Active pixel power control for crosstalk-free all-optical neural interrogation

Domain: physics
Published: 2026-02-11
Editorial outcome: accept

Abstract
All-optical interrogation, based on high-resolution two-photon stimulation and imaging, has emerged as a potentially transformative approach in neuroscience, allowing for the simultaneous precise manipulation and monitoring of neuronal activity across various model organisms. However, the unintended excitation of light-gated ion channels such as channelrhodopsin (ChR) during two-photon calcium imaging with genetically encoded calcium indicators (GECIs) introduces artifactual neuronal perturbation and contaminates neural activity measurements. In this study, we propose an active pixel power control (APPC) approach, which dynamically adjusts the imaging laser power at each scanning pixel, to address the challenge. We aim to achieve simultaneous two-photon optogenetic manipulation and calcium imaging with a single femtosecond laser, while minimizing the cross-talk between manipulation and imaging. To study this technology’s capabilities, we applied it to the larval zebrafish brain in vivo. Our results demonstrate that the APPC approach preserves GECI signal quality while suppressing optogenetic artifacts significantly. This enhances the accuracy of neural circuit dissection and advances the precision of all-optical interrogation, offering a robust framework for probing neural circuit dynamics and causality in vivo with high fidelity, potentially across various model organisms. Importantly, this technology can be seamlessly integrated with commonly used two-photon microscope systems in laboratories worldwide.

Introduction
All-optical interrogation of neuronal circuits represents a paradigm-shifting advancement in neuroscience, successfully applied to various model organisms, including Mus musculus (mice)1–7, Danio rerio (zebrafish)8,9, Drosophila melanogaster (fruit flies)10, and Caenorhabditis elegans (nematodes)11,12. This technique integrates genetically encoded calcium indicators (GECIs) with optogenetic actuators, such as channelrhodopsin (ChR), facilitating non-invasive, in vivo recording and manipulation of neuronal activity. Recent advancements in two-photon microscopy and holographic optogenetics enable neural ensemble recording and optogenetic control at single-cell resolution and sub-millisecond timescales2,5–7,13. Furthermore, closed-loop systems allow real-time extraction of neural activity to guide precise perturbations such as spike rates, timing, and synchrony3,14,15. These capabilities enable targeting of individual neurons based on functional signatures and advance mechanistic studies of circuit connectivity, plasticity, and disease-associated aberrant activity in conditions such as Alzheimer’s disease16,17 and Parkinson’s disease18,19. By elucidating neural circuit dynamics under targeted perturbation, this approach goes beyond merely correlating activity with behavior or cognitive states, establishing causal links; for example, it has been used to demonstrate how emotional states are encoded in neural attractor networks6,20–24. However, a major challenge emerges when a two-photon light source serves dual roles as both reader and stimulator in all-optical interrogation systems: cross-talk between these modules compromises experimental precision, and must be effectively minimized2,5,13,25.

Generally, there are three primary mechanisms underlying the cross-talk between calcium imaging and optogenetic stimulation5,25,26. Type I cross-talk refers to the contamination of calcium signals due to additional GECI signals when the optogenetic stimulation laser excites the GECI. Type II cross-talk involves unwanted excitation of ChR by the imaging laser. Lastly, Type III cross-talk occurs when densely packed neurons, with both somas and dendrites expressing ChR, lead to off-target activation during optogenetic stimulation. Strategies such as image-processing algorithms and time-multiplexed excitation to reduce calcium signal distortion9,27,28 in Type I cross-talk and soma-localized opsin variants to restrict ChR expression to soma membranes5,25 to mitigate Type III cross-talk, but Type II cross-talk remains unresolved. The Type II cross-talk issue is less pronounced in single-photon systems due to GCaMP’s superior light absorption, compared to those of ChRs29–31. However, in two-photon systems, cross-talk is exacerbated: ChR2, the most commonly used light gated ion channel protein-exhibits a 10-fold higher two-photon absorption cross-section, which linearly related to the absorption efficiency, than GCaMP32,33, the most widely used GECI. This indicates that ChR2 has tens of times stronger absorption and enables ChR2 to be activated at <10% of the laser power required for GCaMP imaging34.

Efforts to reduce Type II cross-talk via spectral separation by using red-shifted GECIs32,35–40 or ChRs6,41–43 have achieved substantial achievements. While pairing red GECIs with blue ChRs at 1100 nm can eliminate cross-talk25,44, current red indicators suffer from limited dynamic range and signal-to-noise ratios32,35–39, limiting their broader application. Red-shifted ChRs shift their absorption spectra toward longer wavelengths, reducing activation by imaging light. For example, ChrimsonR exhibits peak two-photon excitation efficiency at 1140 nm, compared to 940 nm for ChR25,45. Combining GCaMP with red-shifted ChRs has been shown to suppress cross-talk2,4,5,7,46. However, imaging conditions must be carefully optimized, as spectral overlap can occur due to broadened absorption profiles of red ChRs towards shorter wavelength. This broadening may result from the complexity of local environment for chromophore, and several distinguish mechanisms in the two-photon excitation such as the enhancement of certain vibronic transitions47–53. Consequently, Type II cross-talk persists even with the use of current red ChRs.

To address the Type II cross-talk, we developed a dynamic imaging power control strategy that actively and precisely modulates excitation power at each scanning pixel using a fast acousto-optic modulator (AOM). By reducing imaging power at designated pixels, we minimized cross-talk while preserving calcium signal extraction efficiency. Our method utilized ChR expression maps generated by standard fluorescent labeling to create an “Active Pixel Power Control” (APPC) scheme that selectively attenuates or blocks laser intensity at ChR+ pixels during two-photon calcium imaging. Specifically, synchronized with resonant scanning, the AOM dynamically adjusted power in real time, minimizing unintended ChR activation while maintaining calcium signal fidelity through spatial summation of pixel fluorescence intensities.

We validated this approach in the larval zebrafish, a vertebrate model ideal for whole-brain imaging and optogenetics, using a single femtosecond laser to combine two-photon calcium imaging (via GCaMP) with holographic optogenetic stimulation via spatial light modulator (SLM). Calcium signals were deconvolved to estimate spike rates3,54, enabling cross-talk quantification through brain-wide activity patterns. The APPC technology achieved nearly cross-talk-free all-optical interrogation without relying on protein engineering or spectral separation, while enabling precise, high-speed in vivo calcium imaging. By controlling laser power at single-pixel resolution, this approach preserved signal quality while offering a scalable and practical solution for circuit-level studies in behaving animals. This advance opens new avenues for probing causal relationships in brain wide dynamics with higher accuracy and creates opportunities for related research in laboratories equipped with standard single laser two photon microscopes.

Results
All-optical interrogation system with precise pixel-specific excitation power control
To achieve active pixel power control (APPC), which provides dynamic, pixel-specific control of imaging laser power based on ChR expression patterns to minimize cross-talk, we developed a two-photon microscope system with a single excitation source and a custom beam control module (Fig. 1a and Fig. S1). The excitation source is a Ti:Sapphire femtosecond laser (Chameleon Ultra II, Coherent) that can be tuned to wavelengths optimal for imaging both GCaMP and the red fluorescent protein fused to the channelrhodopsin. A polarized beam splitter separates the laser beam into independent imaging and optogenetic stimulation paths. In the imaging path, an electrically tunable lens (ETL) with resonant scanners enables high-speed volumetric imaging, capturing a 10-plane brain volume (400×400×120 µm³) at 3 Hz.

