Acetylcholine, a pivotal neurotransmitter in both central and peripheral nervous systems, orchestrates a vast range of physiological processes from muscle activation to cognitive function and emotional regulation. Despite its critical role, the real-time tracking of ACh release alongside other neurochemicals has been severely limited by the lack of sensors capable of multiplex imaging with minimal spectral overlap. Traditional sensors, often fluorescing in the green spectrum, restrict simultaneous visualization due to excitation and emission conflicts with other green fluorescent probes.

The team led by Xie, Miao, Li, and colleagues has adeptly addressed these constraints by engineering a suite of red-shifted genetically encoded GPCR Activation-Based (GRAB) ACh sensors, termed rACh series, with remarkable sensitivity and dynamic range. These sensors exhibit fluorescence emission in the red spectral region, which not only expands the palette for multiplex imaging but also enhances tissue penetration depth and reduces background autofluorescence. Such properties are instrumental for in vivo applications, especially in deep brain regions requiring minimal invasiveness and high fidelity.

Among the newly developed probes, the high-affinity variant rACh1h stands out for its robustness in detecting endogenous ACh release across diverse brain territories, including the nucleus accumbens, a region central to reward processing; the amygdala, integral to emotion and memory; the hippocampus, key for learning; and the cortex, involved in higher cognitive functions. The versatility of rACh1h highlights its potential to unravel region-specific cholinergic dynamics and their implications in neurophysiology and pathophysiology.

Complementing their spectral advantage, these red fluorescent sensors are designed for seamless co-expression with existing green fluorescent neurochemical sensors, enabling researchers to monitor ACh levels in tandem with other signaling molecules such as dopamine, serotonin, or glutamate. This multiplexed approach is particularly critical for dissecting neuromodulator interactions, which underpin complex behaviors and neurological disorders.

The methodological innovations extend to their application modalities. Utilizing fiber photometry, mesoscopic imaging, and two-photon microscopy, the researchers demonstrated that rACh1h provides high spatiotemporal resolution necessary to capture rapid cholinergic fluctuations in freely behaving animals. Such technological adaptability allows for comprehensive interrogation of cholinergic signaling during naturalistic behaviors, advancing our understanding of its role in cognition, motivation, and affective states.

Beyond technical prowess, the implications of this work are profound for neuroscience research and drug discovery. The ability to dynamically monitor multiple neuromodulators concurrently provides a richer and more integrated view of neural circuit function and dysfunction. This insight could spearhead new therapeutic strategies targeting cholinergic and other neuromodulatory systems in diseases such as Alzheimer’s, schizophrenia, and addiction, where neurotransmitter imbalances are hallmark features.

Moreover, the red-shifted sensors’ spectral characteristics mitigate phototoxicity and photobleaching, common pitfalls in fluorescence imaging that can compromise longitudinal studies. This enhancement ensures more reliable data collection over extended timeframes, crucial for chronic experiments investigating developmental or disease progression effects.

The development process, grounded in cutting-edge molecular engineering, utilized directed evolution and rational design to optimize the sensor’s affinity, dynamic range, and photophysical properties. The sensor’s architecture integrates a conformationally sensitive GPCR domain with a red fluorescent protein, harnessing receptor activation-induced structural shifts to translate ACh binding events into fluorescent signals. This allosteric mechanism underpins the sensor’s specificity and responsiveness.

In a series of elegant validation experiments, the authors confirmed that rACh1h responds selectively to physiologically relevant concentrations of acetylcholine, with negligible cross-reactivity to other neurotransmitters or metabolites, thereby ensuring signal fidelity. In vivo experiments showcased the sensor’s ability to report cholinergic activity during behavioral paradigms, including reward, fear conditioning, and spatial navigation tasks, elucidating the temporal dynamics of ACh release in intricate neural networks.

Furthermore, the co-imaging capability was highlighted in experiments where rACh1h functioned simultaneously with green fluorescent dopamine sensors, capturing the orchestration between cholinergic and dopaminergic signaling pathways. These insights underscore the potential for elucidating neuromodulatory crosstalk mechanisms underlying motivation and reinforcement learning.

