Multiplexed Optogenetics with Striped Organic LEDs

Optogenetic stimulation of living systems enables control of neuronal functions with extraordinary cell‐type specificity. The expression of more than one optogenetic actuator grants the possibility to mediate cellular activity via activation and inhibition upon illumination, with a spatial resolution that is hardly matched by electrical and pharmacological paradigms. In addition, delivering light with adequate spatial resolution is of utmost importance to achieve precise control over targeted cells. To this aim, the design strategy and realization of micro‐structured dual‐color organic light‐emitting diodes (OLEDs) are presented with a high degree of light confinement, reaching optical power densities up to 1 mW mm−2, microsecond response speed, and device heat‐up below 3 °C upon constant drive conditions at high brightness. The devices are applied for localized stimulation of Drosophila melanogaster (D. melanogaster) larvae expressing BiPOLES as a bidirectional light‐sensitive actuator. The results suggest the presence of an anterior‐posterior hierarchy for motoneuron signal procession, which is concluded from behavioral observations upon simultaneous and timely controlled activation and inhibition of neuronal activity in different larval segments. Thus, the devices are highly effective in generating complex light patterns for multi‐color optogenetics and lay the basis for cell‐specific multiplexed optogenetics in freely moving animals.


Introduction
[3][4][5][6] The use of organic light-emitting diodes (OLEDs) has emerged as an alternative to the light sources that are traditionally adopted in the optogenetic practice, owing to the possibility of fabrication on flexible substrates, [7][8][9][10] operation at low voltage, [11][12][13] tunable optical properties, [14][15][16][17][18] patterning to cellular scale, [13,[19][20][21][22] and compatibility with neuroimaging techniques such as magnetic resonance imaging. [23]Furthermore, OLEDs can be stacked in order to provide emission of different colors while keeping the number of electrodes in the device minimal for implementation in small and dense architectures. [16,24,25][27] The broad tunability in time and intensity of multicolor OLEDs enables exploring the nonlinear nature of neuronal computation [28,29] and the different expression levels of the two opsins involved, [26] constituting a flexible strategy to either activate or inhibit different neural circuits at different locations.
Recently, we have demonstrated the use of OLEDs for stimulating different bidirectional opsins by utilizing stacked so-called AC-DC OLED architectures that emit two different colors by applying voltages of opposite polarity. [16,24,25]These results were achieved using a large single pixel that did not allow the tuning of spatial light patterns.Here, we engineered dual-color-emitting OLED stripes to lateral dimensions of 200 μm -sufficient to stimulate individual segments in D. melanogaster (segmental width of 400-600 μm [30] ) -and investigated their spatial resolution with the aid of optical simulations.
We model the angularly resolved spectral radiant intensity and simulate the spatial resolution for different device-to-target distances.Our dual-color OLED architecture exerts sub-Lambertian emission and enables optogenetic stimulation with submillimeter resolution (the intensity profile decays below 5% in a space of 320 μm for the red and 500 μm for the blue, including the pixel size), which is within the range of segmental distance of the biological target.Finally, we employ our striped dual-color OLEDs for optogenetic stimulation of D. melanogaster larvae expressing BiPOLES, a bidirectional opsin able to induce wavelengthdependent activation and inhibition of neural activity. [27]Here, the application of uni-color and bi-color stimulation to motoneurons in specific anatomical compartments of Drosophila larvae led to fine spatial and temporal control over their body contraction and relaxation.Our results unlock the possibility of multi-color, high-resolution optogenetics and may in the future be integrated into a flexible platform to form a lightweight implant for highresolution multi-color optogenetics.

Light Confinement in Dual-Color OLEDs
For OLEDs, it is common to assume Lambertian emission, according to which the angular dependency of the radiant intensity I() follows a cosine law: Although this law is often confirmed in ITO-based bottomemitting single cavity stacks, [31] it usually does not hold in top-emitting OLEDs or when multiple reflecting electrodes are incorporated in the device architecture. [24,32]Our devices are fabricated in a top-emitting configuration, but the rationale used here can be applied also to bottom-emitting devices.As described in our previous work, [25] the OLED structure is composed of two pin-OLED units with three electrodes in which the outermost electrodes are set to a common potential.To produce the two emission colors required for the stimulation of BiPOLES, [27] our devices comprise a blue fluorescent stack on top of a red phosphorescent unit, as shown in Figure 1a.
