High-resolution focus-tunable one-photon miniaturized fluorescence microscope for imaging in freely moving animals

The relationship between neural activity, brain function, and corresponding biological behaviors remains a significant challenge in neuroscience. Exploring this relationship needs various optical imaging techniques to acquire real-time data with high spatial resolution. A promising technology recently is wearable miniaturized microscopes (mini scopes), which enable long-term neural activity recording in freely moving animals. However, most one-photon mini-scopes have limitations for imaging in-depth with high resolution and large field of view (FOV). To address this, we developed a one-photon miniaturized fluorescence microscope (1P-miniFM), intended for imaging of live brain neurons in free-behaving animals at subcellular level (~1.2 μm). We conducted specially designed optical path, achieving an imaging FOV of ~700 × 400 μm. In addition, we incorporated an electrowetting lens (EWL) to achieve a wide range of ~300 μm z-axis scanning with little resolution loss. 1P-miniFM is compact (11 × 17 × 24 mm) and lightweight (~2.9 g), causing little impediment to animals’ spontaneous behaviors. With genetically encoded calcium indicator GCaMP6s, we monitored neuron activities in secondary motor cortex (M2) during consecutive pain-related and sensory stimulations. We found that M2 neurons are key components and exhibit distinct variations in the response patterns. 1P-miniFM has potential as an excellent tool to explore relationships between neuron network and animal behaviors.