A tiny microscope weighing just five grams could give neuroscientists an unprecedented view of how individual brain cells control behavior while animals move naturally. Researchers at the University of Colorado Anschutz Medical Campus and the University of Colorado Boulder have developed a head-mounted device that can both record neural activity and selectively activate individual neurons during movement. The technology could help scientists investigate how brain circuits generate behavior and how those circuits become disrupted in conditions such as Alzheimer’s disease, Parkinson’s disease and epilepsy.

The device, called Opto2P-FCM, combines two powerful techniques that have traditionally been difficult to use together in a freely moving animal. Two-photon microscopy provides detailed images of living brain tissue at cellular resolution, while optogenetics uses light-sensitive proteins to control selected neurons. By integrating both capabilities into a miniature instrument, the researchers can observe specific cells as they become active and then stimulate chosen neurons to test how they influence the surrounding network.

“These miniature microscopes have been increasingly used to study the neural basis of behavior in freely moving animals,” said Emily Gibson, PhD, associate professor of biomedical engineering at CU Anschutz and the study’s senior author. “This new microscope allows deeper penetration and higher resolution images in the brain. Importantly, it can also excite select neurons using optogenetics.”

For decades, conventional two-photon microscopes have been regarded as the gold standard for imaging neurons in living brains. These systems use infrared laser light to produce high-resolution images deep within tissue, allowing researchers to track calcium signals and other indicators of neural activity. However, conventional microscopes are generally large, stationary instruments. Animals must often remain immobilized beneath the objective, making it difficult to study brain activity during walking, exploration, social interaction or other natural behaviors.

Miniature microscopes have helped overcome that limitation by moving the imaging system onto the animal’s head. Yet reducing the size and weight of the equipment has often required compromises. Some compact systems provide lower-resolution images, while others can record neural activity but cannot stimulate precisely selected cells. The Colorado team designed Opto2P-FCM to address both problems through a dual-path optical architecture that separates imaging from stimulation.

In the imaging pathway, infrared light is directed through the brain to generate two-photon signals from fluorescent markers inside neurons. These markers can report changes in calcium concentration, which occur when neurons fire. The resulting signals are collected to create detailed images of neural activity at the level of individual cells. A separate optical pathway delivers patterned light for optogenetic stimulation, allowing researchers to activate specific neurons without disrupting the imaging beam or overwhelming the detectors.

This separation is technically important because imaging and stimulation place different demands on an optical system. High-resolution imaging requires precise focusing, efficient light collection and minimal optical distortion. Optogenetic activation, by contrast, requires intense, accurately positioned light capable of reaching the light-sensitive proteins expressed by selected neurons. Designing both functions into a device small enough to be carried by an animal required careful control of the alignment, geometry and weight of every optical component.

“This microscope is a game changer,” said Juliet Gopinath, PhD, professor of electrical, computer and energy engineering and physics at CU Boulder and a study co-author. “Being able to demonstrate a state-of-the-art instrument for both read-out and photo-stimulation of neurons is amazing.” The research team included engineers, physicists and neuroscientists who worked together to solve problems involving miniature lenses, mechanical tolerances, optical alignment and three-dimensional fabrication.

The device weighs approximately five grams, but its small size made construction unusually demanding. Mo Zohrabi, PhD, a senior research scientist at CU Boulder and co-first author, said the team initially thought such an instrument might be impossible to build because of the tight tolerances required by the optics and available 3D-printing technology. Gregory Futia, PhD, a senior research associate at CU Anschutz and co-first author, said that every component had to be designed and accounted for with extreme precision. Even slight misalignment could reduce image quality or prevent the stimulation beam from reaching the intended cells.

The researchers believe the platform could transform experiments designed to distinguish correlation from causation in the brain. Recording a neuron’s activity can show that it is associated with a behavior, but it does not prove that the cell helps produce that behavior. With optogenetic stimulation, scientists can activate a selected population and observe whether movement, decision-making or other actions change. At the same time, the microscope can record how neighboring neurons respond, providing a view of how local circuits coordinate activity.

The current Opto2P-FCM system is a prototype, and the researchers are working on future versions that could be smaller, lighter and faster. They also hope to expand the field of view so that more neurons can be monitored at once. Such improvements could make it possible to track larger networks over longer periods while animals engage in increasingly complex behaviors. By combining cellular-resolution observation with targeted manipulation in a freely moving subject, the technology may help reveal how healthy neural circuits operate and how neurological disease changes the communication between them.