State-of-the-art encapsulation techniques for OLEDs typically feature thick cavity encapsulation glasses filled with an inert gas to protect the organic materials from moisture and oxygen.However, this strategy leads to a significant broadening of the projected image due to the angular spread of light over the large distance between the light source and specimen.In this work, we used a SiOx protection layer [33,34] on top of the OLED in order to glue the encapsulation glass with epoxy directly onto the OLED, reducing steps in refractive indices and obtaining a much thinner encapsulation system.The OLED pixel layout (Figure 1b) features eight striped pixels per substrate with a high aspect ratio (5 mm long and 200 μm wide) and a pixel pitch of 600 μm.The capability of our device to switch the emission color is shown in Video S1 (Supporting Information).In order to investigate light projected through the encapsulation glass and outcoupled to the biological target of interest, we simulated the wavelength-and angledependent glass modes produced by the device (Figure 1c,d).The red-emission profile in Figure 1c features a significant blue shift with increasing viewing angle, with peak intensity at 630 nm at an angle of 21°(Figure 1d).This behavior is not observed in the blue emitting unit, which exhibits preferential emission in the forward direction and nearly no spectral shift (460 nm at 0°).Compared to a Lambertian reference source, both units show significantly reduced light emission at higher angles.
Next, we modeled the red and blue light projected through the encapsulation (glue and glass) and outcoupled into a third-instar Drosophila larva as the biological target by taking into account the in-plane geometry of the OLED.The medium surrounding the larva is constituted by a thin layer of water, to match our experimental setup (see Experimental Section).Therefore, the simulation takes into account the change in refractive index from glass to water and from glass to larva.The results are shown in Figure 1e-j for the cases of a Lambertian light source and red and blue AC-DC subunits at distances of 1.1 mm and 130 μm, representing the cases of illumination through a 100 μm layer of epoxy glue and either a "standard" (thickness of 1 mm) or an ultra-thin (30 μm) glass encapsulation.In the 1 mm case (Figure 1e-g), all emission profiles lead to significant light intensity in between pixels, which will be inadequate for precise optical targeting.The highest pixel crosstalk in our configuration occurs at the center of the architecture, where light coming from multiple pixels adds to a global maximum intensity (Figure S1, Supporting Information).Interestingly, peak intensities occur in between consecutive pixels in the case of the red OLED (Figure 1f) due to the angular light distribution peaking at 21°and not in the forward direction (Figure 1d).
When the glass encapsulation is reduced to 30 μm (Figure 1hj), all configurations show significantly improved light confinement, with only little residual light outside the pixels in the case of the red (Figure 1i) and blue stacks (Figure 1j).Overall, the Lambertian light source (Figure 1e,h) presents the most severe cross-talk between pixels, independently from the distance of the target, while the red (Figure 1i) unit exerts the highest degree of confinement.Based on these observations, a thin-film encapsulation strategy and the sub-Lambertian nature of our devices are both beneficial for high-resolution optogenetic stimulation.Since we focus on the excitation of ventral body wall neurons that sit close to the light source, light propagation inside the animal, which undergoes absorption and scattering, has not been considered in our simulations.However, a comparison of our simulation results to experimentally derived emission profiles on top of the larvae shows that only slight broadening is expected, with blue light showing stronger broadening of the profile due to higher scattering and absorption in biological tissue [35] (Figure S2, Supporting Information).

OLED Properties
Next, we have fabricated the OLEDs incorporating a SiOx capping layer and encapsulated devices with epoxy and a 30 μm flat glass lid.Current-voltage-irradiance characteristics of our OLEDs are shown in Figure 2a.At ± 7 V operation, the red unit exerts higher current density (1272 mA cm −2 ) and irradiance (1085 μW mm −2 ) than the blue unit (622 mA cm −2 and 200 μW mm −2 ).At 5 V, the OLED reaches 349 and 53 μW mm −2 for the red and blue units, respectively.These values represent a big improvement with respect to our previous architecture, [25] in which the SiOx layer was absent, and for which we recorded irradiances of 91 μW mm −2 for the red unit and 23 μW mm −2 for the blue unit at 5 V.This might partially be due to enhanced outcoupling provided by the SiOx-epoxy capping system, which substitutes the low-refractive index air layer of standard OLED encapsulations.
