New miniature microscope stably images brain cells during movement

Researchers at Peking University have developed DUET, a breakthrough dual-color miniature two-photon microscope that overcomes two persistent barriers in neuroscience imaging: motion-induced signal instability and fluorescence channel crosstalk. The work, published in PhotoniX Life, enables stable, high-fidelity recording of distinct neuron populations in freely moving mice during natural behaviors.

Head-mounted miniature microscopes have revolutionized the study of brain function by allowing scientists to observe neuronal activity at single-cell resolution in behaving animals. However, simultaneously imaging multiple cell types has been technically challenging. Existing approaches struggle to deliver multiple excitation wavelengths stably through flexible fibers during vigorous motion, and fluorescence signals from different indicators often interfere with one another, compromising quantitative analysis.

DUET (dual-fiber excitation and temporal multiplexing) addresses both challenges through an integrated hardware and timing-control strategy. The system employs two hollow-core photonic bandgap fibers (PBGFs) with exceptional bend tolerance to independently deliver 920-nm and 1030-nm femtosecond laser pulses for exciting green (GCaMP) and red (tdTomato) fluorescent indicators. A custom 0.276°-wedged dichroic mirror corrects beam-pointing mismatch between the two fiber outputs, while pixel-level temporal multiplexing alternates excitation wavelengths within each pixel dwell time. This approach eliminates both excitation and emission crosstalk without sacrificing the full 8.4 Hz imaging frame rate.

We designed DUET to be motion-robust. By transmitting two excitation wavelengths through independent PBGFs and synchronizing two detection channels at the pixel level, we achieve stable dual-color imaging even when the animal is struggling intensely."

Dr. Muyue Zhai, corresponding author, Peking University

The team validated DUET in demanding behavioral paradigms. During tail suspension tests, the system maintained stable dual-color imaging of cortical neurons with negligible channel crosstalk. In 20-minute open-field sessions, DUET continuously resolved calcium dynamics from 123 excitatory neurons and 10 inhibitory neurons, revealing cell-type-specific activity patterns during natural exploration.

"This level of stability and specificity opens new doors for studying how different neuron types interact during learning, decision-making, and social behavior," said Dr. Aimin Wang, co-corresponding author and professor at Peking University. "Because the dual-fiber architecture is modular, it can be readily extended to three or more colors, or combined with other imaging modalities." The DUET platform is compatible with standard digitizers and control electronics, facilitating adoption by neuroscience laboratories.