Reduction of motion artifacts during in vivo two-photon imaging of brain through heartbeat triggered scanning

Key points Two-photon fluorescence microscopy enables analysis of the structure and dynamic activity of different cell types in the brains of living animals at high temporal and spatial resolution. Our ability to accurately resolve small structures, including dendrites and organelles in vivo, is hampered by pulsations in brain tissue that are caused by vital functions such as breathing and beating of the heart. Here we show that beating of the heart is the major cause of brain pulsations in the cerebral cortex and find that synchronizing imaging scans to cardiac cycles significantly reduces motion artifacts. We introduce a strategy (interlaced scanning) that enables electrocardiogram (ECG)-triggered scanning of large brain volumes, and provide software for implementation of ECG-triggered scanning on conventional two-photon microscopes. Abstract Two-photon imaging of fluorescence in brain enables analysis of the structure and dynamic activity of neurons and glial cells in living animals. However, vital functions such as beating of the heart cause pulsations in brain tissue, leading to image distortion and loss of resolution. We find that synchronizing imaging scans to the cardiac cycle reduces motion artifacts, significantly improving the resolution of cellular structures. By interlacing multiple heartbeat triggered imaging scans, it was possible to image large brain volumes with negligible distortion. This approach can be readily incorporated into conventional microscopes to achieve substantial reductions in motion artifacts during two-photon imaging.