The spectral properties of the OLED with emission peaks at 460 and 607 nm (Figure 2b) will provide precise targeting of BiPOLES (target wavelengths for blue and red stimulation are in the range of 460-490 nm [36] and 590-600 nm, [37] respectively), and the maximum EQEs for the two units are 1.98 %/15.7 % for the blue/red unit, respectively (Figure 2c).In Figure 2d, we show the temperature variation of a single pixel when driving the OLED periodically between positive and negative voltages for 5 s each over 50 consecutive color-switching cycles at high brightness (390 μW mm −2 for the red and 103 μW mm −2 for the blue units, respectively).As a result, the temperature on top of the glass surface eventually increased by 2.9 °C.It is worth noting that the temperature increase exerted by our striped OLEDs remains comparable to the large AC-DC-OLEDs that have been driven at a much lower irradiance of only 7 μW mm −2 for 25 cycles. [25]We attribute this improvement to the significantly reduced pixel size, which leads to enhanced heat dissipation over the substrate and lower drive voltages because losses due to sheet resistance of the transparent electrodes are reduced on the smaller area.We emphasize that at the driving conditions we investigated, the temperature increase stays below the risk of heat-induced behavioral change in D. melanogaster larvae. [38]ext, we measured the transient electroluminescence of the OLEDs to investigate the maximum switching frequency of the individual units.For this measurement, we drove the devices individually in voltage mode by using a square-wave signal with a frequency of 10 kHz and a pulse length of 50 μs and measured the optical light output (Figure 2e,f).The luminescence rise times are  blue = 0.99 ± 0.01 μs and  red = 1.69 ± 0.02 μs and fall times are  blue = 0.81 ± 0.01 μs and  red = 1.36 ± 0.02 μs for the blue and red units, respectively.The dynamics of the red units are mainly limited by the exciton lifetime of the phosphorescent emitter (photoluminescence lifetime of Ir(MDQ) 2 (acac): 1.37 μs), [39] while the dynamics of the blue fluorescent unit are mostly limited by the electrical transient properties of the system.It is worth noting that the devices allow for stimulation frequencies far beyond the limit set by the off-kinetics of BiPOLES ( off,Chrimson = 21.4 ms, [37]  off,GtACR2 = 40 ms). [36]

Targeted Optogenetic Stimulation
Next, we carried out optogenetic stimulation on BiPOLESexpressing 3rd instar Drosophila larvae by illumination of motoneurons projecting from the ventral nerve cord to anterior and posterior segments (denoted as A and P in illustrations of the larvae in Figure 3; expression patterns in Figure S3, Supporting  [36] and Chrimson [37] ).c) EQE as a function of the voltage.d) OLED temperature (orange line) and drive voltage (blue/red bars) during multiple color cycles at 0.2 Hz switching frequency.Current densities used: 276 mA cm −2 for the blue unit and 220 mA cm −2 for the red unit.e-f) Transient electroluminescence of the OLED subunits under 50 μs pulses with 10 kHz driving frequency and 50% duty cycle at 5 V (red) and −8 V (blue) with fall (e) and rise (f) times.
Information) using different spatiotemporal patterns of red and blue light.Figure 3a,b, and Videos S2 and S3 (Supporting Information) show the effect of pulsed red light on the anterior (Figure 3a) and posterior (Figure 3b) segments of the larvae.We used four pulses of 1 sec each (238 μW mm −2 ) with an intermediate black period of 1 sec.Because of the selective stimulation of the Chrimson domain in BiPOLES, the larvae show consistent total body contractions during muscle-stimulating red-light illumination and recovery during the non-stimulating black periods.It is important to note that although the stimulating paradigm is the same for the two anatomical compartments, stimulation of anterior segments results in stronger contractions (72.9% ± 0.1% of the initial length) than in posterior segments (83.5% ± 0.7% of the initial length).