The versatility of this sensor platform extends beyond neuroscience into broader biomedical applications where acetylcholine might play a signaling role, such as the autonomic nervous system and inflammatory processes. The modular design principle promises adaptation to other neurotransmitters by swapping receptor domains, paving the way for a comprehensive toolkit of red fluorescent sensors for multiplex neurochemical imaging.

Importantly, the rACh sensors’ compatibility with two-photon microscopy enables deep-brain imaging with cellular-level resolution, critical for dissecting cholinergic signaling in densely packed neural circuits in vivo. This capability opens avenues to study cholinergic modulation in health and disease at previously inaccessible depths.

The researchers also demonstrated the sensors’ functionality under fiber photometry, a minimally invasive technique suitable for chronic recording in freely moving animals, facilitating the study of neuromodulation in ecological and complex behavioral contexts. This integration of advanced sensor engineering with versatile imaging modalities sets a new standard for in vivo neurotransmitter monitoring.

Looking forward, the implementation of these red-shifted GRAB sensors is anticipated to accelerate discoveries in neuroscience fundamentally. They offer a transformative approach to decipher the neural code through simultaneous multi-neurochemical recordings, potentially revealing how neurotransmitter interplay shapes cognition, emotion, and behavior in real time.

As neuroscientists continue to unravel the brain’s mysteries, tools like the rACh1h represent a critical leap toward a holistic understanding of neurochemical communication networks. This pioneering work underscores the power of molecular innovation combined with optical technology to chart new frontiers in brain research, with far-reaching implications for neuroscience, medicine, and beyond.

Such advancements not only enrich our scientific toolkit but also invigorate the quest for novel therapeutics targeting the cholinergic system and its interaction with other neuromodulators. The rACh sensor suite thus embodies both a technological milestone and a gateway to transformative insights into the neurochemical basis of behavior and brain function.

Subject of Research: Development of red fluorescent genetically encoded acetylcholine sensors for multiplex neurochemical imaging in vivo.

Article Title: Red-shifted GRAB acetylcholine sensors for multiplex imaging in vivo.

Article References:
Xie, S., Miao, X., Li, G. et al. Red-shifted GRAB acetylcholine sensors for multiplex imaging in vivo. Nat Neurosci (2026). https://doi.org/10.1038/s41593-026-02325-w

Image Credits: AI Generated

DOI: https://doi.org/10.1038/s41593-026-02325-w

Cells, primarily composed of aqueous environments with low refractive-index contrast, have long puzzled scientists striving to reveal their intricate internal architectures using conventional microscopy. Traditional fluorescence microscopy, despite its specificity, suffers from phototoxicity and photobleaching, rendering it less ideal for prolonged imaging sessions. Conversely, Intensity Diffraction Tomography (IDT), a label-free approach, circumvents these issues by exploiting the inherent refractive-index distributions of cellular structures, but it too grapples with environmental instabilities and optical aberrations over extended periods.

The innovation by Prof. Chao Zuo and his team leverages an iterative ptychographic algorithm at the heart of AO-FIDT to disentangle three-dimensional refractive index distributions from fluctuating wavefront aberrations. By directly extracting these parameters from unlabeled intensity images, the system dynamically adjusts its point spread function in real time, correcting distortions that would otherwise obscure or degrade fluorescence data. This synchronization of aberration correction across both imaging modalities enables sustained, high-fidelity 3D reconstructions.

At its core, AO-FIDT redefines the synergy between label-free imaging and molecular specificity. While IDT provides comprehensive structural maps by harnessing intrinsic optical properties, fluorescence microscopy complements this by labeling specific biomolecules. Traditionally, merging these modalities faced significant hurdles, especially in maintaining image stability during prolonged acquisitions. AO-FIDT’s adaptive optics framework ensures that both modalities remain corrected and aligned, preserving clarity and spatial accuracy throughout extended experiments.

