Developments in miniaturized microscopes have enabled visualization of brain activities and structural dynamics in animals engaging in self-determined behaviors. However, it remains a challenge to resolve activity at single dendritic spines in freely behaving animals. Here, we report the design and application of a fast high-resolution, miniaturized two-photon microscope (FHIRM-TPM) that accomplishes this goal. With a headpiece weighing 2.15 g and a hollow-core photonic crystal fiber delivering 920-nm femtosecond laser pulses, the FHIRM-TPM is capable of imaging commonly used biosensors (GFP and GCaMP6) at high spatiotemporal resolution (0.64 μm laterally and 3.35 μm axially, 40 Hz at 256 × 256 pixels for raster scanning and 10,000 Hz for free-line scanning). We demonstrate the microscope's robustness with hour-long recordings of neuronal activities at the level of spines in mice experiencing vigorous body movements.

The maintenance of a constant ATP level (‘set-point’) is a vital homeostatic function shared by eukaryotic cells. In particular, mammalian myocardium exquisitely safeguards its ATP set-point despite 10-fold fluctuations in cardiac workload. However, the exact mechanisms underlying this regulation of ATP homeostasis remain elusive. Here we show mitochondrial flashes (mitoflashes), recently discovered dynamic activity of mitochondria, play an essential role for the auto-regulation of ATP set-point in the heart. Specifically, mitoflashes negatively regulate ATP production in isolated respiring mitochondria and, their activity waxes and wanes to counteract the ATP supply-demand imbalance caused by superfluous substrate and altered workload in cardiomyocytes. Moreover, manipulating mitoflash activity is sufficient to inversely shift the otherwise stable ATP set-point. Mechanistically, the Bcl-xL-regulated proton leakage through F1Fo-ATP synthase appears to mediate the coupling between mitoflash production and ATP set-point regulation. These findings indicate mitoflashes appear to constitute a digital auto-regulator for ATP homeostasis in the heart.DOI: http://dx.doi.org/10.7554/eLife.23908.001

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