In addition to the present stimulation sequence, we next introduced pulses of inhibitory blue light on the posterior (Figure 3c; Video S4, Supporting Information) and anterior (Figure 3d; Video S5, Supporting Information) segments of the larvae with a delay of 350 ms with respect to the onset of the red light pulses.The presence of a delay between excitatory and inhibitory signals ensures that initial response is partially developed in the larvae, to be later modulated according to the relative position of the inhibitory and excitatory illumination.In analogy to the previous stimulation sequences, all light pulses lasted 1 s (238 μW mm −2 of red light and 134 μW mm −2 of blue light) with a 1 s interval between consecutive pulses of the same color.
In Figure 3c, larvae first undergo optogenetic activation of their anterior body-wall muscles by illumination with red light and, hence, start contracting.With the onset of blue light, which predominantly targets the GtACR2 domain of BiPOLES and thus leads to inhibition of neuronal activity, the strength of the contraction becomes reduced compared to the case shown in Figure 3a (81.3% ± 0.6% of the initial length).The overall shape of the contraction and relaxation, however, is similar to the case without inhibition.In order to further analyze the larval response upon delayed inhibition in posterior segments, Figure S4a-c (Supporting Information) shows a direct comparison of this experiment with the one of anterior red illumination.In particular, Figure S4b,c (Supporting Information) compares the head and tail speeds following muscle contractions and relaxations of the larvae undergoing the two stimulation paradigms.While there is no particular difference in the movement of the head, the larvae are able to move the tail freely when only red light is used, but stop tail movement at the onset of the inhibitory blue light and are only able to move again when the red illumination is released.
Subsequently, we inverted the locations for activation and time-delayed inhibition (see Figure 3d).As in our previous experiments, the larvae start contracting when red light is present at posterior segments, but the contraction is stopped at 89.6% ± 0.9% of the initial length as soon as blue light is shone onto anterior segments and subsequently held to a minor extent (92.8% ± 1.1% of the initial length) until the red light goes off and the larvae can release the residual contraction.In contrast to any of the previous experiments, the contraction and relaxation strength and dynamics, in this case, are highly modulated by the presence of blue light in anterior segments.The direct influence of the anterior blue stimulation is further shown in Figure S4d-f (Supporting Information) in direct comparison to only posterior stimulation with red light.Upon red light stimulation of the posterior segment, the larvae display consistent spikes in head and tail speeds (Figure S4e,f, Supporting Information).At the onset of blue light, the anterior part of the larvae experiences a push to a more relaxed state, and the whole body elongates accordingly (Video S5, Supporting Information).This effect is translated into spikes in the head and tail speed followed by paralysis until the red light is turned off.Afterward, the larvae release the residual contraction and fully recover the ability to move when the blue light is turned off.The statistical significance of the individual stimulation sequences supports all previously discussed observations (Figure S5a,b,d,e and Table S1, Supporting Information).To the best of our knowledge, although BiPOLES has been used for optogenetic control of D. melanogaster before, this is the first time that localized bidirectional modulation is achieved in a small animal model.In the following, we fully exploit the ability of AC-DC OLEDs by alternating between activation and inhibition of neurons in the same compartment using bi-color light emission from the same pixel (Figure 4; Videos S6 and S7, Supporting Information).In particular, the experiments involve the simultaneous use of excitatory red and inhibitory blue light in different anatomical regions with subsequent switching of the stimulation color after 0.5 s. Figure 4a,c,e shows the length, head, and tail speeds of larvae undergoing first blue stimulation of the anterior body and simultaneous red stimulation of the posterior body, before switching to red stimulation of the upper body and simultaneous blue stimulation of the lower body.In the first part of the cycle, the larvae are strongly hindered from contracting (Figure 4a, p = 0.04 (Figure S5c and Table S1, Supporting Information)), with heads and tails barely moving (Figure 4c,e), thanks to the intervention of blue light during red stimulation.When the colors are switched, larvae contract (80.9% ± 0.8% of the initial length) as demonstrated by the spikes in head and tail speeds (Figure 4c,e), and eventually release the contraction when the OLED is turned off.