Subject of Research: Miniature two-photon microscopy, optogenetics, neural circuits and brain activity during natural movement

Article Title: New Miniature Microscope Allows Scientists to Watch and Control Brain Cells During Natural Movement

News Publication Date: Aug. 11, 2026

Web References: University of Colorado Anschutz Medical Campus; University of Colorado Boulder; Optica study

References: Optica, “New Miniature Microscope Allows Scientists to Watch and Control Brain Cells During Natural Movement,” article publication date: Aug. 7, 2026

Keywords

Miniature microscope, two-photon microscopy, optogenetics, neuroscience, brain imaging, neural circuits, freely moving animals, biomedical engineering, applied physics, neurological disease, brain research

Expansion microscopy has spent the past decade changing the rules of super-resolution imaging. Instead of relying solely on increasingly sophisticated optics, the technique physically enlarges biological specimens so that molecules separated by nanometers become easier to distinguish with ordinary fluorescence microscopes. Now, researchers have introduced a method designed to push that concept toward one of the most difficult goals in microscopy: seeing the shapes of individual proteins directly.

The approach, described by Ahmed H. Shaib, Mahmoud M. Alawieh and Stefan O. Rizzoli in Nature Protocols, combines one-step nanoscale expansion microscopy with fluctuation-based super-resolution analysis. The resulting workflow, called ONE microscopy, is intended to make molecular-scale imaging more accessible to laboratories that do not have access to cryo-electron microscopes or specialized high-end optical systems.

Traditional light microscopy is limited by diffraction, a physical effect that causes light from two nearby objects to blur together when they are too close. Even modern super-resolution methods can require complex instruments, intense labeling strategies or extensive computational processing. Expansion microscopy takes a different route. Researchers anchor biological molecules within a swellable polymer network and then expand the material, increasing the physical distance between fluorescent labels.

The expansion step does not automatically reveal every molecular detail. A protein’s structure can still be represented by only a small number of fluorescent signals, and conventional images may contain noise, background fluorescence and motion-related fluctuations. ONE microscopy addresses these limitations by analyzing changes in fluorescence intensity over time. These fluctuations contain information about the presence, position and behavior of labeled molecules that may not be obvious in a single frame.

In practical terms, the method links chemical preparation, physical enlargement and computational analysis into a single workflow. Samples are embedded in a polymer gel, labeled to identify the proteins of interest and then expanded. After expansion, researchers acquire image sequences using conventional fluorescence equipment. Specialized software analyzes the temporal variation in the recorded signals, extracting spatial information beyond what a standard diffraction-limited image would provide.

This combination is significant because it shifts the focus from simply locating a protein to examining its overall shape. For many biological questions, knowing that a protein is present is not enough. Its size, orientation and structural organization can determine how it interacts with membranes, vesicles, organelles or neighboring proteins. Directly observing those features could help researchers investigate molecular machines in their native cellular environments rather than relying exclusively on purified samples or averaged structural models.

The protocol is designed to work across a broad range of biological materials, including purified proteins, cultured cells and tissues. That flexibility could make the technique useful for researchers studying protein organization at multiple scales. A purified protein might provide a controlled test of shape reconstruction, while cells and tissues could reveal how the same protein is arranged amid the crowded and complex environment of living biology.

A major part of the reported advance is the accompanying software package. Fluctuation-based imaging can be powerful, but its usefulness depends on reliable data processing, and computational analysis has often been a barrier for non-specialist users. The authors present the software as stable and user-friendly, with the goal of making the analysis more efficient, reproducible and practical for laboratories using standard fluorescence microscopes.

ONE microscopy does not replace cryo-electron microscopy, which remains capable of resolving structures at atomic or near-atomic scales under appropriate conditions. Nor does it eliminate the challenges associated with labeling, gel chemistry, image quality and sample preparation. Expansion can introduce distortions, and successful imaging depends on preserving the relationship between the fluorescent labels and the underlying structures. Nevertheless, the method offers a complementary strategy: rather than averaging thousands or millions of molecules, it aims to examine individual protein shapes through expanded, fluorescence-labeled specimens.

The researchers describe the workflow as a practical framework for protein imaging on conventional equipment. By combining physical separation of fluorophores with information extracted from fluorescence fluctuations, the method brings nanoscale structural analysis closer to routine biological imaging. Its broader impact may come not from replacing existing forms of super-resolution, but from making a previously specialized capability more reproducible and attainable for laboratories investigating how individual proteins operate inside cells and tissues.

Subject of Research: One-step nanoscale expansion microscopy for visualizing individual protein shapes using conventional fluorescence microscopes.

Article Title: ONE microscopy.

Article References: Shaib, A.H., Alawieh, M.M. & Rizzoli, S.O. ONE microscopy. Nature Protocols (2026). https://doi.org/10.1038/s41596-026-01399-x

Image Credits: AI Generated

DOI: https://doi.org/10.1038/s41596-026-01399-x

Keywords: expansion microscopy, ExM, nanoscale imaging, super-resolution microscopy, fluctuation-based analysis, protein structure, fluorescence microscopy, single-protein imaging, cryo-electron microscopy, biological imaging

When designing microscopes, optical engineers have long faced a trade-off between speed, field of view and resolution. But