A technique for two-photon fluorescence imaging with cellular resolution in awake, mobile mice is described. The method uses a spherical treadmill with an air-supported Styrofoam ball to allow mice to move while their heads are restrained. This setup minimizes brain motion, enabling high-resolution imaging of neural activity. The apparatus combines an upright two-photon microscope with a spherical treadmill, allowing mice to run while their heads remain still. Image sequences show minimal brain motion, primarily in the focal plane, making motion correction algorithms effective for postprocessing. Calcium transients from large neuronal and astrocytic populations were measured with a low false positive error rate (<5%). The technique allows for the imaging of neural and astrocytic activity in behaving mice, with motion correction using a Hidden Markov Model (HMM) algorithm. The method enables the study of neural circuits in awake animals, overcoming previous limitations of anesthetized preparations. The spherical treadmill allows for stable imaging of brain activity during running, with minimal motion artifacts. The technique provides a new capability for in vivo two-photon microscopy of the mammalian brain, enabling the study of neural activity in awake, behaving mice. The results show that brain motion is limited to ~2-5 μm, with minimal out-of-plane motion. The method allows for the imaging of calcium dynamics in neurons and astrocytes, with high spatial resolution and minimal motion artifacts. The technique is suitable for studying neural activity in awake mice, with the potential for further applications in understanding neural circuits and behavior. The method provides a reliable way to image neural activity in awake mice, with minimal motion artifacts and high spatial resolution. The results demonstrate the feasibility of imaging neural activity in awake, behaving mice, with the potential for further applications in neuroscience research.A technique for two-photon fluorescence imaging with cellular resolution in awake, mobile mice is described. The method uses a spherical treadmill with an air-supported Styrofoam ball to allow mice to move while their heads are restrained. This setup minimizes brain motion, enabling high-resolution imaging of neural activity. The apparatus combines an upright two-photon microscope with a spherical treadmill, allowing mice to run while their heads remain still. Image sequences show minimal brain motion, primarily in the focal plane, making motion correction algorithms effective for postprocessing. Calcium transients from large neuronal and astrocytic populations were measured with a low false positive error rate (<5%). The technique allows for the imaging of neural and astrocytic activity in behaving mice, with motion correction using a Hidden Markov Model (HMM) algorithm. The method enables the study of neural circuits in awake animals, overcoming previous limitations of anesthetized preparations. The spherical treadmill allows for stable imaging of brain activity during running, with minimal motion artifacts. The technique provides a new capability for in vivo two-photon microscopy of the mammalian brain, enabling the study of neural activity in awake, behaving mice. The results show that brain motion is limited to ~2-5 μm, with minimal out-of-plane motion. The method allows for the imaging of calcium dynamics in neurons and astrocytes, with high spatial resolution and minimal motion artifacts. The technique is suitable for studying neural activity in awake mice, with the potential for further applications in understanding neural circuits and behavior. The method provides a reliable way to image neural activity in awake mice, with minimal motion artifacts and high spatial resolution. The results demonstrate the feasibility of imaging neural activity in awake, behaving mice, with the potential for further applications in neuroscience research.