Next, we focus on the selection of an optical modulator for power control. The AOM modulates light power by applying sound waves to an AOM crystal, causing the light to be diffracted. The bandwidth of the AOM is limited by both the beam diameter and the acoustic velocity in the crystal. To enhance bandwidth, we reduce the beam diameter to 0.5 mm. The AOM exhibits a rise time (τ) of 85 ns, corresponding to an 11.8 MHz bandwidth, which is compatible with a pixel dwell time of 180 ns (5.6 MHz bandwidth). This configuration allows for single-pixel resolution modulation. The Electro-Optic Modulator (EOM) is also widely applied to control laser power by rotating light polarization and responds quickly to electric fields. However, its bandwidth is limited by the control signal, and larger EOMs require high-voltage amplifiers, which typically have limited gain bandwidth (≤1 MHz). Given this limitation, we have opted for the AOM for rapid control of laser power. The AOM is synchronized with the resonant scanner to dynamically regulate the excitation power, while a pair of dispersion prisms compensates for the wavelength-dependent dispersion induced by the AOM. Using the 0th-order beam (5–100% power range), we calibrated the relationship between AOM driver voltage and output power (Fig. 1b). We then confirm the second-order relationship of excitation power and fluorescence intensity, which has a maximum excitation contrast ratio of ~400:1 (Fig. 1c). After adjusting the time delay (see Methods and Fig. S2), we applied APPC imaging to a uniform fluorescent plate, demonstrating robust continuous control of excitation power at designated pixels (Fig. 1d). Next, we tested the APPC on larval zebrafish and verified its ability to efficiently turn off the two-photon excitation of specific target neurons (Fig. 1e, f). When applied to live, large-volume brain imaging in larval zebrafish, the AOM turned off the scanning laser in the eye region during functional imaging sessions (Fig. S3a), effectively mitigating laser-induced agitation (Fig. S3b-d).

In the optogenetics stimulation path, we programmed the SLM to generate scanning spot arrays targeting neuronal soma. Co-calibration of imaging and stimulation modules using fluorescent plates ensured spatial precision (see Methods).

Cross-talk between imaging and stimulation for optogenetic targeted neurons
We first assessed the cross-talk of ChR2, the most widely used optogenetic tool across various animal models, that has largely overlapped absorption spectrum with GCaMP6s. This stringent condition enabled us to validate that the proposed APPC method can effectively minimize cross-talk. Our strategy was to perform multi-plane volumetric imaging (3 Hz) under standard zebrafish imaging conditions9,55 while applying APPC to adjust laser intensity specifically in ChR2+ pretectal neurons. Next, we analyzed the calcium trace and evaluated pretectal neuron activity at different laser powers for the following APPC pattern updating (Fig. 2a). In this study, we utilized a transgenic fish line: Tg(KalTAu508; UAS-ChR2(H134R)-mCherry; UAS-GCaMP6s)9,56,57, in which the Gal4 variant protein KalTA4 drives expression of both ChR2-mCherry and GCaMP6s in specific neuron populations. Structural imaging confirmed colocalization of green (GCaMP6s) and red (mCherry-tagged ChR2) fluorescence in KalTA4u508+ pretectal neurons (Fig. 2b).

For calcium imaging, the laser power on pretectal ChR2+ neurons was set to 5 mW, 10 mW, and 15 mW by APPC, the commonly used imaging power for zebrafish9. As the imaging power decreases, the signal-to-noise ratio (SNR) of calcium traces become lower while the ΔF/F remain the same (Fig. S4). We determined that 5 mW was the minimum laser power required to maintain an acceptable SNR for measuring calcium traces (ΔF/F) in individual ChR2+ neurons (Fig. S4)54. Next, we performed three trials (100 s each) for every power condition, resulting in nine trials presented in a randomized sequence. Calcium traces (ΔF/F) from each neuron were normalized to their corresponding 15 mW trials (see Methods). Representative traces are shown in Fig. 2c. Our results demonstrated that the normalized mean ΔF/F decreased with reduced imaging power, indicating diminished neuronal activity caused by cross-talk (Fig. 2d). Specifically, the mean ΔF/F value at 15 mW excitation is more than double that at 5 mW. To further quantify neuronal activity, we applied the CNMF algorithm to deconvolve calcium traces and estimate “spike values”, which approximates spiking frequency54 (see Methods and Fig. S5). Compared to 15 mW excitation, neuronal activity (spike values) was significantly reduced at 5 mW and 10 mW excitation (Fig. 2g). On the contrary, in control experiments using ChR2- larvae (Tg(KalTA4u508; UAS-GCaMP6s)), neither mean ΔF/F nor spike values in KalTA4u508+ neurons varied across three excitation power conditions (Fig. 2f, 2i). Notably, at 5 mW excitation, both metrics were statistically indistinguishable between ChR2+ and ChR- neurons (Fig. S6), suggesting the absence of detectable cross-talk at low excitation power. This finding is crucial for the effectiveness of the APPC method, as it selectively attenuates laser intensity in ChR2+ neurons to minimize cross-talk while maintaining the optogenetically stimulated calcium signal from ChR2+ neurons at an acceptable SNR.

ChrimsonR, a widely used red-shifted channelrhodopsin, is favored due to the separation of its single-photon excitation spectrum from that of GCaMP58. To test whether two-photon calcium imaging at 920 nm excitation would induce significant cross-talk in ChrimsonR expressing neurons, we conducted a similar experiment in the Tg(KalTA4u508; UAS-GCaMP6s; UAS-ChrimsonR-mKate2) larvae, using APPC to control the imaging power on ChrimsonR+ neurons58. The results showed that despite spectral separation of single-photon excitation, neurons expressing ChrimsonR exhibited similar behaviors of cross-talk as ChR2 for two-photon imaging: the reduced artefactual neuronal activity was observed under low-power excitation (Fig. 2e). Spike values at 5 mW decreased significantly compared to 15 mW and 10 mW (Fig. 2h), indicating the presence of residual cross-talk at high excitation power, which likely arises from ChrimsonR’s strong two-photon excitation at 920 nm (Fig. S7). Again, both mean ΔF/F and spike values did not differ significantly between ChrimsonR+ and ChR- neurons at 5 mW illumination (Fig. S6). Taken together, these results indicate that localized low-power imaging effectively mitigates cross-talk. It is important to note that ChrimsonR was fused with mKate2, which exhibit higher fluorescence intensity than the mCherry marker fused to ChR251, thus complicating direct comparisons of channel expression levels based solely on fluorescent protein signals. A plausible explanation for the unexpectedly high cross-talk with ChrimsonR may be attributed to the elevated membrane expression of ChrimsonR, combined with sufficient two-photon excitation efficiency at 920 nm, collectively leading to the substantial cross-talk observed in our ChrimsonR-expressing neurons5.

In summary, we determined that 5 mW is a nearly cross-talk-free power for both ChR2 and ChrimsonR under our experimental conditions. The calcium traces of ChR+ neurons could be reliably segmented due to their strong responses to optogenetic stimulation (Fig. S4). We validated our holographic optogenetic stimulation on multiple neurons (Fig. S8) and found that downstream neurons typically exhibit significantly weaker or distinct responses compared to upstream neurons (Fig. S9). Under low signal-to-noise ratio (SNR) conditions, downstream neurons with weak responses cannot be reliably identified or analyzed using the CNMF algorithm54 (Fig. S10). Therefore, it is essential to set the laser power in ChR- regions to 15 mW to ensure the quality of calcium signal data, which is crucial for accurately dissecting neural circuits.

Interrogation of neural circuits with minimized cross-talk via active pixel power control
We first investigated how cross-talk at the ChR+ neurons affects their downstream neurons. To identify these downstream neurons, we applied two-photon holographic optogenetic stimulation targeted at pretectal u508+ neurons while simultaneously performing volumetric whole-brain imaging. Concurrently, we utilized APPC on the target u508+ neurons to control laser power for subsequent cross-talk evaluation (Fig. 3a). In these experiments, we systematically reduced the imaging power at the ChR+ pixels from 15 mW to 10 mW, 5 mW, and “0 mW,” while maintaining 15 mW imaging power at other pixels to ensure high-quality calcium signals from downstream neurons. For this, we used larvae pan-neuronally expressing nucleus-localized GCaMP6s, while restricting ChR2 or ChrimsonR expression to the u508+ neurons Tg(elavl3:Hsa.H2B-GCaMP6s; KalTA4u508; UAS:ChR2(H134R)-mCherry)9,56,59 or Tg(elavl3:Hsa.H2B-GCaMP6s; KalTA4u508; UAS:ChrimsonR-mKate2)58. We demonstrated that 920 nm optogenetic stimulation effectively activated neurons expressing either ChR2 or ChrimsonR (Fig. 3b). However, we observed that the colocalization of GCaMP6s and ChRs was very low in both strains of zebrafish larvae. Consequently, we performed holographic optogenetic stimulation on all pretectal ChR+ neurons. Noticed that we used APPC to block ChR+ neuropil and prevent unintended crosstalk stimulation (Fig. S11a–d). Here, we present the case of activating a single neuron co-expressing ChR and GCaMP6s in Fig. 3c, d and Fig. S11e–g, showing successful activation of the target upstream neuron and identification of its downstream connections. The case of activation of multiple neurons is shown in (Fig. S11h–k). Our study then focused on the calcium responses from downstream neurons. The putative downstream neurons were identified by their highly correlated activity patterns with the targeted ChR+ u508+ neurons60. Since the stimulation light could scatter onto the adjacent retina and potentially introduce visual artifacts, we conducted control experiments to exclude sensory neurons responding to this scattered light. Specifically, we positioned the optogenetic stimulation light on randomly selected ChR- neurons in a neighboring region (15–20 µm) from the ChR+ neurons (Fig. 3c and Fig. S12) as an “off target control”. The “off target control” neurons did not show neuronal activities respond to holographic optogenetic stimulation at ChR+ neurons, indicating that there are no direct or indirect connections between them.

