinson’sdisease. In the year 2009, Gradinaru et al. (2009) proposed an integrated ChR2-NpHR optogenetic device, equipped with a fiber-coupled laser diode as light source.An animal experiment was conducted on freely moving rodents which were parkin-sonian. The ChR2-NpHR optogenetic device was implanted into the animal subjects,and the optical stimulation was set as switchable: activation (ChR2, 473 nm) orinhibition (NpHR, 561 nm). The therapeutic effects of deep brain stimulation (DBS)within the subthalamic nucleus (STN) were studied by performing activation/inhi-bition to corresponding afferent axons. This device enabled a better understanding ofthe working principle of DBS and meanwhile demonstrated the feasibility to useoptogenetic devices to analyze abnormal brain circuity and subsequent brain illness.

Apart from the applications of Parkinson’s disease, epilepsy can also be poten-tially investigated using optogenetic technologies. A closed-loop optogenetic systemwas developed, aimed to provide effective treatment for temporal lobe epilepsy(TLE) (Krook-Magnuson et al. 2013). A fiber-coupled laser was used as light source,equipped with both blue light and yellow light (blue 473 nm, yellow 589 nm), so asto achieve optical neural activation and inhibition within a single system. Thisoptogenetic system was used to simultaneously stimulate (activate and inhibit) twodifferent types of hippocampal neurons in a mouse, while in situ electroencephalog-raphy (EEG) recording electrodes were placed to record neural signals of seizure. Asophisticated seizure detection algorithm was implemented in Matlab running on aPC. Once the neural signals of seizure was detected by the EEG electrodes, theseizure signals would be transmitted via front-end amplification component and

4 H. Zhao

analog-to-digital (ADC) component to the PC terminal. Then the optogenetic stim-ulator would be then triggered to deliver light with specific wavelength (473 nm or589 nm) to region of interest in the hippocampus area of the mouse. This optogeneticstudy illustrated a promising detection and controllability of seizures from TLE.

Besides this, visual prosthesis is another emerging application for optogenetictechnologies. Bi et al. (2006) conducted an experiment on a mouse in which themajority of photoreceptors of its retinal ganglion cells had been degenerated. Duringthe experiment, the ChR2 expression was injected into the retinal ganglion neuronsof the subject, which enabled the retinal ganglion neurons to receive and code lightsignals. This initial success leads to further clinical explorations. In 2014, a clinicaltrial on patients to use optogenetic technologies to explore the possible treatment forretinitis pigmentosa (RP) has been approved by the US Food and Drug Administra-tion (FDA). In 2016, the optogenetic clinical trial was performed for the first time ona patient (called the RP RST-001). As the first human experiment ever, thisoptogenetic clinical trial was a milestone for the field of optogenetics.

Optogenetics demonstrated its potential to be a promising tool forneuromodulation, and it has been widely utilized for complex brain illnesses, suchas Parkinson’s disease, visual prosthesis, and epilepsy. More explorations of usingoptogenetics to address broad diseases and health issues will be further investigatedby neuroscientists and clinical neurologists. Meanwhile, there is an increasing needfor neural engineers to develop high-quality optogenetic implants that can be easy touse.

Design Requirements

This chapter would highlight the significant progress that has been made toward aminiaturized, lightweight, integrated, multi-site optogenetic implant. In the processof compelling this review, the author has observed obvious difference in the descrip-tions about the performance of optogenetic implants published in peer-reviewedjournal articles. It is not rare that some key features of these published implants weremissed from the articles altogether. In an attempt to optimize this situation, the authorsuggests the following list of design requirements, which identify a reasonableminimum expectation for the peer-reviewed description of any optogenetic implantspublished later, and also proposes a conception of an “ideal” optogenetic implantthat might be realized by neural engineers in the future.

Miniaturized Size

The size of optogenetic implants must be small, which could only cause minimalinjury of subjects. The ideal length of shaft of the optogenetic implants should bearound 4 mm, which can have a good matching of the thickness of the human brain,so as to potentially achieve multi-site multilayer stimulation. The width and thick-ness (or diameter) should be fitted within the scale of micrometer, for example, 200–

Optogenetic Implants 5

400 μm, which can reduce the area of tissue damage meanwhile still keep thesharpness and robustness of the tip of the implant. The components used on theimplants, such as light sources, drive, and control circuitry, should be fabricated withminiaturized size as well, to increase the illuminance intensity and further reduce theimplant dimensions.