In the protocol shown in Figure 4b,d,f, red stimulation of the upper body and simultaneous blue stimulation of the lower body is followed by blue stimulation of the upper body and simultaneous red stimulation of the lower body.This time, the contraction occurs in the first part of the cycle, since the red anterior stimulation is dominating the behavioral changes (81.2% ± 1.1% of the initial length).However, in contrast to the sole activation of anterior segments (Figure 3a), the extent of the contraction is here limited by the presence of the blue light, as was also observed in Figure 3c for delayed inhibition.In the first part of the cycle, both head and tail retract, as visible from the peaks in their speeds, and release when the second half of the cycle begins (Figure 4d,f), allowing the larvae to relax again.Statistical analysis of changes in larval length during illumination supports the discussed observations (Figure S5c,f and Table S1, Supporting Information).No optogenetic response was found in control experiments on whiteeyed w 1118 larvae, a mutant line of Drosophila not expressing the light-sensitive actuator (Figure S6 and Table S2, Supporting Information).
In all our stimulation sequences, anterior stimulation (both activation and inhibition) dominated the behavioral response as compared to stimulation in posterior segments.This was observed for both activations with delayed inhibition (Figure 3c,d) as well as simultaneous stimulation of anterior and posterior segments (Figure 4).We suggest that our observations are caused by the anatomy of the larval locomotor system, where motoneurons extend from the ventral nerve cord into the individual segments that regulate local muscular activity [40][41][42][43] .Hence, optical stimulation leads to the activation or inhibition of all motoneurons posterior to the stimulated segment.

Conclusion
We fabricated 200 μm-wide, dual-color organic LEDs and modeled the light confinement in between pixels to elicit spatially resolved bidirectional stimulation in small anatomical compartments of D. melanogaster larvae.Our AC-DC OLEDs benefit from the presence of stacked optical microcavities, which lead to forward-shaped emission.As a result, the light sources provide enhanced lateral resolution for optogenetics at the microscale.Furthermore, the device performance was significantly improved compared to our previous large-area devices, [25] which is attributed to improved current conduction over the smaller pixel areas and reduced device heat-up and furthermore supported by improved light outcoupling.
Our OLED was carefully designed to allow light delivery to specific segments of Drosophila larvae and to selectively activate and inhibit motoneurons in those regions.In particular, we modulated initiated neuronal activity by complementing the activation with delayed inhibition on separate anatomical compartments.Furthermore, we explored the effect of competing activation and inhibition by simultaneous red and blue illumination of distant segments of larvae.From this, we confirmed the presence of an anterior-posterior hierarchy in the occurrence of behaviors since the effects induced from light shone in anterior segments dominate over stimulation in posterior segments.
In conclusion, we show that finely patterned OLED light sources can provide segment-specific modulation of neuronal activity in living Drosophila larvae and thus enable simultaneous multi-site activation and inhibition as well as switching between the two modes without the need for complex optical elements.In the future, our devices could be used to tune the activity in dense neural networks, including one of neuromodulatory neurons, for example, to study their role in mechano-nociceptive behavior [44] or in the olfactory system. [45]Furthermore, the electrodes of our OLEDs could be adapted to form a three-terminal device that allows simultaneous driving of blue and red sub-units.This would be interesting for independent optogenetic activation of two distinct neuronal populations, which is possible by combining the red light-sensitive BiPOLES with a second, blue lightsensitive cation channel. [27]More generally, our platform enables spatially resolved, multiplexed optogenetics by harnessing the possibility to tailor OLEDs in color, shape, size, and emission profile.Furthermore, a combination of dual-color micro-structured OLEDs with flexible substrates could unlock the possibility of applying them as lightweight implants for larger models, where cell-specific stimulation can be used to locally and dynamically control sensory or motor neural pathways.