Only neurons that responded specifically to activation of “target” pretectal ChR+ neuron, and not generally to light (Fig. S13), were selected as downstream neurons (Fig. 3d). These neurons exhibited strong correlations with the optogenetic stimulation applied to the targeted ChR+ neuron across various imaging conditions (Fig. 3d). In zebrafish expressing either ChR2 or ChrimsonR in the u508+ neurons, analysis of the downstream neurons revealed that their artefactual activity diminished as the imaging power applied to the upstream neurons was reduced (Fig. 3e–h). Importantly, these results demonstrate that applying 15 mW imaging power to ChR+ regions caused strong cross-talk, which affected downstream neurons and perturbed the neuronal circuit. Furthermore, neuronal activity levels were statistically indistinguishable when the upstream neurons were scanned at 5 mW versus “0 mW” (Fig. 3e–h). This led to the conclusion that 5 mW is the “near cross-talk free” power for upstream neurons, effectively minimizing cross-talk and providing a solution for negligible interference within the neuronal circuit. This means that in a strain of zebrafish expressing calcium indicators and ChRs in upstream neurons, alongside calcium indicators in downstream neurons, “near cross-talk free” optical interrogation can be achieved using the APPC approach.

To extend our method to different imaging scenarios, we investigated how to optimize imaging conditions to minimize cross-talk. A particularly important consideration is the extent to which ChRs can be activated by out-of-focus scanning light in 3D volumetric imaging. First, we modeled the activation of ChR2 molecules during imaging (see Methods) and assessed the extent of ChR2 molecule activation under various scanning conditions. Our simulation indicates that the ChR2 molecule can be effectively activated at a typical low power density of ~0.2 mW/µm² with a stimulation duration of 1 ms, consistent with numerous in vivo studies5,7,9,34 (Fig. 4a and Fig. S14). The excitation probability is proportional to laser power density and stimulation time. However, during calcium imaging, the power density at the focal point can be 50 times higher than the typical activation power density, significantly reducing the stimulation time required for ChR activation3,9,46. At an excitation power of 15 mW, a channelrhodopsin-2 (ChR2) molecule has an activation probability of ~8.1% under each scan during imaging. This probability decreases to ~0.9% at an excitation power of 5 mW, given a pixel dwell time of 68 ns under our imaging conditions (Fig. 4a). The trend is consistent with our previous in vivo experiment results. In addition, a recent study also demonstrated that rapid raster scanning can lead to power-dependent cross-talk45. Therefore, by controlling imaging power at the pixel level (e.g., 5 mW), we can effectively mitigate cross-talk. Suitable imaging power can be further adjusted according to the change of FOV. A previous study has shown that setting stationary out-of-focus light with a high numerical aperture (NA) can activate more ChRs across the entire membrane compared to focusing on the soma equator33. While dendrites and axons can contribute to photoactivation, their distribution and relationship with the neuronal membrane potential are complex. Therefore, to effectively simulate the scanning scenario, we have simplified our model to concentrate primarily on the main source of cross-talk, which is the ChR located at the soma membrane. For this simulation, we used a neuronal soma diameter of 10 µm, which is consistent with the sizes of neurons we observed in larvae zebrafish. The imaging light was scanned above or below the soma equator at varying distances and power levels (Fig. 4b). Our simulation indicated that the total photocurrent decreases as the distance between the focal plane and the equator increases (Fig. 4c, Fig. S15, and Fig. S16). Given the symmetry of the photocurrent generated by the out-of-focus plane, we focused only on the scenarios where the out-of-focus plane was positioned above the equator. To validate our simulation results in vivo, we performed quasi two plane imaging to validate out off focus activation on ChR2+ neurons in larva zebrafish (Tg(KalTAu508; UAS-ChR2(H134R)-mCherry; UAS-GCaMP6s))59. One imaging plane was set at the equatorial (central) plane of the neuron, while the other was placed at an offset latitudinal plane located at distances varied from 0 µm to 14 µm using the ETL, exceeding the typical sampling gap9,55. And the two planes were scanned sequentially (offset plane first) under ETL control, ensuring that the equatorial plane was not simultaneously excited with the offset plane.

With APPC, we applied 5 mW of imaging power to the equatorial plane and 15 mW to the offset plane. Since our previous results (Figs. 2 and 3) suggested that 5 mW imaging power does not induce detectable cross-talk, the calcium signal recorded at the equatorial plane alone was used as the near cross-talk–free reference. To evaluate out-of-focus activation, we measured calcium signals at the equatorial plane while the offset imaging plane was excited at a higher power (15 mW). These signals were then compared with the reference condition, in which the offset plane was not excited, to quantify cross-talk originating from out-of-focus activation. We found that out-of-focus light at 10 µm or farther away from the equatorial plane leads to a negligible cross-talk (Fig. 4d). This experiment mimics the case of the neuronal equator located in the gap between imaging planes and demonstrated the in vivo experiment results are consistent with our simulation model.

Next, we evaluate the impact of 10-plane volumetric calcium imaging on cross-talk as more imaging planes may induce more severe cross-talk. In our experimental setup, the imaging planes are positioned at distances of 2, 5, 8, 10, and 14 µm, with the equatorial plane of monitored neuron situated at the 6th plane from the top. All the offset imaging planes are at 15 mW while applying 5 mW imaging at target neuron at the 6th plane using APPC to avoid high cross-talk baseline. In the control group, the imaging power for the other 9 planes was turned off (single-plane imaging). Consistent with previous results, cross-talk from out-of-focus excitation becomes non-negligible when the distance between planes is smaller than 8 µm, as shown in Fig. 4f and Fig. S17, indicating the further imaging plane provide minimum contribution. Taking together, these results provide critical guidance for determining optimal depth intervals in 3D volumetric imaging protocols.

To further investigate the performance of APPC, we examined its potential to spatially prevent excitation of ChR at the cell membrane and in the axons and dendrites near the soma. Given that ChR is localized at the cell membrane while GCaMP is situated within the cytoplasm or nucleus, we propose that restricting the imaging laser to the sub-soma region may significantly reduce cross-talk. We conducted numerical simulations with varying sizes of restrictions on the soma, as illustrated in Fig. S16. When scanning at the equatorial plane, most ChR activation occurs in the equator. Consequently, confining excitation inside this region markedly diminishes activation across the entire neuron, as depicted in Fig. 4g. We then performed in vivo experiments to validate our simulation results, where we scanned the whole neuron by 5 mW and 15 mW laser, and 15 mW laser confined to sub-soma region with a diameter of 4 µm due to neuron’s irregular shape. The results presented in Fig. 4h and Fig. S18a demonstrate that laser confinement effectively reduces cross-talk caused by the activation of ChR at the equator and adjacent axons and dendrites. Although this approach results in a decrease in the calcium signal collected of 73%, it still reflects overall cellular activity (Fig. S18b).

Methods
Animals
All procedures conformed to the guidelines by the animal ethics committee of HKUST. Animals were kept under a standard 14:10 light cycle at 28 °C. Zebrafish larvae carrying mutations in the mitfa allele (nacre)61 were used for all experiments. Larvae were kept in Danieau’s solution, and fed with paramecia (Paramecium multimicronucleatum, Carolina Biological Supply Company) on 5 to 7 days post fertilization (dpf).