Device Materials

The choice of the substrate materials is vital for the development of optogeneticimplants. As an optical-based implantable device, the opto-thermal effect is a keyconcern. It would be encouraging if the substrate materials can hold great thermalconductivity. Flexibility is another important factor. The substrate materials shouldbe with reasonable flexibility, as high rigidity could lead to tissue damage and otherside effects. While maintaining the flexibility, the sharpness of the implant still needsto be guaranteed. Biocompatibility is another feature that needs to be taken intoconsideration, and any toxic risks should be excluded. Besides this, the idealsubstrate materials should be compatible with the most of available fabricationprocess. In short, a satisfactory choice of substrate materials should be with out-standing flexibility, biocompatibility, thermal conductivity, and easy access to stan-dard fabrication technologies.

Fabrication Process

Currently, most of the optogenetic implants are developed using custom-designedfabrication technologies, and the majority of them are relatively time- and moneyconsuming, which could cause limitations for wider access and reproduction. Itwould be encouraging if there was a commercial available fabrication technologythat can be used for the development of optogenetic implants. In the past severalyears, complementary metal-oxide-semiconductor (CMOS) process has been uti-lized to fabricate implants for electrical neural recording and stimulation. Using theCMOS process, the shaft area of the implant can be used to fit the active CMOSelectronics in, and this strategy holds the feasibility to be transferred for fabricationof new type of optogenetic implants.

Thermal Effect

Thermal effect is a practical and critical factor for any type of implantable devices,particularly for optogenetic implants that with light sources integrated on the deviceitself. The heat dissipation from the light source is the primary source to contribute tothe thermal effect. Thus suitable thermal analysis and management are required toregulate the thermal increase of optogenetic implants within 2 �C and ideally within1 �C. In order to monitor the temperature increase, it would be helpful if in situ

6 H. Zhao

temperature sensors can be developed and integrated into the optogenetic implants toachieve real-time monitoring of the thermal effect.

Power Consumption and Power Delivery

Power consumption is another important factor for the development of optogeneticimplants. Current optogenetic implants are powered with battery or wireless trans-mission; power consumption is a key concern for either approach. The optoelec-tronic components incorporated in the optogenetic implants usually requirerelatively high voltage and strong current; this will further add limitations to thepower budget of the implants. Some approaches of low power circuit and systemdesign (such as energy harvesting, power gating, asynchronous circuit, etc.) perhapscan be considered to be adopted for the optimization of the power consumption ofoptogenetic implants. Moreover, in order to get rid of the tethered setting ofexperimental subjects, the technology of wireless power transmission can be con-sidered to be applied into optogenetic implants.

Light Sources

To ensure the accuracy and stability of light delivery from optogenetic implants, theperformance of the light sources does matter. Also the choice of the suitable lightsources will influence the characteristics of Implant Size, Device Materials, Fabri-cation Process, Thermal Effect, and Power Consumption (i.e. all the factors pre-sented above). Typical light sources for current optogenetic implants are laser and(micro) light-emitting diode (LED). LEDs seem to hold better stability for lightdelivery, while lasers can provide coherent light with lower divergence than LEDs’(Zhao 2017). The emergence of the technology of vertical-cavity surface-emittinglaser (VSCEL) provides another potential choice for light sources of optogeneticimplants. Compared to most of conventional lasers and LEDs, VSCELs holdnarrower bandwidth, higher emission efficiency, and lower energy consumption. Itwould be a promising option for light sources in optogenetic implants when suitableblue VSCELs are available in the commercial market.

Neural Inhibition

As described above, optogenetics not only can activate neurons (usually usingCHR2) but also can inhibit neural activities (using NpHR). However, most of theoptogenetic implants in the field only hold the capacity for neural activation usingblue light, and few of them can achieve neural inhibition using yellow light. There isa demand to develop a device that can achieve two-way control simultaneously.Suitable dual-wavelength (or multi-wavelength) light sources can be a possibleapproach to meet the requirement.

Optogenetic Implants 7

Intensity Programmability

The programmability of light intensity is a useful feature for optogenetic implants,which could affect the thermal effect, the energy efficiency, system safety, andpenetration depth. Most of the existing devices change the light intensity by man-ually adjusting the supply voltage/current, or the pulse duration of the stimulation. Itwould be meaningful if more sophisticated modulation scheme (in hardware and/oralgorithm) can be incorporated into the optogenetic implants to realize finer pro-grammability of light intensity.

Spatial-Temporal Resolution

An “ideal” optogenetic implant should be with high spatial resolution, and amodality of “high-density” should be realized so as to accomplish high-resolutionmulti-site multilayer stimulation. Meanwhile, in order to ensure the precision andefficiency of the optical stimulation, the resolution of temporal control of the opticalstimulation should be kept in a reasonable range.

Closed-Loop System

In conventional electrical neural stimulations, neural recording electronics can beintegrated into the implantable devices to record the in situ local signals of neuralactivities. But for most of existing optogenetic implants, no neural recording func-tion is achieved. To develop the new generation of optogenetic implants, appropriateneural recording electronics can be considered to be integrated so as to monitor localneural activities in real time and afford effective feedback information for opticalstimulators. The development of this type of closed-loop optogenetic implants couldenhance the stimulation accuracy, energy efficiency, and overall performance.