Experimental Section
OLED Simulation, Fabrication, and Characterization: Optical simulations of the spectral radiant intensity emitted into the encapsulation glass were carried out using transfer matrix calculations, [46] and spatial light distributions were calculated using a MATLAB routine developed in-house.The MATLAB script makes use of the geometrical layout of the device and the spectral radiant intensity of each subunit to compute the light pattern that was projected into a horizontal plane sitting at a distance from the OLED.The region of interest (ROI) corresponding to the whole substrate area (25 mm × 25 mm) was divided into a mesh with units of 0.01 mm × 0.01 mm, for which the projected light intensity was calculated for different distances between the OLED and the biological target.In order to take a jump in the refractive index at the outcoupling plane from glass into water and the Drosophila larvae into account, Snell's law and Fresnel coefficients were applied.The refractive index of Drosophila was set to 1.4, in agreement with the refractive index of similar species. [47]ll OLEDs were fabricated by thermal evaporation in a vacuum tool (Kurt J. Lesker Co.; base pressure of 4 × 10 −8 mbar) on pre-cleaned glass substrates.Thickness and deposition rates of the organic materials were measured via quartz crystal monitors and doping was assured by co-evaporation.A 2-methyl-9,10-bis(naphthalen-2-yl)anthracene (MADN) matrix doped with 1.5 wt.% 2,5,8,11-tetra-tert-butylperylene (TBPe) was used as blue emitter system.N,N'-di(naphthalene-1-yl)-N,N'-diphenylbenzidine (-NPD) was doped with 10 wt.% iridium(III)bis(2-methyldibenzo-[f,h]chinoxaline)(acetylacetonate) (Ir(MDQ) 2 (acac)) to obtain the red emission layer.2,2′,7,7′-tetrakis-(N,N-di-methylphenylamino)−9,9′-spirobifluorene (Spiro-TTB) doped with 4 wt.%2,2′-(perfluoronaphthalene-2,6-diylidene)-dimalononitrile (F6-TCNNQ) was used as HTL, whilst 4,7-diphenyl-1,10-phenanthroline (BPhen) doped with Cesium (Cs) was chosen as ETL.BPhen and -NPD were used as hole-blocking and electron-blocking layers (HBL/EBL), respectively.The transparent electrodes consisted of 15 nm of silver (Ag) evaporated on 2 nm of gold (Au) to reduce sheet resistance and improve transmittance.The reflective back electrode consisted of 35 nm of Ag evaporated on top of 35 nm of aluminum (Al).All samples were covered by 50 nm of thermally evaporated SiO x as a capping layer and then encapsulated under Nitrogen atmosphere with 30 μm flat glass lids (SCHOTT, D 263 T) and epoxy resin (Norland NOA68) directly after fabrication, before use in ambient conditions.The thickness of the epoxy was measured with a Vernier caliper to be 100 μm after curing (averaged over ten samples).All devices had pixels with active areas of measured 1.02 mm 2 (nominally, 5 mm × 0.2 mm).
Current-voltage-Irradiance characteristics (J-V-I) were measured using a source measure unit (SMU2450, Keithley Instruments) whereas the irradiance was recorded with a calibrated Si photodiode (Thorlabs).EQE and spectra of the OLEDs were obtained in an integrating sphere (LMS-100, Labsphere Inc.) equipped with a calibrated spectrometer (CDS-600, Labsphere Inc.).The frequency response experiment was carried out by using a signal generator (Keysight 33210A) with which a pulse train of five (red light) or eight Vpp (blue light) at 10 kHz was exerted across the OLED.A series resistor of 50 Ω was used to determine the current in the circuit.A photodiode (SM1A6T, Thorlabs) and a current amplifier (DLPCA-200, FEMTO) were used to measure the irradiance.Data was collected by using an oscilloscope (HMO3004, Rohde & Schwarz).Device temperature was tracked every 1 s with an infrared thermometer (Optris CT laser LT) and data was smoothened over five adjacent points to reduce noise.
Drosophila Rearing and Optogenetic Stimulation: The following fly strains were used: OK371-GAL4, [48] UAS-BiPOLES [27] (gift from Peter Soba), UAS-CsChrimson (tagged with mVenus, BDSC#55136), CS (Canton special), and w 1118 [49] , and all were raised in the dark at 25 °C on a conventional cornmeal-agar medium supplemented with 0.5 mm all-transretinal (ATR).All optogenetic measurements with Drosophila larvae were recorded underneath a stereo microscope (Nikon SMZ25, P2-SHR Plan Apo 0.5×/0.075NA objective) with an sCMOS camera (Andor Zyla 4.2 PLUS).A 695 nm long-pass filter was mounted in front of the camera to avoid overexposure due to OLED illumination.Using this filter, only a small portion of light from the red OLED was detected by the camera.Larvae were imaged under infrared light using an LED bar light source (LU-MIMAX LSB-Series; 850 nm peak).Videos were acquired by the sCMOS camera using Nikon NIS-Elements software at a frame rate of 25 Hz.For optogenetics and control experiments, third instar larvae were taken out of the vials in dim green light, gently washed in DI water, and confined in an agarose channel (1.5% w/v in DI water) located on top of the OLED, which was driven in current mode.To prevent larvae from escaping from the channel, water was added at the two ends of the agarose channel to surround the larvae.Before starting optogenetic experiments, the animals got accustomed to their new environment in the dark.Larval tracking of the head, tail, and middle point was performed in every frame in ImageJ using the plugin MTrackJ.Larval length was calculated as the sum of the distances from head to midpoint and midpoint to tail.Normalization of the length was performed with respect to the mean of the first black period.Statistics and significance were calculated via pair sample t-test using Origin Pro.