The following transgenic lines were used for this study: Tg(KalTA4u508; UAS:GCaMP6s; UAS:ChR2(H134R)-mCherry)9,57, Tg(KalTA4u508; UAS:GCaMP6s)56, Tg(KalTA4u508; UAS:GCaMP6s; UAS:ChrimsonR-mKate2)58, Tg(elavl3:Hsa.H2B-GCaMP6s; KalTA4u508; UAS:ChR2(H134R)-mCherry)59, and Tg(elavl3:Hsa.H2B-GCaMP6s; UAS:ChrimsonR-mKate2)59.

Zebrafish preparation
6-8 dpf larvae were embedded in 2% low melt agarose (Invitrogen) on 90 mm round (Thermo Scientific (cat# 101VR20)) plastic petri dish. The agarose surrounding the tail and the eyes was removed by surgical blade (Paragon p303) to allow for tail and eye movements. Fish were allowed to recover overnight prior to the experiment.

Two-photon imaging microscope
A home-built two-photon light scanning microscope controlled by a C# based custom software is modified from previous setup. The laser source is a Ti:Sapphire femtosecond laser (Chameleon Ultra II, Coherent), repetition rate: 80 Mhz, and can be tuned between 680 nm and 1080 nm. The laser beam first passes through the half-wave plate to adjust polarization direction. Then, a polarized beam-splitter separates the laser beam into two paths, one for imaging and the other for optogenetic stimulation.

At imaging path, the laser beam first pass through a 4f system of scale factor x0.467 (AC254-075-AB-ML F=75mm, Thorlabs and AC254-035-AB-ML F=35mm, Thorlabs) in order to not overfill the AOM (CAOM-080-010-TEC-740-1020-AF-A17, Castech) aperture (1.0 mm). After passing through the AOM, the laser beam is expanded by a factor of x3 through a 4f system (AC254-050-AB-ML F=50mm, Thorlabs and AC254-150-AB-ML F=150mm, Thorlabs). Two dispersion compensating prism (SF10 25.4 × 25.4 mm Ultrafast Prism, Edmund optics) is set and optimized the dispersion compensation at 920 nm. The first prism is fixed, and the second prism is located at a linear translation stage with actuator (CONEX-LTA-HL, Newport). The working direction of the linear translation stage is perpendicular to the prism base plane. For each wavelength used, the position of the prism is adjusted by maximizing the fluorescent intensity from the fluorescent plate. The total system group dispersion was estimated from the optical components used to calculate the distance between two prisms when the laser worked at 920 nm. The pulse duration after the objective is 118 fs, measured by pulseCheck autocorrelators (NX150, A.P.E.) to verify the dispersion compensation.

The laser beam with 4 mm in diameter (propagate a large distance and thus expanded) is then expanded by a factor of x4 through a 4f system (AC254-050-AB-ML F=50mm, Thorlabs and AC254-200-AB-ML F=200mm, Thorlabs) to full fill the ETL (EL-16-40-TC-NIR, Optotune AG), which is used for multi-plane functional imaging. Then a 4f system (AC254-200-AB-ML F=200mm, Thorlabs, and AC254-050-AB-ML F=50mm, Thorlabs) reduces the beam diameter to 4 mm to avoid overfilling the scanner mirror. A resonant scanner (CRS 8 kHz, Cambridge technology) for high-speed scanning is conjugated with a set of XY galvo scanners (6215H, Cambridge technology) by a 1:1 relay (AC254-100-AB-ML F=100mm, Thorlabs). Finally, two lenses (Scan lens SL50-CLS2 F=50mm, Thorlabs, and Tube lens TTL200MP F=200mm, Thorlabs) are used to correct the aberration and expand the laser beam to fulfill the objective back pupil (objective lens (20x, N.A. 1.0, XLUMPLFLN20XW, Olympus Corporation)). The ETL, resonant scanner, XY galvo scanners, and objective pupil plane are all conjugated. The control voltage of ETL was calibrated by comparing the 3D stack of samples obtained by moving the objective with an actuator and shifting the focal plane using the ETL. We fulfill the ETL aperture to minimize the change in focal power required for shifting the focal plane by the same distance along the Z-axis. Only the upper one tenth of the image plane is slightly tilted, but each plane remains parallel to one another at the same distance.

In the fluorescence collection path, a dichroic mirror (FF705-Di01-25*36, Semrock) is set between the tube lens and objective to reflect the fluorescence light, which then passes through two relay lenses (AC254-150-AB-ML F=150mm, Thorlabs and AC254-040-AB-ML F=40mm, Thorlabs) into the PMT (H11461-03, Hamamatsu). Two filters (FF03-525/50-25, Semrock, and FF01-600/52-25, Semrock) are installed on a flipper which can switch the filter to collect different fluorescence signals. Voltage signals are converted from PMT output current using a high-speed current amplifier (DHPCA-100, Femto) and digitized using an oscilloscope device (PCIe-9105H, ART technology).

At stimulation path, the laser beam passes through a set of galvo scanners (6215H, Cambridge Technology). Then the light beam is expended by two lenes (AC254-50-AB-ML F=50mm, Thorlabs, and AC254-250-AB-ML F=250mm, Thorlabs) to fill the spatial light modulator (HSP1K-500-1200-PC8, Meadowlark Optics). Then the SLM is conjugated to the back focal plane of objective by a demagnify relay (AC508-250-AB-ML F=250mm, Thorlabs, and AC254-150-AB-ML F=150mm, Thorlabs). The photon stimulation light combines with imaging light by a polarized beam splitter (CCM1-PBS252/M; Thorlabs) between tube lens and dichroic mirror.

The LCR projector and camera were employed to provide visual stimuli and record animal behavior, similar to our previous work60. We used the spatial visual stimulator module (DLP® LightCrafter™ 4500 modified by EKB Technologies Ltd) to display stimuli, with 385 nm light source. The LightCrafter (LCr) projected images onto a Teflon fabric screen which is positioned inside the petri dish, under water, and 1 cm in front of the fish head, with light intensity of ~400 nW/cm². The prey-like stimuli consisted of UV dots measuring 4°×2°, moving in an elliptical path of 8°×4° at a speed of 2 Hz for 2 s against a dark background.

Pixelwise adjustment of laser intensity
The orientation of the AOM is initially adjusted to achieve maximum diffraction efficiency. Here we adopt the 0th order light and measured the intensity modulation dynamic range, which spans from 5 to 100%. To enable dynamic adjustment of laser intensity on a pixel-by-pixel basis, we developed a GUI that allows for the custom generation of figures using fluorescence images of ChR+ neurons as the pattern for APPC. These figures are encoded into voltage signals and transmitted to the AOM driver (CARD-FE2-080-24D04C-1DA5H-AF, Castech). To synchronize the AOM with the imaging scanning beam, the clock signal generated by the resonant scanners serves as the reference clock signal for the DAQ card (PCIe 9105_H, ART technology). The AO port is configured to generate a series of output signals upon receiving a trigger from the resonant scanner clock, thereby eliminating time delay accumulation (Fig. S3). Finally, we captured the fluorescence image from a fluorescence plate with the loaded figure to finely tune the delay by aligning the fluorescence pattern between two adjacent lines.

In vivo calcium imaging and holographic optogenetics stimulation
Two-photon multiple-plane imaging was performed using resonant-galvanometer raster scanning (30 Hz frame rate, 512×512 pixel per frame, 400 µm field-of-view, 12-14 µm between imaging plane, 10 planes, 3 Hz volumerate) typical for whole brain imaging in zebrafish60. An objective lens (20x, N.A. 1.0, XLUMPLFLN20XW, Olympus Corporation) was used for all experiments. The femtosecond laser (Chameleon Ultra II, Coherent) was operated at 920 nm wavelength for calcium imaging and 1020 nm for mCherry and mKate imaging. In all the in vivo experiments, the imaging power applied to the sample ranged from 0.5 to 15 mW. At the beginning of the imaging, we first imaging at the very low power, 0.5 mW, to outline the eye contours from the fluorescence signal of the pigments on retina. Then we manually selected the eye region and included it in the APPC pattern, where the laser power was set to be the minimum. For pixel level power control of ChR expression region, the APPC pattern was generated based on the ChR expression pattern verified by fused fluorescent protein. We set the pixel intensity threshold to adjust the pattern region flexibility. We then manually refined the pattern for each plane. The imaging power at these regions were determined based on the experiment requirement.