Algorithms design to control the closed-loop system is also important. Suitablealgorithms such as threshold detection and spike classification need to be considered.Driven by the advanced technology of artificial intelligence (AI), appropriate AIalgorithms such as machine learning may be possible to be embedded into thenew-generation optogenetic implants to further improve the precision, efficiency,and overall performance of the closed-loop system.

Integrated and Intelligent Devices

So far, almost all the conventional optogenetic implants are controlled by variousexternal instruments, which have to tether the subjects on the laboratory benches.Also most of these devices are not developed using standard hardware and softwareplatforms, which constraint their wider access from researchers in the community.Besides this, the relatively bulky size and high power consumption of these external

8 H. Zhao

controlling devices further limit their broad and long-term usage. Driven by thetechnology of intelligent hardware particularly microchip technologies, perhaps thenew-generation optogenetic implants can be developed using integrated microchipplatforms with intelligent control algorithms and standard communication protocolsembedded, which can be potentially adopted by the broad communities.

Diagnostic Function

Due to the nature of implantable devices, it is extremely challenging to assess theconditions particularly the integrity of the implant during and after implantation. Ifany fracture of implant shaft or component failure occurs, it would be harmful for thepatients and may cause unpredictable side effects. If the feasibility is available, itwould be promising that a self-diagnostic sensing function can be implemented andintegrated into the optogenetic implant so as to monitor the functioning status of theimplant and its key components (i.e., light sources, recording electrodes)continuously.

Technology Developments

In the past 10 years, various explorations toward miniaturized, multi-site, multilayer,intelligent optogenetic implants have been made. In these existing optogeneticimplants, based on the approaches of light delivery and settings of the systems,they can be classified into two different types of systems: (1) discrete optogeneticimplants and (2) integrated optogenetic implants. For discrete systems, usually anexternal waveguide structure such as optical fibers is used to achieve light coupling(with lasers or LEDs) that the light can be delivered to the targeted area of the brainwith some distances. In comparison, light sources (usually LEDs) are directlyembedded on the integrated optogenetic implants, and there is no extra light cou-pling devices required. In this section, some typical systems of these two types ofimplants are respectively introduced.

Discrete Optogenetic Implants

In the past years, there are different types of discrete optogenetic implants that havebeen developed. In Fig. 2, LeChasseur et al. (2011) developed a glass fiber-basedoptogenetic implant in 2011, and the functionalities of optical neural stimulation andelectrical neural recording were both achieved, in which an optical core wasdesigned for optical stimulation and a hollow core was developed for electricalrecording, so as to realize a hybrid optical-electrical stimulation-recording system.The light intensity provided by this system was about 10 mW/mm2, and an alumi-num coating was used as an optical shield to minimize the optical loss during thestimulation process. External instruments such as dichroic mirrors, photomultiplier

Optogenetic Implants 9

tube (PMT) detectors, shutters, and band-pass filters were applied into this systemfor effective light coupling.

At the same period, Wang et al. (2012) proposed a MEA (microelectrode array)-based optogenetic implantable system, which achieved simultaneous optical neuralstimulation and electrical neural recording. A 6 � 6 Utah array structure was used toconstruct this system which equally mixed with fiber-based optical stimulation sitesand neural recording electrodes. Each microelectrode had a length of 1 mm and thepitch in between was about 400 μm. A single-site stimulation can be achieved with30-site concurrent neural recording during each operation.

Besides conventional fiber-based system, LED-coupled discrete optogeneticimplants were also explored in the similar period. Still in 2012, Stark et al. (2012)developed a LED-based fiber coupling implantable system optogenetic stimulation.A 50-mm-long optical fiber was used for light guide, which included four-steppedsections (Fig. 3a). Particularly, the last section was the effective optical shaft, with a

Fig. 2 Diagram of the opto-electro optical fiber-coupleddiscrete optogenetic implantin LeChasseur et al. (2011).(This figure is reprinted withpermission)

10 H. Zhao

length of ~5 mm and a diameter of 60–70 μm. A 12-degree shaft tip was constructedto ensure the sharpness. The fabricated probe was reproduced by extra five times tocomplete a multi-diode stimulation array.

