Neurons communicate by generating brief electrical impulses known as action potentials, or spikes. These signals travel along the cells and trigger communication with neighboring neurons, allowing networks of interconnected cells to process information. Conventional calcium imaging has enabled scientists to observe the activity of large numbers of neurons, but it does so indirectly. When a neuron fires, calcium ions flow into the cell, producing a chemical signal that can be detected through fluorescent indicators. Because calcium concentrations rise and fall relatively slowly, however, calcium imaging usually records activity over timescales of seconds or longer and may miss the individual spikes that carry information through neural circuits.

Voltage imaging offers a more direct alternative. Researchers can introduce genetically encoded voltage indicators into neurons, causing the cells to produce fluorescent proteins whose brightness changes when the electrical potential across the cell membrane changes. When a neuron fires, the indicator responds to the rapid shift in voltage, allowing the electrical event to be observed optically. In principle, this makes it possible to follow the timing and sequence of individual neural impulses. In practice, voltage signals are extremely brief and often faint, making it difficult to image them across a large three-dimensional brain at the speed and resolution required to distinguish individual cells.

To overcome this limitation, the MIT team modified a light-sheet microscope, an instrument designed to image large biological samples rapidly while reducing light exposure. A light sheet illuminates only a thin plane of tissue, and the microscope records the fluorescence emitted from that plane before moving through successive layers. Combining those images produces a three-dimensional representation of the sample. The researchers accelerated both parts of the process: they increased the acquisition speed of the camera and used a technique called remote refocusing to shift the imaging plane rapidly without mechanically moving the specimen or the main optical components.

The resulting instrument was able to scan the entire brain of a larval zebrafish 200 times per second, completing one full volume every five milliseconds. This rate is fast enough to capture many of the electrical events that conventional whole-brain imaging would blur or miss. The zebrafish is particularly useful for this kind of experiment because its larval brain is small and relatively transparent, allowing researchers to image neural activity throughout the organism without the need to physically remove tissue. Its nervous system also contains many of the major functional structures found in vertebrates, making it a valuable model for studying how brain-wide circuits operate.

For their demonstration, the researchers engineered larval zebrafish to express a genetically encoded voltage indicator called Positron2-Kv. The indicator did not produce usable signals in every neuron, but approximately one-quarter of the neurons showed fluorescence changes strong enough for analysis. Even this partial coverage allowed the team to observe activity across many brain regions at once. In fish that were resting, the microscope detected individual voltage spikes as well as rapid bursts of activity. These recordings provided a direct view of the timing of electrical signals and offered information that would be difficult to obtain from slower calcium measurements.

The system also revealed how activity spread through the brain after the fish received ultraviolet light. Soon after the stimulus, neurons in the optic tectum became active. This brain region receives visual information from the retina and performs early stages of visual processing. The activity then propagated across the tectum, moving from one side of the structure to the other. Because the microscope recorded the activity throughout the brain rather than in a single visual-processing area, the researchers could also observe stimulus-independent sequences in groups of neurons located in the cerebellum and hindbrain. These patterns suggest that spontaneous brain activity is organized across distributed networks, even when the animal is not responding to an obvious external signal.

The ability to observe voltage signals across a complete brain could change the way researchers formulate questions about neural computation. Brain functions rarely depend on isolated groups of neurons; instead, they emerge from interactions among circuits that may be separated by considerable anatomical distances. A neuron in a sensory region may influence cells involved in movement, attention, or internal state within milliseconds. If experiments examine only one region at a time, important participants in these network-wide processes can be overlooked. Whole-brain voltage imaging could help scientists identify coordinated activity patterns first and then investigate how specific neurons and connections contribute to them.

The new microscope is not yet a complete solution to the challenges of brain-wide recording. The researchers aim to increase the proportion of neurons that produce strong voltage-indicator signals while improving spatial resolution, imaging speed, and data analysis. Fluorescence must be collected quickly enough to distinguish a faint electrical response from background noise, and the enormous data volumes generated by high-speed three-dimensional imaging require sophisticated computational methods. The team is also exploring whether the approach can be adapted for other experimental organisms, including mice, whose larger and more complex brains present additional optical and technical obstacles.