For optogenetics stimulation, the holograms were calculated by compressive sensing weighted Gerchberg–Saxton algorithm62 and loaded on SLM which controlled by the custom software in MATLAB. A large beam diameter is applied to achieve an effective excitation NA (~0.5) for axial profile compression. A spot array is then created on each target neuron, consisting of spots that are 10–12 µm in diameter with a 1 µm gap between them, and an average power density of 0.15–0.3 mW/µm². The spot array is scanned over a range of 2 µm to ensure that the entire soma area is stimulated. To mimic natural neuronal response, we first provided visual stimuli and extract the calcium signal from the anterior pretectal u508 neurons. Then we adjust the optogenetic stimulation laser power to trigger similar calcium response. Each optogenetic stimulation lasted for 1 second, with a total of 5 stimulations per session, separated by 30-s intervals. In the experiments that aimed at identifying the downstream neurons, we selected approximately 5–10 anterior pretectal u508 neurons on either the left or right side.

Before each optogenetic experiment, we calibrated the alignment between the imaging light and the photo-stimulation light. We bleach two dark spots on a fluorescent microscope slide (FSK3, Thorlabs) using both imaging light and photo-stimulation light. After measuring the distance between the spots, we included a coordination shift in the hologram calculation.

Calcium imaging data processing
Image analysis was performed using custom-written scripts in Matlab (Mathworks). Non-rigid motion correction for each imaging plane was performed using the NoRMCorre algorithm63. For neuron-based analyses, the locations of regions of interest (ROIs) and their fluorescent signal were extracted using the CaImAn algorithm, which is > 5 µm from the dead pixels because they were not able to keep precise information. ROIs with the SNR, as computed by CaImAn, below 1.5 were excluded from further analysis. Specifically, the deconvolveCa function returned the baseline, denoised calcium trace, noise level estimated from the spectral density and deconvolved signal (spike train). We calculated ΔF/F based on the mean baseline value and calcium trace recorded. The mean ΔF/F value and spike value were calculated during the entire recording. Then we computed the inferred spike value as the summation of the spike value normalized by the baseline. Spike values below one standard deviation of the noise level were discarded.

Data presentation and statistical analyses
All values are presented as mean ± standard deviation (s.d.), unless otherwise specified. Statistical analyses were conducted using appropriate tests, as indicated in the figure legends. The statistical tests were performed using GraphPad Prism, version 9.5.1, with a p value of < 0.05 considered statistically significant. Data normality was first checked using the Shapiro–Wilk normality test. Normally distributed data were analyzed using paired, unpaired one-way ANOVA test. Non-normally distributed data was analyzed using paired, unpaired non-parametric test (Friedman test with Dunn’s multiple comparison test). In the statistical results presented in Figs. 2d–i and 3e–h the neurons depicted were selected based on their distribution, as they exhibited strong cross-talk. Specifically, there was no statistically significant difference in spike values below laser power of 5 mW between the ChR+ and ChR- groups, leading us to consider cross-talk negligible at this power level. Neurons with mean ΔF/F or spike values at 15 mW imaging power exceeding the mean plus one standard deviation at 5 mW were classified as exhibiting strong cross-talk and included in Figs. 2d–i and 3e–h.

A regressor for single optogenetic stimulation was generated by averaging the calcium trace corresponding to optogenetic stimulation. The downstream neurons depicted in Fig. 3c–h were selected based on the criterion that the correlation between the calcium trace and the target optogenetic regressor exceeded 0.39, while the correlation with the control optogenetic regressor remained below 0.05.

ChRs modeling
A ChR2 molecule has an absorption rate of σ2 I(t)2, and the ground state lifetime is given by τ = 2 / (σ2 I2), where σ2 is the two-photon absorption cross-section (260 GM)33. The laser intensity I which can be expressed by ref. 64: I = g(2) Pavg / (fp τp λ hc) (1), where g(2) is the factor of 0.59 for a hyperbolic-secant-squared pulse shape65, Pavg is the total time-average laser power, fp is the pulse train repetition rate, τp is the pulse duration. In our calculations, we used fp = 80 MHz and τp = 120 fs. h is the Planck’s constant, c is the light speed, λ is the wavelength.

The laser is attenuated in the sample due to scattering and absorption. We determined the attenuation length (AL) from the brain z-stack images, where the average fluorescence signal of the brightest 0.1% of pixels attenuates by 1/e2 66 (Fig. S19a). The calculated AL for 7–8 days post-fertilization (dpf) zebrafish larvae is approximately 201 µm (Fig. S19b). At a depth of 140 µm, the laser power reaching the pretectal u508 neurons was estimated to be 50% of the initial power, based on the calculated AL, and this value was used for subsequent photocurrent calculations.

The probability of activating a ChR2 molecule under stationary illumination of time Δt is: P = 1 - e^(-fp τp σ2 I^2 Δt) (2).

Next, we consider the activation of a ChR2 molecule by a moving focused beam, which has a Gaussian distribution in the lateral direction. Thus, the laser intensity can be expressed as33: I(r,z) = g(2) Pavg / (fp τp h λ c) * (ω0^2 / ω(z)^2) * e^(-2r^2 / ω(z)^2) (3), where ω is the beam waist. At the focal plane, ω = ω0 and ω0 can be determined by measuring the two-photon excitation point-spread function from image 0.2 µm microspheres. The two-photon excitation waist ωTPE was obtained by fitting the two-photon excitation point-spread function by the Gaussian distribution. The ω0 can then be calculated from ω0 = ωTPE / sqrt(2). Or ω0 can be estimated by ω0 ≈ λ / (2NA). In the perifocal region, the ω can be expressed by ref. 34: ω(z) = ω0 * sqrt(1 + (λΔz / (πω0^2))^2) (4).

To simulate the activation of ChR2 molecules on the neuron, we modeled the neuronal soma as a sphere with a diameter of 10 µm, generating 25,000 points representing ChR2 molecules randomly distributed on the surface, corresponding to approximately 200 proteins/µm². We simulated the focused beam scanning and calculated the photocurrent of each ChR2 molecules by combining equ. (1)-(4) and integrating over time. The kinetic information can refer to Rickgauer’s work33. For ChR2, the photocycle can be completed before the next frame even for a 30 Hz frame rate. Thus, we only considered the ChR2 activation during single frame scanning. The maximum photocurrent for each ChR2 molecule was normalized to 1. The total photocurrent for the entire neuron was then obtained by summing the photocurrent contributions from each individual ChR2 molecule.