Two years later, Schwaerzle et al. (2014) proposed another LED-based discreteoptogenetic implant, based on a polyimide substrate on which the LED chip wasembedded (Fig. 3b). The polyimide materials demonstrated outstanding bendabilityand flexibility. On the polyimide substrate, a silicon housing was constructed to fix

Fig. 3. (a) A single four-step fiber-coupled discrete optogenetic implant (Stark et al. 2012). Thelength of the implant is 50 mm, and the last shank is 5 mm long with a 12� tip. (This figure isreprinted with permission.) (b) Schematic diagram of the fiber-coupled LED optical implant(Schwaerzle et al. 2014). (This figure is reprinted with permission)

Optogenetic Implants 11

the LED chip on the substrate. An optical fiber (5 mm long) was used for lightcoupling.

Although abovementioned discrete optogenetic implants demonstrated differentmerits, all these devices only realized single-site single-layer stimulation. To achievemultilayer setting, Zorzos et al. (2012) proposed 3D waveguide structure-baseddiscrete optogenetic implant as shown in Fig. 4. A number of optical probes wereconstructed in parallel with different lengths so as to achieve the setting of multilayerstimulation. In addition, light coupling was achieved using external light couplingdevices.

Schwaerzle et al. (2017) further developed an optical-electro optogenetic implantusing a silicon substrate. The implant held two identical probe shafts (~8 mm long).Two laser diose chips were bonded on each shaft to attain two optical stimulationsites, while four electrical recording sites were placed on every shaft. Each laserdiode coupled with a waveguide for light guiding.

There are also some other discrete optogenetic implants developed in recentyears, and a list of recently published discrete optogenetic implants is summarizedin Table 1. Although all of these discrete optogenetic implants held various advan-tages, there are several limitations that need to be overcome. Single-site stimulationis an obvious drawback. It is challenging to apply this type of devices to conductmulti-site even multilayer stimulation, which largely limit their wider applications.Some devices used array setting (by reproducing several probes) to achieve “multi-site” stimulation, but the penetration depth was still fixed that cannot be used for

Fig. 4 Diagram of the 3Dwaveguide optogenetic array(Zorzos et al. 2012). (Thisfigure is reprinted withpermission)

12 H. Zhao

Table

1Listof

recently

publisheddiscreteop

togenetic

implants.(Thistableismod

ified

with

perm

ission

from

Zhao20

17)

Developers/

year

Light

source/

wavelength

Size

No.

ofSti.

sites

Light

intensity

Pow

erconsum

ption

Max

Sti.

frequency

Electrical

recording

Fabricatio

nprocess

Sub

strate

material

LeC

hasseur

etal./2

011

Laser/

488nm

Diameter,2

00μm

;shafttip

diam

eter,

10μm

110

mW/m

m2

––

Yes

Custom

fabricated

–

Wangetal./

2012

Laser/

473nm

Shaftlength

(L),1mm;

spacing,

400μm

15mW/m

m2

–40

kHz

Yes

Custom

fabricated

–

Zorzos

etal./2

012

Laser/

473nm

Apertures,9

�30

μm1�

2514

8�

56mW/

mm

215

00mW

–No

Custom

fabricated

Silicon

Schwaerzle

etal./2

017

Laser/

650nm

ShaftL,8

mm;W,2

50μm

;laserdiod

edimensions,

300�

300�

100μm

3

2�

296

.9mW/m

m2

12.82mW

100kH

zYes

Custom

fabricated

Silicon

Rub

ehn

etal./2

011

Laser/473,

593nm

Shaftlength,7

mm;width,2

00μm

1–

21mW

(for

blue

light)

–Yes

Custom

fabricated

Polyimide

Wuetal./

2013

Laser/

473nm

Shaftlength,5

mm;width,2

00μm

194

00mW/

mm

250

mW

25Hz

Yes

Custom

fabricated

Silicon

Son

etal./

2015

Laser/

473nm

Diameter:150μm

10.9mW

(~51

mW/

mm

2)

––

Yes

Custom

fabricated

Silicon

Stark

etal./

2012

LED/470

,58

9,63

9nm

ShaftL,5

mm;diameter,60–

70μm

(blue);

LEDdimension

s,1.6�

0.6mm

21�

640

mW/m

m2

(bluelig

ht)

Current:

60mA

–No

Custom

fabricated

–

Schwaerzle

etal./2

014

LED/

460nm

Totalleng

th:5mm

Diameter,1

25μm

;LEDdimension

s,27

0�

220�

50μm

3

11.71

mW/m

m2

Current:

30mA

–No

Custom

fabricated

Polyimide

Schwaerzle

etal./2

0159

LED/

460nm

Totalleng

th:5mm

Diameter,1

25μm

;LEDdimension

s,27

0�

220�

50μm

3

1�

91.28

mW/m

m2

Current:

30mA

–No

Custom

fabricated

Polyimide

Optogenetic Implants 13

multilayer stimulation. Moreover, for discrete optogenetic implants, external lightcoupling devices were usually required, but almost all of these devices had relativelycumbersome structures that would be difficult to use the implants for broad appli-cations particularly in untethered settings. In addition, the low coupling efficiency oflight guides significantly constrained the light emission efficiency and overall energyefficiencies.