The system’s efficacy was demonstrated using the self-developed ZRICON 3000 dual-modal microscope, which employs a ring-shaped illumination strategy meticulously matched to the numerical aperture. This configuration achieved an unprecedented lateral resolution of 175 nanometers and an axial resolution of 775 nanometers, capturing volumetric data at 7.5 volumes per second across sizeable cellular samples. By supplementing IDT with axial fluorescence stacks, the researchers further enhanced image fidelity and delineation of subcellular components.

One of the most compelling showcases of AO-FIDT’s robustness is its stability in the face of environmental perturbations, such as thermal drift and mechanical vibrations intrinsic to laboratory settings. These factors often induce wavefront distortions that disrupt the fidelity of fluorescence and diffraction tomography data, complicating long-term monitoring of live cells. AO-FIDT circumvents these issues by real-time aberration tracking and correction, enabling continuous visualization of dynamic organelle behavior without signal degradation.

The implications of this technology are vast. Cells’ dynamic processes—ranging from organelle morphology shifts to intracellular trafficking—unfold over hours or days, necessitating imaging modalities that combine resolution, specificity, and temporal continuity. AO-FIDT answers this call by providing a non-invasive, adaptable platform capable of capturing this complexity, empowering researchers to decode subtle phenotypic changes correlated with health, disease, or therapeutic interventions.

Looking ahead, the research team envisions integrating super-resolution techniques such as Structured Illumination Microscopy (SIM) with AO-FIDT. SIM offers enhanced spatial resolution and minimal phototoxicity, promising to reveal biomolecular arrangements with unprecedented detail. By combining SIM’s capabilities with AO-FIDT’s dual-modal and adaptive optics strengths, future systems could visualize complex subcellular architectures—like mitochondrial cristae—and their alterations in real time, deepening our understanding of cellular physiology.

AO-FIDT represents a paradigm shift in computational microscopy, exemplifying how advanced algorithms coupled with adaptive optics can transcend traditional hardware constraints. This innovation aligns with a broader trend toward computational methods that refine image quality and resolution post-acquisition or in real time, enhancing accessibility and usability in biomedical laboratories worldwide.

Professor Zuo emphasizes the unique virtue of this computational approach, stating that the method achieves sustained, high-resolution imaging without necessitating complex hardware modifications or extensive post-processing. This not only streamlines experimentation but also democratizes access to cutting-edge imaging, potentially accelerating discoveries across cell biology, pharmacology, and nanomedicine.

Beyond basic research, AO-FIDT’s non-invasive, label-free modality holds promise for translational applications including drug screening, cellular diagnostics, and in situ monitoring of therapeutic responses. The ability to track cellular morphology and molecular markers over time with minimal perturbation could revolutionize personalized medicine and cellular therapies.

In sum, AO-FIDT stands as a transformative technology in the microscopy landscape, bridging the gap between structural clarity and molecular insight with unmatched stability over extended periods. This dual-modal, adaptive optics-enabled strategy not only refines the visualization of cellular secrets but also opens new horizons for long-term live-cell studies, potentially catalyzing breakthroughs in biology and medicine.

Subject of Research:
Imaging analysis

Article Title:
Adaptive Optics-Assisted Long-Term 3D Fluorescence and Intensity Diffraction Tomography for High Spatiotemporal Resolution Cellular Imaging

News Publication Date:
7-Aug-2025

Web References:
http://dx.doi.org/10.1002/lpor.202570059

References:
Zhou, N., Zhang, R., Zhu, R., Zhou, Z., Wu, H., Chen, Q., Sun, J., & Zuo, C. (2025). Adaptive Optics-Assisted Long-Term 3D Fluorescence and Intensity Diffraction Tomography for High Spatiotemporal Resolution Cellular Imaging. Laser & Photonics Reviews, 19(15). DOI: 10.1002/lpor.202570059.

Image Credits:
Ning Zhou et al., Nanjing University of Science and Technology, Nanjing, China

Keywords

Cell biology, Microscopy, Imaging, Computational biology, Optics, Biomedical engineering, Biotechnology, Nanotechnology