Peer review rounds
[{"round": 1, "reviewer_comments": {"reviewer_1": "In this manuscript entitled“Toward Crosstalk-free All-optical Interrogation of Neural Circuits”, the authors present an power-modulated two-photon imaging and optogenetic stimulation system designed to mitigate the undesired activation of opsins by the imaging laser. By precisely reducing the imaging power at target neurons, they aim to decrease the probability of inadvertent opsin activation during all-optical physiology experiments. The proposed strategy seems impractical. Here are several important questions to be resolved.\n1. The authors employ a single-wavelength laser (e.g., 920 nm) for both optogenetic activation and two-photon imaging. The advantage for this design is unclear, whereas the drawbacks are readily apparent. First, 920 nm deviates from the peak two-photon absorption wavelengths of widely used opsins such as C1V1 and ChrimsonR, thus reducing stimulation efficiency for these tools. Second, using ChR2 is not a good choice for crosstalk free two photon all-optical experiment since the cross-section curve cannot be separated from GCaMP6s by excitation wavelength. Third, using 920 nm to activate opsins introduces another severe form of crosstalk—this wavelength also effectively excites GECIs, leading to significant fluorescence contamination in the imaging channel. Because the excitation wavelength is identical, this crosstalk is spectrally indistinguishable from the calcium imaging signal. This issue is likely exacerbated when spatial light modulation (SLM) is used for multi-point stimulation, potentially resulting in fluorescence artifacts several times stronger than the real signal. In extreme cases, this may saturate the PMT and overwhelm the recorded data. Given that the stimulation duration is on the order of 1 second, such crosstalk would likely compromise signal interpretation during activation. The authors must clarify how they addressed this issue.\n2. In mouse cortex, due to the presence of dendrites and axons, the physiological resolution is worse than the optical resolution. Although the authors touch on this in Fig. 4, the problem should be even more pronounced in zebrafish, where neurons are smaller and densely packed. In line 308, the authors assume that “ChR2 expression is confined to the soma membrane” and perform simulations accordingly. However, the manuscript lacks experimental validation: specifically, it does not show whether a neuron located in the middle plane would be activated if the imaging beams are applied to adjacent upper and lower planes at the target area. (And I also should mention that no volumetric imaging data is given.) In lines 63–64, the authors mention that only ~10% of the full imaging power is sufficient to activate ChR2, which implies a high potential for strong out-of-focus activation. Thus, the probability of inadvertently activating neurons in the intermediate plane is significant. The authors should present experimental data to support or refute this possibility. Moreover, to convincingly demonstrate the effectiveness of their method under such conditions, the authors are encouraged to reference and follow a more detailed approach as seen in “Synaptic connectivity to L2/3 of primary visual cortex measured by two-photon optogenetic stimulation”(https://doi.org/10.7554/eLife.71103). It is also important to emphasize that labelling density is a critical factor in such systems and should be carefully discussed.\n3. The authors state that even low imaging power is sufficient to extract meaningful calcium signals from target neurons under their APPC strategy. However, if a ChR2-expressing neuron is present in the field of view but not directly targeted, and exhibits a weak response due to indirect network activation, the reduced imaging power will compromise the ability to detect this signal. The authors should acknowledge this limitation, which also implies that their approach is only suited for sparsely labeled neural populations.\n4. Although the optical layout includes an SLM, the manuscript does not present any experiments involving multi-neuron or patterned stimulation. This makes it difficult to evaluate the effectiveness of the proposed APPC method under more complex activation scenarios. If the SLM is not utilized in practice, its inclusion in the schematic is misleading. Furthermore, as mentioned earlier, crosstalk artifacts may become even more severe during simultaneous multi-site activation. This potential issue must be addressed.\nAdditional Issues to be Addressed:\n1. The authors should ensure correct citation throughout the manuscript. For instance, reference [29] does not use an image-processing algorithm to address crosstalk.\n2. In lines 113–114, a brief discussion comparing AOMs and EOMs for laser intensity modulation would be valuable. See this ref，\"A large field-of-view, single-cell-resolution two- and three-photon microscope for deep and wide imaging\" (https://elight.springeropen.com/articles/10.1186/s43593-024-00076-4).\n3. In Fig. 1a, the translation direction of the pulse compression stage appears to be incorrect. Authors should verify their understanding of the principles behind prism-based pulse compression and group velocity dispersion (GVD) compensation.\n4. What is the dispersion difference between the imaging and activation beams? Why is the pulse compressor placed after the beam splitter rather than before?\n5. In Fig. 1f, it would be helpful to include curves corresponding to different modulation powers, or at least indicate the imaging power applied to the target neuron.\n6. The authors should also discuss the potential step response effect of the ETL and how it was managed in their setup for volumetric imaging. Still, volumetric imaging data should be given.\n7. All numerical values and units should be formatted properly. For example, \"um\" should be written as \"µm\".", "reviewer_2": "In this submission, a novel strategy for combining imaging and optogenetic excitation is presented that minimizes crosstalk, which is a common problem that is often addressed by spectral separation between imaging and optogenetic stimulation channels, or ignored while hoping for the best. The proposed method involves fast modulation of the two-photon excitation imaging laser to tune it to low power when it is situated above a sparse set of optogenetically tagged neurons in the brains of zebrafish. The proposed strategy is clever and useful and the work is well performed and presented.\nBelow is feedback that I believe should be addressed before considering acceptance of this work for publication.\nThe strategy of minimizing power when scanning over cells with optogenetic channels requires accurate knowledge of where those cells are in space, which can change as a result of tissue drift. For best performance, tissue drift would need to be measured in real time and corrected for in real time using real-time 3D registration methods. The use of registration with NoRMCorre is mentioned in the Methods but it is unclear whether this ran in real time or offline. While not necessary to implement for the current proof-of-principle demonstration, this requirement should be addressed.\nThe use of 5 mW to minimize activation of ChR2 is carefully motivated in three ways: empirical observations that this renders pretectal neurons statistically similarly active as non-opsin-expressing pretectal neurons, physics-based calculation, and inspection of downstream activity. This is great, but there is a missed opportunity: the remaining stimulation at 5 mW (of which there is, of course, some) can, in principle, be extrapolated from the excitation seen at 15, 10, 5, ... mW. This could be a linear or nonlinear extrapolation. Given that this is a methods paper, it would be nice to include an attempt at such extrapolation or, at the minimum, to include it in the Discussion as a possibility to further enhance the method.\nIn line 258, it is stated that 5 mW is in the 'crosstalk free' zone, but I would be careful stating that because it might depend on factors like the point spread function, possibly the concentration of the opsin, unknown tissue properties, and indeed the authors calculate that a tiny bit of crosstalk is still expected. A bit more cautious, to avoid misinterpretation, would be to say 'near crosstalk free' or 'minimal crosstalk'.\nThe comparison between different pretectal neurons to reinforce that 5 mW is in the crosstalk free zone is a bit shaky because comparing across neurons is not always easy and 'confirming the absence of detectable crosstalk' (line 169) seems a bit too strong. Rephrasing this as being suggestive or indicative or supportive might be more justified. That said, as written above, I appreciate the multiple approaches for testing crosstalk.\nA major point to emphasize is that decreasing the power at opto-labeled neurons does also decrease the signal to noise ratio for those neurons, even if ∆F/F remains similar. That is, the imaging data for these neurons will be noisier than for the other neurons where the power reduction is unnecessary because they lack opsin expression. This should be discussed in the Discussion and maybe mentioned in the results.\nOverall, the Discussion is more of a summary instead of something more nuanced where the caveats of the method are also discussed. Another caveat, in addition to the above, is that the method works in two-photon point scanning microscopes but would not work in light sheet or light field microscopes, which deserves mentioning. It would also not work in multiplexed two photon microscopes like light beads microscopy. None of these are criticisms or disadvantages, it would just be good to broaden the discussion a bit.\nOne more point that deserves mentioning is that this method is described for samples containing sparse expression of ChR2. If many or all neurons are labeled, then the method would be equivalent to scanning at low laser power and adding power for the targeted neurons through the SLM, or perhaps through the scan laser, but in most cases the dwell time would be too short, however, the laser could spiral a little longer at the targeted neurons with increased power. In some sense, this is an equally valid use of the method, which sits somewhere in between a caveat and an additional opportunity of the method that could be highlighted.\nThe end of the introduction (line 98), 'accelerates therapeutic development', sounds a little cliche and it's a bit indirect. The study is valuable it itself and I don't think it needs to be justified or reinforced in this way unless specific examples are laid out.