Integrated Optogenetic Implants

Compared to the discrete optogenetic implants, the light sources (usually LEDs)were directly bonded on the integrated optogenetic implants. This setting canpotentially have some advantages: smaller size, higher optical emission efficiency,and multi-site/multilayer stimulation. Moreover, the implantable system can beintegrated on a single probe and no external devices/components required. Severalexplorations of integrated optogenetic implants have been made in recent years.

One example is in 2014; Fan et al. (2014) has proposed an integrated optogeneticimplant using SU-8 as substrate material (Fig. 5a). The implant was 4.2 mm long and0.86mmwide. Amicro-LED (with dimensions of 550� 600� 200 μm3) was positionedat the tip of the implant as a stimulation site. The total power consumption of thisintegrated implant was 130 mW, producing a light intensity of about 0.9 mW/mm2.

At the same period, Cao et al. (2013) developed another integrated implant usingpolyimide as a flexible substrate (Fig. 5b). Micro-LED (1000� 200� 600 μm3) waspositioned at the tip of the implant as a stimulation site, and there were three neuralrecording sites in the surrounding area to achieve concurrent opto-electro neuralstimulation and recording within a single integrated implant. The length of shaft theimplant is about 12 mm and a width about 900 μm. This implant provided relativelylow light intensity (only ~0.7 mW/mm2).

Although these two devices made integrated implant become possible, there weresome limitations with the designs. First, the utilization of the bulky LEDs leads tohigh challenge to accomplish multi-site multilayer stimulation, and a micro-LEDwith miniaturized profile would be a more appropriate choice. More crucially, thelight intensity generated by both abovementioned integrated implants were low (0.9and 0.7 mW/mm2, respectively), which are difficult to be used for most of applica-tions which require higher light intensity.

To resolve these issues, McAlinden et al. (2013) developed a sapphire-basedintegrated implant embedded with custom-designed miniaturized micro-LEDs(Fig. 6a) so as to achieve multi-site and multilayer stimulation. The implant held alength of 7 mm and a width of 80 μm, and five stimulation sites were evenlydistributed along the implant shaft (1 mm long). The diameter of each stimulationsite (micro-LED) was only about 40 μm, which can produce a 600 mW/mm2

maximum light intensity of implant. Under such high light intensity, this optogeneticimplant demonstrated strong thermal dissipation performance, and maximum ther-mal increase was constraint in the range of 1.5 �C.

14 H. Zhao

Building upon this multi-site high-intensity integrated implant, a more advancedoptogenetic implantable array was proposed by Scharf et al. in 2016. This implantutilized silicon as the substrate, as displayed in Fig. 6b. This implantable array consistedof six optical probes, on which each probe had 16 evenly distributed micro-LEDs thatcan be individually controlled. In total, this array was with 96 stimulation sites. Themaximum light intensity generated by this system was ~400 mW/mm2, and thetemperature increase was constraint averagely around ~0.5 �C when light intensitywas regulated to 150 mW/mm2 with 50 ms stimulation duration.

In the similar period, Wu et al. (2015) proposed a silicon-based optical-electrointegrated implantable optogenetic array; the functionalities of concurrentstimulation-recording, multi-site/multilayer stimulation, and individual control ofstimulation sites were all achieved. This array consisted of four branches; eachbranch contained three optical stimulation points and eight electrical recordingelectrodes. The length of each probe shaft was about 5 mm, and width was 70 μm.

Fig. 5 (a) SU-8-based optogenetic implant using an off-the-shelf Samsung LED, developed by Fanet al. (2014). (This figure is reprinted with permission.) (b) Flexible polyimide-based integratedoptical implant (Cao et al. 2013). (1) The assembly of the implant using a PCB. (2) A scanningelectron microscope (SEM) imaging of the LED and recording electrode. (This figure is reprintedwith permission)

Optogenetic Implants 15

Driven by a current of 13 mA, this optogenetic integrated array can generate fairlyhigh light intensity (353 mW/mm2).

The optogenetic stimulators developed by McAlinden et al. (2013), Scharf et al.(2016), and Wu et al. (2015) explored different approaches to achieve multi-sitehigh-intensity integrated implants; particularly Wu et al. implemented simultaneousoptical stimulation and electrical recording. Through promising, all of these devicesstill had some drawbacks, in particular external bulky control (printed circuit board)

Fig. 6 (a) Sapphire-based integrated optogenetic implant using GaN LED (McAlinden et al.2013). (This figure is reprinted with permission.) (b) Silicon-based high-density optogenetic array(Scharf et al. 2016). (This figure is reprinted with permission)

16 H. Zhao

PCB were required, which add inconveniences for wider applications, especially infreely-moving experiments.