Despite those limitations, the demonstration represents a significant step toward observing the brain as an integrated electrical network. By measuring voltage directly from neurons distributed throughout an entire organism, the method could help researchers connect rapid neural events with sensory responses, spontaneous activity, behavior, and internal mental states. Future experiments may use it to investigate how brain-wide circuits support learning, decision-making, movement, or states such as daydreaming. The broader goal is to understand not only what individual neurons do, but how thousands of cells coordinate their electrical activity to produce the unified functions of a living brain.

Subject of Research: Brain-wide voltage imaging of neuronal activity in larval zebrafish

Article Title: Voltage imaging of neurons distributed across entire brains of larval zebrafish

News Publication Date: 14-Aug-2026

Web References: https://doi.org/10.1038/s41592-026-03179-7

References: Nature Methods, DOI: 10.1038/s41592-026-03179-7

Keywords

Voltage imaging, genetically encoded voltage indicators, neurons, zebrafish, whole-brain imaging, light-sheet microscopy, neuroscience, neural networks, brain activity, Positron2-Kv

For years, neuroscientists have been grappling with the challenge of linking brain activity to behavior in a precise and cell-type-specific manner. The tiny head-mounted microscopes known as miniscopes have offered a window into neural activity in living, behaving animals, but their capacity to distinguish different neuronal identities was heavily constrained. Researchers could typically differentiate no more than two neural populations in a single experiment, forcing repetitive trials targeting different neuronal groups sequentially. This piecemeal approach not only consumed valuable time and resources but also complicated data interpretation due to animal-to-animal variability and prevented tracking the dynamic changes of neuronal populations over extended periods.

Traditional methods to circumvent this limitation involved post-experimental analysis of brain tissue. Scientists would euthanize the animal, slice the brain into thin sections, and use color-coded fluorescent markers to identify various neuronal subtypes under high-resolution microscopes. While this technique permitted finer cellular distinction, it suffered from critical drawbacks: the difficulty of matching neurons imaged during behavior with their post-mortem counterparts introduced significant data loss and precluded longitudinal studies of neuronal activity, as the living brain’s dynamics were no longer accessible.

Neuroplex ingeniously bridges these gaps by synergizing miniscope imaging with advanced spectral confocal microscopy in the same living animal. Initially, neuroscientists introduce a set of fluorescent markers designed to label up to nine different neural circuits or cell types, each with a unique color signature. The mice are then implanted with a tiny lens and outfitted with a miniscope that records neural activity in real-time as the animals freely navigate their environment. Although the miniscope itself cannot distinguish the specific fluorescent colors, it captures the functional activity of the entire labeled population.

Subsequently, the miniscope is carefully detached, and the mouse is placed under a high-end confocal microscope — in this case, the ZEISS LSM 980. Unlike miniscopes, this confocal system features spectral detection capabilities, allowing precise differentiation of the diverse fluorescent tags associated with each neuronal population. The same neurons observed via miniscope are imaged again through the identical lens, this time revealing their molecular identities according to color labels. These images are then computationally aligned and co-registered using anatomical landmarks and a custom Python-based alignment tool developed through collaboration with MetaCell. This innovative computational framework integrates the functional activity recorded in vivo with the molecular identity of each neuron, providing an unprecedented resolution of brain circuit dynamics.

Dr. Zhe Dong of MetaCell, a co-author on the study, highlights the critical role of computational sophistication in this breakthrough. By crafting a robust workflow for imaging, registration, and data analysis, MetaCell transformed complex, multi-dimensional biological data into interpretable outputs with enhanced accuracy, reproducibility, and researcher confidence. Such computational rigor is essential for making sense of the multifaceted datasets generated by Neuroplex, enabling scientists to simultaneously monitor and analyze multiple neuronal populations over time.

