Scientists at the Max Planck Florida Institute for Neuroscience (MPFI), in collaboration with ZEISS and MetaCell, have developed a powerful new imaging pipeline called Neuroplex. Published in eLife , the technique allows simultaneous monitoring of the activity of up to nine distinct neuronal populations in freely moving mice, dramatically accelerating the pace of scientific exploration into how the brain controls behavior.
The Challenge
For years, neuroscientists linking brain activity to behavior have faced a fundamental limitation: miniscopes, the tiny head-mounted microscopes used to observe neural activity in behaving animals, could capture neural activity, but couldn't reliably distinguish more than two different types of brain cells at a time.
"To understand the brain, we need to link patterns of activity in specific neurons to behavior," stated lead author Dr. Mary Phillips. "We can readily use labels to color-code different populations of neurons, but when using miniscopes to correlate neural activity to behavior, we couldn't distinguish more than two of these populations. This made it difficult to compare the activity across multiple cell types and circuits to understand how specific circuits regulate behavior."
To work around this, researchers were forced to test one cell type at a time, repeating the same behavioral experiments, but labeling distinct neuron types each time. This iterative process, however, was slow and costly. It also prevented direct comparison of different neuron types within the same animal, muddying conclusions due to differences among individual animals. As an alternative, scientists delineated different neuron types after the behavioral experiment by removing and slicing brain tissue, color-coding different neuron types, then imaging the processed brain tissue using microscopes that can distinguish multiple colors. However, matching the cells imaged with a miniscope in a living animal to those in post-mortem, processed brain tissue was challenging and low-throughput, resulting in significant data loss. Additionally, this approach destroyed the ability to track the activity of identified cell types over time to determine how their activity changes with learning, aging, or during disease progression.
The Solution: Neuroplex
To overcome these challenges, the MPFI team, together with collaborators at ZEISS and MetaCell, developed Neuroplex, an imaging pipeline that combines the two complementary imaging approaches in the same living animal. Researchers first label up to nine different neural circuits or cell types using a spectrum of differently-colored fluorescent tags. They then use a tiny lens and a head-mounted miniscope to record the neural activity of the entire labeled population in freely moving, behaving mice. After miniscope imaging, which cannot distinguish among the fluorescent tags, the miniscope is gently removed, and the mouse is positioned under a confocal microscope capable of distinguishing many different colors.
In this case, scientists used the ZEISS LSM 980, a confocal microscope with spectral detection capabilities to distinguish each of the different color tags. With the confocal microscope, the same neurons visualized with the miniscope are imaged through the same lens, but this time the color-coded tags are visualized, identifying which neurons belong to which specific type. Finally, the images from the miniscope and the confocal are co-registered using anatomical landmarks and a custom Python-based alignment tool that the scientists developed with MetaCell. The result is that the team can map each neuron's color identity directly onto its functional activity record.
"As part of MetaCell's contribution to this project, we helped take the complex data collected and turn it into a practical computational workflow that enables imaging, registration, and analysis with greater accuracy, reproducibility, and confidence. Neuroplex shows how carefully designed computational tools can help researchers make sense of complex biological imaging data and study multiple neuronal populations at once and over time," says Dr. Zhe Dong, co-author and Data Scientist at MetaCell.
As proof-of-principle, the researchers retrogradely targeted nine brain regions that receive projections from the medial prefrontal cortex, a brain area important for decision making. This allowed them to use a distinct fluorescent marker to distinguish neurons projecting from the prefrontal cortex to nine other brain regions. They recorded the activity of the neurons across all nine circuits simultaneously as animals interacted socially, sniffing, approaching and following.
"Neuroplex allowed direct comparison of neural activity patterns across cell circuits during social behavior, overcoming long-standing challenges in miniscope recordings and dramatically expanding the efficiency and reproducibility of data collection," explains senior author Dr. Ryohei Yasuda.
The scientists found that approximately 75% of active neurons could be assigned to one of the nine specific cell types, and the automated program built to assign a neuron to a specific group performed with 90% accuracy and few false positives.
"Because Neuroplex is performed entirely in the living animal through the same implanted lens, it enables scientists to measure how different populations of neurons change their activity over time. Researchers can identify cell populations prior to behavior and monitor the same neurons over weeks or months, enabling studies of learning, aging, and disease progression over time," described Dr. Phillips.
What Comes Next
The team is already working on even more improvements to the technique to increase the accuracy of color code identification. Additionally, they hope to make Neuroplex accessible to all labs, including those that may not have access to high-end spectral confocal systems. Their goal is to disseminate this approach widely to the neuroscience community by using standard filter-based widefield microscopes, bring the core benefits of the approach to the entire research community.
"The increase in data collection efficiency for cell-type- or circuit-specific functional data will accelerate our understanding of the neural computations underlying behavior," says Phillips. "Beyond basic research, we expect this approach to accelerate understanding of circuit-specific functional changes in disease models, particularly in neurodevelopmental or neurodegenerative disease models, which benefit from longitudinal studies examining disease progression."
To disseminate the approach, the team has also developed tutorials for scientists who wish to use Neuroplex in their own research. In addition, the approach will be featured in a ZEISS webinar with first author Dr. Mary Philips on July 14th to share the technique and resources with the scientific community. Register here for more details.
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 Link
This research was funded by National Institutes of Health Grants R35-NS-116804 (RY) and F32MH120872 (M.L.P.) This content is solely the authors' responsibility and does not necessarily represent the official views of the funders.
About Max Planck Florida Institute for Neuroscience
The Max Planck Florida Institute for Neuroscience (MPFI) is a not-for-profit basic scientific research institution located in Jupiter, Florida. It is the first Max Planck Society institute established in the United States. MPFI is committed to exploring the structure and function of neural circuits that underlie sensory perception, learning, and memory.
About MetaCell
MetaCell is a life science technology partner that helps pharmaceutical, medtech, and advanced R&D teams turn complex scientific workflows into secure, usable, production-ready software. For the past 15 years, its scientists and software engineers have built custom tools, platforms, and data experiences across imaging, biomarkers, omics, analytics, and cloud-based collaboration for leading organizations including Johnson & Johnson, Pfizer, the Allen Institute, Yale, Stanford, and CZI Biohub.
About ZEISS
ZEISS Research Microscopy Solutions is the leading provider of light, electron, X-ray microscope systems, correlative microscopy and software solutions leveraging AI technologies. The portfolio comprises of products and services for life sciences, materials and industrial research, as well as education and clinical routine applications. The unit is headquartered in Jena. Additional production and development sites are located in Germany, UK, USA, China and Switzerland. ZEISS Research Microscopy Solutions is part of the Industrial Quality & Research segment.