At the same time, Kim et al. (2013) explored a different approach to develop amultilayer multifunctional integrated optogenetic implant (Fig. 7). This four-layerdesign was based on a soft polymer substrate that maintained high flexibility of thesystem, in which different layers performed the different functions (L1, electricalneural recording; L2, optical detection; L3, optogenetic neural stimulation; and L4,temperature sensing). In particular, this implant used a resolvable substrate that canbe easily dissolved with specific dissolving liquid, and this strategy can potentiallyimprove the system safety and reliability. The implant also demonstrated good lightintensity (avg. 17.7 mW/mm2, max ~40 mW/mm2) and thermal dissipation perfor-mance (1 �C thermal increase). This design was also compatible with wireless powertransmission setting. Although promising, this design was not without shortcomings.The length of the implant was only about 1 mm that would be challenging to be usedfor deeper stimulation. More crucially, the fabrication process was fairly compli-cated, which is costly, labor-intensive, and hard to be adopted by other researchers.

Table 2 summarized the recently developed integrated optogenetic implants. Theintegrated optogenetic implants that made direct interaction with brain tissuesbecome possible. Compared to the discrete optogenetic implants, the integrated

Layer #1

Layer #2

Layer #3

Layer #4

releasablebase

200 µm

200 µm

microelectrode

temperaturesensor

injection microneedle

multifunctional, integrated system

µ-IPD

µ-ILEDs

Fig. 7 Multilayermultimodal integratedoptogenetic implant using aflexible resolvable base (Kimet al. 2013). (This figure isreprinted with permission)

Optogenetic Implants 17

design got rid of the external (bulky) waveguide structures so as to reduce the overallsize of the system and also can potentially improve the efficiency of optical emis-sion. Moreover, the integrated optogenetic implants would facilitate the multi-site/multilayer stimulation. Despite the success of these integrated implants, there arestill some challenges that need to be overcome. To integrate neural recordingcircuitry into the implant for closed-loop applications is a trend that can achievemuch better system performance (Zhou et al. 2019). To ensure relatively high-intensity optical stimulation is another concern. Besides this, suitable thermal man-agement for this type of integrated designs (directly embedded emitters on) is vital.More importantly, the integrity and operation stability of the implants during andafter implantation are of great concern.

In the recent years, a different approach to develop integrated optogenetic implantis to use commercially available microchip CMOS design process. During 2014–2018, Zhao et al. (2014, 2015, 2017, 2018) proposed different versions (both open-loop and closed-loop) of microchip-based optogenetic implants, as shown in Fig. 8.In these microchip designs, multi-site and multilayer stimulation, multiple function-alities (optogenetic stimulation, electrical recording, and thermal sensing), high lightintensity, and miniaturized profile have been all achieved. Moreover, intelligentelectronics have been developed to realize active control (of stimulation/recordingsites individually), two-way communication, and power management. Sophisticatedlogic control system, sensitive data converters, light drivers, recording front end, andsensing circuitry have all been implemented in the chips to act as an intelligentstandalone platform. This microchip-based design approach for optogeneticimplants may provide a possibility toward the development of new-generationintegrated intelligent multimodal optogenetic implants. Details of this type ofoptogenetic implants can be found in the next section “Design Example: HUBINOptrode – A Microchip-Based Optogenetic Implant”.

Design Example: HUBIN Optrode – A Microchip-BasedOptogenetic Implant

Open-Loop HUBIN Optrode

The HUBIN optrodes (optrode: optical probe) named from the first name of theleading developer Dr. Hubin Zhao, which include both open-loop and closed-loopversions (Fig. 8a, b). In the first-introduced open-loop design (Zhao et al. 2014,2018), there are 2 main chunks of the implant: the head section of the optical probewhich includes all active control circuits and the probe shaft (4.4 mm) to maintain all18 stimulation sites. The length of probe shaft (4.4 mm) matches the thickness of thebrain cortex, and each of the three stimulation sites (1 main site and 2 backup sites) isclassified into a stimulation cluster to achieve six stimulation clusters that match withthe number of cortex layers. Thus, multi-site multilayer stimulation can be readilyrealized.