To illustrate the power of Neuroplex, the team focused on nine distinct brain regions receiving projections from the medial prefrontal cortex, a cerebral hub pivotal for decision making and social behavior. Using retrograde labeling techniques, they tagged neurons projecting from the prefrontal cortex to these diverse areas, each with a distinct fluorescent color. As the mice engaged in dynamic social interactions — sniffing, approaching, and following conspecifics — the researchers recorded the simultaneous activity of all nine neuronal circuits. This experiment marked a monumental advance, providing direct comparative insights into how interconnected neuronal networks orchestrate behavior in real time.

Results from these experiments were striking. Approximately 75% of the active neurons were successfully classified into one of the nine specified cell types, with the automated neuron classification algorithm achieving around 90% accuracy and minimal false positives. This high-fidelity mapping of functional data onto cellular identity illustrates Neuroplex’s enormous potential for unraveling the complex choreography of neuronal ensembles underpinning behavior. Moreover, the non-destructive nature of this approach makes it possible to longitudinally trace identified cell populations, opening avenues to study learning, memory, aging, and disease progression across extended timescales in living animals.

One of the most exciting aspects of Neuroplex is its ability to track changes in neuronal activity patterns as animals experience new environments, learn novel tasks, or undergo pathological transformations. These longitudinal studies, which were previously impractical or impossible, may provide vital clues about the mechanisms driving neurodevelopmental and neurodegenerative diseases. Understanding how different neuronal circuits adapt or deteriorate during disease could inform targeted interventions and therapies, propelling translational neuroscience research forward.

Looking ahead, the MPFI team is already pushing the boundaries of this technology. They are refining Neuroplex to further improve the precision and reliability of color code identification, thereby enhancing the resolution and robustness of circuit-specific data. Recognizing the importance of accessibility, they are also working to democratize this approach so that laboratories without access to expensive spectral confocal systems can adopt it. By developing variations compatible with standard filter-based widefield microscopes, their goal is to bring the core advantages of Neuroplex to a broader scientific community worldwide.

The potential ramifications of these advancements cannot be overstated. Neuroplex dramatically accelerates data collection efficiency for cell-type-specific functional studies, which will significantly deepen our understanding of how neural computations give rise to behavior. Beyond basic research insights, this technology holds promise for accelerating discoveries in disease modeling, especially where circuit-level dysfunction evolves over time. Early and precise observation of such changes could revolutionize the way we study neurological and psychiatric disorders.

To facilitate widespread dissemination of their breakthrough, the team has developed comprehensive tutorials available to scientists aiming to incorporate Neuroplex into their own research endeavors. Furthermore, upcoming webinars hosted by ZEISS, featuring Dr. Mary Phillips, aim to share practical knowledge and resources with the broader neuroscience community. These educational efforts underscore the team’s commitment to fostering collaboration and innovation across the field.

In summary, Neuroplex represents a transformative step in systems neuroscience. By overcoming longstanding technical barriers through a harmonious blend of cutting-edge imaging technology and sophisticated computational tools, this approach enables unprecedented insights into the intricate neural orchestra directing behavior. As Neuroplex continues to evolve and become more accessible, it promises to unlock new frontiers in brain research, ultimately enriching our understanding of the mind and its myriad functions.

Subject of Research: Animals

Article Title: Functional imaging of nine distinct neuronal populations under a miniscope in freely behaving animals

News Publication Date: 12-May-2026

Web References:

• Original publication in eLife
• Neuroplex tutorials by ZEISS
• ZEISS Webinar Registration

References:
Mary L. Phillips, Nicolai T. Urban, Taddeo Salemi, Zhe Dong, Ryohei Yasuda (2026) Functional imaging of nine distinct neuronal populations under a miniscope in freely behaving animals. eLife 15:RP110277. DOI: 10.7554/eLife.110277.3

Image Credits: Mary Phillips

Keywords: Neuroscience, Microscopy, Imaging, Ethology, Neural Circuits, Miniscope, Confocal Microscopy, Spectral Imaging, Neural Activity, Cell Type Specificity, In Vivo Imaging, Computational Neuroscience