\nRegarding the cost of two-photon lasers and the complexity of the optical pathways (line 91), fixed wavelength lasers are now not that costly anymore compared to variable wavelength lasers, and the optical setup used in the manuscript also contains some additional complexity and cost, so here I also feel that this justification is a bit unnecessary because the technical advancement stands on its own and is useful regardless of what other strategies might be available.\nRegarding the 10-fold higher absorption cross-sections than GCaMP (line 62-63), some readers might not understand this, and it would be good to explain a little more intuitively what the effect of this is (possibly with some quantitation).\nLines 64-70, in the comparison to other opsins, the fact that they were developed using single-photon effects as benchmarks is true but not all that relevant if they work with two-photon stimulation. Similar to the above comments, I feel that contrasting the new method against the perceived disadvantages of these other methods does not add that much and might sound a bit defensive, which is not necessary for a technique which is clearly useful and valuable.\nThe use of 'sophisticated' (line 337) seems unnecessary, this is already clear and doesn't need to be advertised.\nWith these comments addressed, in my opinion, this will be a very nice methods paper.", "reviewer_3": "Yan et al. describe the design and application of active pixel power control (APPC), a novel method for diminishing unwanted excitation of neurons (“crosstalk”) expressing opsins such as ChR2 and ChrimsonR during all optical interrogation of neuronal circuits with two-photo calcium imaging. This is particularly challenging within in vivo preparations with overlapping and dense expression of opsins and genetically encoded calcium indicators. While conceptually simple, proper implementation of APPC requires careful testing and calibration, which the authors describe. Moreover, they clearly demonstrate the activation of neurons expressing commonly used opsins during two-photon calcium imaging and quantify the contribution of crosstalk in the zebrafish larvae preparation, which is the strength of this manuscript. However, concerns regarding whether this method is superior to alternatives approaches and will actually be adopted by other users limits my enthusiasm for the manuscript.\nMajor comments:\n1) It is unclear to what extent this brute force method will be adopted over less cumbersome solutions to overcome crosstalk that utilize the rapidly growing palate of genetically encoded calcium indicators (GECIs) such as described by Forli et al. (Cell Reports 2018) and others. This includes the use of more recently developed red GECIs such as PinkyCamp (Fink et al, bioRxiv, 2025) and SomaFRCaMPi ( Zhou et al, Plos Biology, 2025), yellow GECIs such as jYCaMP1 (Mohr et al., Nat. Methods, 2020), and red chemigenetic calcium indicators (Ferrants et al., Nat Methods, 2024), all of which can be efficiently excited outside of the range of ChR2 (>1050 nm, Fig. S7) and have very good signal to noise properties in neurons in vivo.\n2) It would be very challenging to implement APPC in the setting where the opsin expression is not restricted to the soma as activation of axons in the neuropil should make a significant contribution to the crosstalk and would be difficult if not impossible to eliminate using APPC. Authors should discuss this limitation.\n3) Fig. 2a is confusing and the caption stating “a, Experimental strategy for minimizing crosstalk of optical interrogation of ensembles” does not contain sufficient details . The schematic in Fig. 2a. indicates that APPC is utilized to modulate the power delivered to selected neurons. My understanding from the text in the Results section corresponding to the experiments in Fig. 2 is that the authors performed two-photon microscopy using standard raster scanning to measure the amount of crosstalk induce during calcium imaging without APPC, which does not seem to be consistent with the schematic. If my understanding is incorrect, the authors should spend more effort clarifying which type of excitation is used in each experiment.\n4) Likewise, the schematic and caption for Fig. 3a is also not clear. Is it a single neuron that is being stimulated with holographic excitation? Can stimulation of a single neuron provide such reproducible and extensive downstream activity? Or is it a larger spot containing multiple neurons? Or a collection of multiple spots each containing an individual neuron? How do you choose or define the off target neuron(s)? What is the relationship between the off target neuron(s) and the on target neuron(s) in this experiment? Are they always separated by a fixed distance for each experiment or are they functionally defined in some way? Are there no direct or indirect connections between on target and off target neurons such that strong optogenetic stimulation of the on target neurons does not induce any activity in the off target neuron?\n5) If raster scanning with very modest laser powers (≤15 mW) with short dwell times (180 ns) is sufficient to excite opsins in these experiments and cause significant downstream activation of neurons within the network, why do the authors and other groups go to such great extent to achieve efficient 2P excitation of opsins in their experiments (using galvanometer and SLM to scan an array of large spots, providing continuous excitation throughout the soma lasting for 1 second, which is 6 orders of magnitude longer than pixel dwell time) in order to activate ChR2+ expressing neurons? If cross talk is so prevalent at the parameters used for raster scanning during calcium imaging, couldn’t APPC also be used to activate targeted ChR2+ expressing neurons at desired time points and eliminate the need for holographic excitation at the modest imaging acquisition rates used in these experiments? It would be important to clarify this apparent contradiction.\nMinor comments:\n1) line 75-76: “dual femtosecond laser systems for spectral separation are cost-prohibitive and technically constrained”. While this might have been the case early on after the first introduction of pulsed 920 nm solid state lasers, there is no longer a significant separation in cost or specifications. In fact many if not most new laboratories setting up both two-photon optogenetics and calcium imaging, and even just calcium imaging without optogenetics, are now using two solid state pulsed lasers instead of a single Ti:Sapphire laser.\n2) line 117: “relationship of excitation power and excitation intensity” should read “relationship of excitation power and fluorescence intensity\"\n3) line 183: “did not differ significantly between ChrimsonR+ and opsin- neurons under 5 mW 183 illumination” is slightly confusing as under can also mean less than. Perhaps it would be better to state “did not differ significantly between ChrimsonR+ and opsin- neurons at 5 mW 183 illumination”\n4) line 194: “ChR+ neurons could be reliably extracted due to their strong responses” could be more clearly stated as “ChR+ neurons could 193 be reliably segmented due to their strong responses”\n5) line 200-201: “This demonstrates that APPC is critical for minimizing crosstalk while ensuring an acceptable SNR for calcium imaging of all neurons in the circuit.” This is confusing or misleading. At this point in the manuscript (prior to Fig. 3), the authors have not shown any data regarding the impact of APPC on calcium signals, and this is perhaps not what the authors meant to imply here.\n6) line 273: “Under 15 mW excitation, a ChR2 molecule has an activation probability of ~8.1%”. Is that 8.1% each time the line scan traverses the cell or 8.1% per frame or per movie? These and other similar probabilities described in the paper should be clearly defined.\n7) Fig S4. In the shot-noise-limited regime, the SNR should increase as the square root of the excitation power. Here, when the power is increased by a factor of 3, say from 5 mW to 15mW, the SNR increases by a factor of >3, instead of a factor of √3, which is expected in the case of no crosstalk. This is another indication that raster scanning during two-photon calcium imaging is indeed inducing opsin excitation and crosstalk, consistent with the findings described in Fig. 2. Perhaps the authors could discuss this briefly."}, "author_response": "Responses to reviewers’ comments\nThe authors sincerely thank the reviewers for their thoughtful and constructive feedback. We\nhave carefully considered each comment and revised the manuscript accordingly, addressing\nall concerns either in the revised text or in our responses. We greatly appreciate the reviewers’\ninsightful suggestions, which have significantly enhanced the clarity and overall quality of our\nwork. Below, we provide a detailed point-by-point response, together with a description of the\ncorresponding revisions made.\nRESPONSES TO THE COMMENTS FROM REVIEWER #1:\nGeneral:\n“In this manuscript entitled “Toward Cross-talk-free All-optical Interrogation of Neural\nCircuits”, the authors present an power-modulated two-photon imaging and optogenetic\nstimulation system designed to mitigate the undesired activation of opsins by the imaging laser.\nBy precisely reducing the imaging power at target neurons, they aim to decrease the probability\nof inadvertent opsin activation during all-optical physiology experiments. The proposed\nstrategy seems impractical. Here are several important questions to be resolved.”\nResponse:\nWe thank the reviewer for the careful evaluation of our manuscript and for highlighting\nimportant concerns about the practicality of our approach. In response, we have revised the\nmanuscript to clarify the rationale and technical feasibility of our power-modulation strategy,\nand we have strengthened the discussion to address the key questions raised. These revisions\naim to provide clearer context for how the method can be applied in practice and to better\ndelineate its advantages and limitations.