18 H. Zhao

Table

2Listof

recently

publishedintegrated

optogenetic

implants.(Thistableismod

ified

with

perm

ission

from

Zhao20

17)

Ref./Y

ear

Dim

ension

s

No.

ofSti

sites

Light

intensity

Max

power

Con

trol

electron

ics

Diagn

ostic

sensing

Therm

alincrem

ent

Therm

alsensing

Electrical

recording

Sub

strate

material

Cao

etal./

2013

Shaft,1

2mm;width

(W),90

0μm

;μL

ED,

1000

�60

0�

200μm

3

10.7mW/

mm

2Pow

er:

14.5

mW

External

instruments

No

–No

Yes

Polyimide

Fan

etal./

2014

Length(L),4.2mm;W,

0.86

mm;μL

ED,

550�

600�

200μm

3

10.95

mW/

mm

2Pow

er:

>21

6mW

–No

0.5

� Cincrease

with

7mW

power

and

2.74

Vinpu

tvo

ltage

No

No

SU-8

Fan

etal./

2016

Shank

L,5

mm;W,

0.9mm;μL

ED,

550�

600�

200μm

3

11.5mW/

mm

2Voltage:

3.6V

External

instruments

No

1� C

increase

with

3.6Vinpu

tvo

ltage

No

Yes

Polycrystallin

ediam

ond

McA

linden

etal./2

013

L,7

mm;shaftL,

1mm;W,8

0μm

;μL

ED,4

0μm

diam

eter

560

0mW/

mm

2–

External

instruments

No

1.5

� Cincrease

with

600mW/

mm

2and20

0ms

pulse

No

No

Sapph

ire

Scharf

etal./2

016

TotalL,3

mm;shaftL,

750μm

;μL

ED,2

5μm

diam

eter

1640

0mW/

mm

2Current:

5mA

External

PCB

control

boards

No

0.5

� Cincrease

with

150mW/

mm

2radiance

50mspu

lse;

max:4

� C

No

No

Silicon

Wuetal./

2015

Shank

L,5

mm;W,

70μm

;μL

ED,

11�

13μm

2

3�

435

3mW/

mm

2Current:

5mA

External

PCB

control

boards

No

<1.0

� Cincrease

with

3.4Vvo

ltage

No

Yes

Silicon

Kim

etal./

2013

ShaftL,1

mm;W,

~400

μm;thickn

ess,

~20μm

;LED

4~4

0mW/

mm

2Pow

er:

40mW

External

flexible/

rigid

No

1.0

� Cwith

17.7

mW/m

m2

radiance

and

Yes

Yes

Platin

um,

silicon

,po

lymer

(con

tinued)

Optogenetic Implants 19

Table

2(con

tinue

d)

Ref./Y

ear

Dim

ension

s

No.

ofSti

sites

Light

intensity

Max

power

Con

trol

electron

ics

Diagn

ostic

sensing

Therm

alincrem

ent

Therm

alsensing

Electrical

recording

Sub

strate

material

dimension

s,50

�50

μm2

control

boards

10mspu

lse;

max:10

� CZhaoetal./

2018

ShaftL,4

400μm

;W,

200μm

μLED

dimension

s,20

�20

μm2

6–18

1256

mW/

mm

2Pow

er:

6.04

mW

In-built

activ

eelectron

ics

Yes

0.8

� Cwith

6mW

power

Yes

Yes

Silicon

20 H. Zhao

This implant not only achieved the functional of optical neural stimulation butalso implemented an advanced self-diagnostic sensing function. To achieve precisestimulation, each stimulation site (LED) is individually controlled by a dedicatedstimulation control block. The stimulation circuitry demonstrated high spatial reso-lution with 50–100 μs minimum optical switching time. The purpose of the diag-nostic sensing circuitry is to evaluate the integrity of the implant and long-termusability of the emitter of the implant during and after implantation. After long-timeobservations and tests, Zhao et al. (2014, 2015, 2018) found that there are two mainissues associated with the implant integrity and normal functionality of emitters:implant breakage and contact corrosion of light emitters. If the breakage occurs, anopen circuit would be formed at the south of the light emitter; if the contactcorrosions are formed, the serial resistance of the emitter would be significantly

Fig. 8 (a) System architecture of the microchip-based open-loop HUBIN optrode (Zhao et al.2018). (b) A multimodal closed-loop design HUBIN optrode (Zhao et al. 2015). (This figure isreprinted with permission)

Optogenetic Implants 21

increased. Zhao et al. further found that both abnormal scenarios can be reflected bythe voltage value at that point if the corresponding I-V profile can be characterized.