Imaging of 3rd instar larval brain explants as well as in vivo imaging was conducted in ice-cold, Ca 2+ and Mg 2+ -free Rinaldini's solution [50] using an inverted microscope (Zeiss Axio Observer Z1, Germany) with a 10× objective connected to a Visitron Systems LEj light source (Visitron Systems GmbH, Germany) and a Photometrics Evolve 512 EMCCD camera (Teledyne Photometrics, USA).Fluorescence signals were obtained utilizing a 450-490 nm bandpass excitation and 515 nm long pass emission filter.Images were acquired using VisiView 2.1.4(Visitron Systems GmbH, Germany).

Figure 1 .
Figure 1.Optical properties of the dual-color micro-stripes.a) Layer structure of the OLEDs (see Experimental Section for full chemical names of materials) and b) photograph of a fabricated device.c) Spectral radiant intensity of the glass modes in the AC-DC OLEDs and d) angular dependency of the radiation.e-j) Simulated light confinement of OLED emission in the plane of the biological target (here a Drosophila larva).Normalized intensity of a patterned Lambertian emitter (e,h), of the red OLED subunit (f,i), and of the blue OLED subunit (g,j) for 1 mm (e-g) and 30 μm (h-j) thick glass encapsulation.Dashed lines: outline of the pixels and the biological target.

Figure 2 .
Figure 2. Optoelectronic properties of the organic micro-stripes.a) JV (solid) and irradiance-voltage curves (dotted) of the devices.b) Integrated emission spectra of the OLEDs matching the action spectra of BiPOLES (GtACR2[36] and Chrimson[37] ).c) EQE as a function of the voltage.d) OLED temperature (orange line) and drive voltage (blue/red bars) during multiple color cycles at 0.2 Hz switching frequency.Current densities used: 276 mA cm −2 for the blue unit and 220 mA cm −2 for the red unit.e-f) Transient electroluminescence of the OLED subunits under 50 μs pulses with 10 kHz driving frequency and 50% duty cycle at 5 V (red) and −8 V (blue) with fall (e) and rise (f) times.

Figure 3 .
Figure 3. Activation and delayed inhibition of motoneurons in Drosophila larvae using dual-color micro-stripes.a,b) Normalized length of the larvae under repeated 1 s-long pulses of red light (238 μW mm −2 ) in the anterior (a) and posterior (b) segments.c,d) Normalized length of the larvae under pulses of red light (238 μW mm −2 ) in the anterior (c)/posterior (d) segments followed by delayed pulses of blue light (134 μW mm −2 ) in the posterior (c)/anterior (d) segments.Sketches of the larvae illustrate the location and timing of the pulses; A: anterior; P: posterior.Line: mean; shaded area: standard error of the mean (s.e.m.).N = number of larvae.

Figure 4 .
Figure 4. Multiplexed stimulation of the larvae under repeated 0.5 s-long switching cycles of red (238 μW mm −2 ) and blue (134 μW mm −2 ) pulses of light applied simultaneously at anterior and posterior segments of the larvae.Drosophila larvae were initially stimulated with blue light in the anterior (a,c,e)/posterior (b,d,f) and red light in the posterior (a,c,e)/anterior (b,d,f) segments before switching to the opposite color.a,b) Normalized length, c,d) head speed, and e,f) tail speed.Sketches of the larvae illustrate the location and timing of the pulses; A: anterior; P: posterior.Line: mean; shaded area: s.e.m.N = number of larvae.