\nMajor Comment 1:\n“The authors employ a single-wavelength laser (e.g., 920 nm) for both optogenetic activation\nand two-photon imaging. The advantage for this design is unclear, whereas the drawbacks are\nreadily apparent. First, 920 nm deviates from the peak two-photon absorption wavelengths of\nwidely used opsins such as C1V1 and ChrimsonR, thus reducing stimulation efficiency for these\ntools. Second, using ChR2 is not a good choice for cross-talk free two photon all-optical\nexperiment since the cross-section curve cannot be separated from GCaMP6s by excitation\nwavelength. Third, using 920 nm to activate opsins introduces another severe form of cross-\ntalk—this wavelength also effectively excites GECIs, leading to significant fluorescence\ncontamination in the imaging channel. Because the excitation wavelength is identical, this\ncross-talk is spectrally indistinguishable from the calcium imaging signal. This issue is likely\nexacerbated when spatial light modulation (SLM) is used for multi-point stimulation,\npotentially resulting in fluorescence artifacts several times stronger than the real signal. In\nextreme cases, this may saturate the PMT and overwhelm the recorded data. Given that the\nstimulation duration is on the order of 1 second, such cross-talk would likely compromise signal\ninterpretation during activation. The authors must clarify how they addressed this issue.”\nResponse:\nWe appreciate the reviewer’s insightful questions and fully agree that 920 nm is not the optimal\nexcitation wavelength for red-shifted channelrhodopsins (ChRs) such as C1V1 and ChrimsonR.\nWe also acknowledge that, in the original manuscript, we did not clearly describe the goal and\nrationale of this study, which may have caused confusion.\nOur primary objective was to develop a technology and general framework to suppress cross-\ntalk in two photon optical interrogation, applicable to both single- and dual-laser systems.\nTo evaluate this, we first focused on the most challenging case: using a single laser at 920 nm to\nexcite both Channelrhodopsin-2 (ChR2) and GCaMP6s, the widely used light-sensitive ion\nchannel and genetically encoded calcium indicator, respectively. Using a stringent condition in\nwhich the 920 nm laser coincides with the peak two-photon excitation of both ChR2 and\nGCaMP, we demonstrated that the proposed active pixel power control (APPC) method can\nstill effectively minimize cross-talk arising from spectral overlap. The experimental results,\npresented in Figure 2d, 2g, Figure S6 and Figure 3e, 3g, provide evidence that careful control\nof excitation power, based on the analysis on calcium traces of ChR2+ neurons, can effectively\nsuppress cross-talk. We appreciate the reviewer’s comments and have revised the manuscript\nto clarify the objective of this first set of studies.\nIn the second set of studies, we used a single excitation laser at 920 nm for imaging GCaMP\nand stimulating ChrimsonR, a red-shifted ChR with an excitation peak at 1130 nm. The\npurposes were threefold: (1) to demonstrate that strong cross-talk between optogenetic\nmanipulation and calcium imaging still occurs even when the optimal excitation wavelength of\nChrimsonR is far from that of GCaMP6s; (2) to show that the proposed APPC approach\neffectively suppresses such cross-talk for both short-excitation-wavelength and red-shifted\nChRs; and (3) to illustrate that, with cross-talk minimized, accurate two-photon optical\ninterrogation can be achieved using the single-laser systems commonly available in most\nlaboratories worldwide. The experimental results, presented in Figure 2e, 2h, Figure S6 and\nFigure 3f, 3h provide evidence supporting the effectiveness of the APPC-based technology and\nframework in minimizing cross-talk in two photon optical interrogation. In addition, the APPC\napproach can also be applied in dual-laser systems, using a second laser to optimally stimulate\nred-shifted ChR, such as ChrimsonR, while applying APPC to the 920 nm imaging laser for\nGCaMP excitation. We regret that the purposes of these studies were not clearly described in\nour previous manuscript. The reviewer’s comments have helped us revise the manuscript and\nclarify these important issues.\nTo further elaborate on the cross-talk between optogenetic manipulation and calcium imaging\nunder two-photon excitation, we have added the following discussion in the revised manuscript:\nIn general, under multiphoton excitation, the absorption spectrum of ChRs is greatly broadened,\nand red-shifted ChRs can exhibit a pronounced blue shift during two-photon excitation. A ChR\nconsists of a seven-transmembrane (7TM) ion channel protein (opsin) and a covalently bound\nchromophore (retinal), which undergoes photoisomerization upon light absorption to control\nthe ion channel opening1,2. Specifically, the retinal chromophore that includes a β-ionone ring\nand forms a protonated Schiff base (PSB) with a lysine residue in the 7TM domain. Upon\nphoton absorption, the proton from the PSB is transferred to an adjacent carboxylate group.\nHowever, this process is strongly influenced by nearby counterions1,2. Specifically, the negative\ncharges of these counterions alter the electrostatic potential at the PSB and stabilize the ground\nstate, thereby increasing the energy gap between the ground and excited states. The complex\nlocal chromophore environment around the PSB introduces particular uncertainties under two-\nphoton excitation, for the following reasons:\n1. Interactions between the retinal and nearby counterions can lead to the formation of new\nstates that provide additional transition pathways, thereby broadening the excitation\nspectrum3-4.\n2. Certain vibronic transitions may be enhanced in two-photon excitation (TPE) as previously\nascribed5. And thus red-shifted ChRs, such as Chrimson/ChrimsonR, exhibit blue-shifted\nabsorption spectrum compared with twice their one-photon excitation spectrum. This\nphenomenon also appears in many orange and red fluorophores5–7.\n3. TPE can access higher excited singlet states that are not readily reachable via one-photon\nprocesses due to resonance enhancement effect8. For example, ChrimsonR shows an\nincreased absorption below 900 nm9. If counterions stabilize these higher states, TPE may\nmore efficiently excite the retinal, further contributing to enhanced excitation at blue-\nshifted wavelengths5.\n4. Because the 7TM constrains retinal chromophore and lead to loss of centrosymmetry,\nparity restrictions could be relaxed, permitting otherwise forbidden transitions10–12.\nWe extracted published two-photon absorption spectra of commonly used ChRs from the\nliterature, as summarized below. Consistent with theoretical predictions, ChR2, ChrimsonR,\nC1V1, and other ChRs exhibit markedly broadened two-photon absorption spectra9,13–16. For\nexample, although the excitation peak of ChrimsonR, the most red-shifted ChR, is at 1130 nm,\nit still retains ~45% excitation efficiency at 920 nm16. Similarly, the red-shifted C1V1 exhibits\n~35% excitation efficiency at 920 nm. These findings indicate that excitation of GCaMP at\n920 nm can still generate cross-talk if laser power is not carefully controlled, as observed in\nour study. Consistent with our results, other in vivo investigations have also reported cross-talk\nunder 920 nm imaging light when using red-shifted ChRs such as C1V1, ChrimsonR, and\nReaChR16–19.\nFor the third question, we fully agree with the reviewer that holographic light at the same\nwavelength as used for calcium imaging inevitably elevates the fluorescence baseline or\nbackground, thereby posing a challenge for the accurate extraction of calcium responses. We\nappreciate the reviewer pointing out the issue and regret that it was not discussed clearly in the\nprevious manuscript.\nAlthough the elevated baseline can interfere with calcium recordings, this issue can be\nmitigated by subtracting the fluorescence baseline, as demonstrated in previous studies20. In\nthis approach, signals from inactive pixels, ideally those where neither GCaMP nor ChR are\nexpressed, are treated as baseline fluorescence generated by holographic stimulation targeting\nneurons co-expressing both ChR and GCaMP. Therefore, subtracting this baseline from the\nGCaMP signal yields an undistorted calcium trace. In our study, it was not even necessary to\nidentify the inactive pixels. The baseline fluorescence is extracted from the signal acquired\nduring the scanning flyback period at the edge of the line, when the imaging laser power is\nturned off by the APPC. Subtracting this baseline from the GCaMP signal results in an\nundistorted calcium trace. Based on our experimental results, we were able to activate up to 10\nneurons without saturating the PMT, and we estimate the upper limit to be around 25 under our\nexperimental conditions, which is adequate for most in vivo optogenetic applications21–23.\nIndeed, the PMT may become saturated in more complex applications, such as the\nsimultaneous activation of hundreds of neurons. In such cases, integrating APPC into a dual-\nlaser system, using 920 nm for GCaMP excitation and a longer wavelength for stimulation of\nred-shifted ChRs, offers an effective strategy to eliminate baseline fluorescence and\nsubstantially suppress cross-talk. In contrast, for single-laser systems, specialized approaches\nare required to address baseline fluorescence. Here, we propose two potential strategies that\nexploit the short duration of holographic stimulation (<100 ms) relative to the slow dynamics\nof GCaMP fluorescence (~seconds)20,24, thereby enabling accurate recovery of calcium signals:\n1. Dynamic PMT gain adjustment: Temporarily reduce the PMT gain by lowering the driving\ncurrent during holographic stimulation.\n2. Mechanical shuttering of the PMT: Place a mechanical shutter, which achieves millisecond\ncontrol, in front of the PMT during the holographic stimulation period to prevent excessive\nlight from reaching the detector. For example:\na. Electronic Shutter (Newport): https://www.newport.com/p/76992\nb. Optical Shutters (Thorlabs):\nhttps://www.thorlabs.com/newgrouppage9.cfm?objectgroup_id=927\nWe are grateful for the reviewer’s thoughtful comments. In response, we have carefully\nrevised the manuscript as detailed below and clarified the issues raised.\nReferences:\n1. Zhang, F. et al. The microbial opsin family of optogenetic tools. Cell 147, 1446–1457\n(2011).\n2. Deisseroth, K. & Hegemann, P. The form and function of channelrhodopsin. Science\n(1979) 357, (2017).\n3. A. Cembran, F. Bernardi, M. Olivucci, and M. Garavelli, “The retinal\nchromophore/chloride ion pair: Structure of the photoisomerization path and interplay\nof charge transfer and covalent states,” Proc. Natl. Acad. Sci. U. S. A., vol. 102, no. 18,\npp. 6255–6260, 2005"}]

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