Figure 9 illustrated the circuit designs of stimulation control circuitry and diag-nostic sensing circuitry. In the stimulation circuitry, the signals of LEDON andLEDOFF determined the working status of the stimulation site. A pulse widthmodulator (PWM) was implemented to regulate the stimulation duration with highspatial resolution. A Read signal is fetched in real time to monitor the operationstatus of the specific emitter. In the diagnostic sensing circuitry, sophisticated,miniaturized digital-to-analogue converter (DAC) and ADC have been implementedto characterize corresponding I–V profiles of normal operation status and twoabnormal operation statuses (implant breakage, and contact corrosion). The dynamicrange of the ADC is >100 dB; given the input of the DAC (VSensing) to achievevoltage scanning, corresponding output of diagnostic sensing, VDia, can be corre-spondingly detected and recorded by the ADC. If the voltage profile of an opencircuit is detected, then implant breakage happens, and the implant should be entirelyswitched off and taken out by the clinician; if the recorded voltage is obviouslyhigher than the voltage of normal working status, the contact corrosion of the emittercould be existed, and the malfunctioned emitter should be shut down and bereplaced. There are 18 stimulation control blocks and diagnostic sensing blocks inthis integrated open-loop implant to individually control and diagnosis each stimu-lation site, and all of these blocks have been implemented on a microchip along withother logic control circuitry, commutation and power circuitry; a detailed chip layoutcan be found in Fig. 10.

Closed-Loop HUBIN Optrode

Building upon the open-loop HUBIN optrode, a closed-loop HUBIN optrode hasalso been proposed. In this closed-loop optogenetic implant, while maintaining thefunctionalities of optical neural stimulation and self-diagnostic sensing, concurrentelectrical neural recording and in situ thermal sensing have been further achieved(Zhao et al. 2015). Figure 8b demonstrates the overall architecture of the proposedclosed-loop HUBIN optrode, and Fig. 11 illustrates the block diagrams of thestimulation control circuitry and diagnostic sensing circuitry. In the stimulationcircuitry, an 8-bit DAC has been utilized to achieve high-resolution regulation ofthe magnitude of light intensity that the drive current of the light emitters can bemodulated in 256 levels. Combining the original pulse width modulation schemeand this new magnitude modulation method, this implant holds great programma-bility and controllability of light intensity and ensures the precision of light delivery.This will also be beneficial for suitable thermal management. Moreover, an H-Bridgehas been implemented into the stimulation circuitry so as to achieve biphasic opticalstimulation for the first time. Furthermore, the diagnostic circuitry has still been keptand integrated into the H-Bridge, which can significantly improve the operationalsafety and functioning reliability of this closed-loop optogenetic implant. Moreimportantly, electrical neural recording circuitry has been integrated into this implant

22 H. Zhao

Fig.9

Circuitdiagramsof

theop

en-loo

pHUBIN

optrod

e(Zhaoetal.201

8).(a)

Schem

aticof

astim

ulationcontrolb

lock.(b)Schem

aticof

diagno

sticsensing

block.

(c)Schem

aticof

aminiaturizedDAC.(d)Diagn

ostic

sensingcompo

nent.(Thisfigu

reisreprintedwith

perm

ission

)

Optogenetic Implants 23

so as to detect and record local field potentials (LFPs), which can provide effectiveinformation to the optical stimulator. This could improve stimulation efficiency andaccuracy and overall system performance. In addition, thermal sensors have beenimplemented on each stimulation site. This resistor-based sensor has been placedwithin the stimulation site, providing in situ real-time thermal monitoring. Thissetting can further improve the system thermal management and operation safety.

Exploration: Scalable Architecture of HUBIN Optrode

Based on the development of HUBIN optrodes, a scalable architecture has beenexplored for possible future use (Fig. 12). Owing to the merits of individual

Fig. 10 Compact chip layout of the open-loop HUBIN optrode (Zhao et al. 2018). The 18 localcontrol blocks consume 90 � 900 μm die area in total. (This figure is reprinted with permission)

24 H. Zhao

miniaturized control blocks, this type of HUBIN optrodes can be potentially scalablewith up to 49 stimulation sites evenly distributed along the 4-mm-long optrode shaft.Neural recording sites can also be readily increased. This scalable architecture hasdemonstrated the feasibility to develop an ultrahigh-density integrated optogeneticimplant, using commercially available microchip design process.

Fig. 11 (a) Block diagram of the optical stimulation circuitry. IDrive is the LED drive currentgenerated via the DAC and TCA, and it then go through the LED via P1 and N2. An H-Bridge,which is adopted, is utilized for μLED emissions. (b) Block diagram of the diagnostic sensingcircuit. It consists of DAC module, TCA module, analogue DEMUX & MUX, shared H-Bridge,ADC module, and supplementary cells (S-to-P, P-to-S, and local counter). The diagnostic sensingcircuit receives global commands from the FSM. A local counter is utilized for the timing control ofthe ADC readout

Optogenetic Implants 25

Conclusion

In the recent years, significant progress toward the development of new-generationoptogenetic implants has been made. An “ideal” optogenetic implant should holdminiaturized profile, intelligent electronics, multilayer stimulation sites, and multi-modal functionalities. Driven by microchip, AI, and advanced material technologies,more explorations of new-generation optogenetic implants (such as the IntegraBrainProject) will be firmly conducted in the next 5 to 10 years.

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28 H. Zhao