Improved deep two-photon calcium imaging in vivo |
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| Institution: | 1. Institute of Neuroscience, Technical University of Munich, Munich, Germany;2. Munich Cluster for Systems Neurology (SyNergy) and Center for Integrated Protein Sciences (CIPSM), Munich, Germany;1. Department of Biomedical Engineering, Vanderbilt University, Station B, Box 351631, Nashville TN 37235, United States;2. Laboratory for Functional Optical Imaging, Departments of Biomedical Engineering and Radiology, Columbia University, 120th Street and Amsterdam Avenue, New York, NY 10027, United States;1. Department of Neuroscience, Mortimer Zuckerman Mind Brain Behavior Institute, Columbia University, New York, NY 10027, USA;2. Kavli Institute for Brain Science, Columbia University, New York, NY 10027, USA;3. Department of Statistics, Columbia University, New York, NY 10027, USA;4. Grossman Center for the Statistics of Mind, Columbia University, New York, NY 10027, USA;1. Department of Anesthesiology and Critical Care, Penn Medicine, University of Pennsylvania, Philadelphia, PA, USA;2. Feil Family Brain and Mind Research Institute, Weill Cornell Medical College, New York, NY, USA;3. Molecular Neurobiology Program, Skirball Institute, Department of Physiology and Neuroscience, New York University School of Medicine, New York, NY, USA;4. Department of Anesthesiology, Columbia University Medical Center, New York, NY, USA;5. Department of Anesthesiology, New York University School of Medicine, New York, NY, USA;1. Princeton Neuroscience Institute, Princeton University, Princeton, NJ 08544, USA;2. Bezos Center for Neural Circuit Dynamics, Princeton University, Princeton, NJ 08544, USA;3. Department of Molecular Biology, Princeton University, Princeton, NJ 08544, USA;4. Janelia Research Campus, 19700 Helix Dr., Ashburn, VA 20147, USA;5. Howard Hughes Medical Institute;1. Division of Neurology, The Children’s Hospital of Philadelphia, Philadelphia, PA, 19104, USA;2. Medical Scientist Training Program (MSTP), The University of Pennsylvania Perelman School of Medicine, Philadelphia, PA, 19104, USA;3. The Neuroscience Graduate Group, The University of Pennsylvania Perelman School of Medicine, Philadelphia, PA, 19104, USA;4. Department of Neurology, The University of Pennsylvania Perelman School of Medicine, Philadelphia, PA, 19104, USA;5. Department of Neuroscience, The University of Pennsylvania Perelman School of Medicine, Philadelphia, PA, 19104, USA;1. Neurotechnology Center, Department of Biological Sciences, Columbia University, New York, NY 10027, USA |
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| Abstract: | Two-photon laser scanning calcium imaging has emerged as a useful method for the exploration of neural function and structure at the cellular and subcellular level in vivo. The applications range from imaging of subcellular compartments such as dendrites, spines and axonal boutons up to the functional analysis of large neuronal or glial populations. However, the depth penetration is often limited to a few hundred micrometers, corresponding, for example, to the upper cortical layers of the mouse brain. Light scattering and aberrations originating from refractive index inhomogeneties of the tissue are the reasons for these limitations. The depth penetration of two-photon imaging can be enhanced through various approaches, such as the implementation of adaptive optics, the use of three-photon excitation and/or labeling cells with red-shifted genetically encoded fluorescent sensors. However, most of the approaches used so far require the implementation of new instrumentation and/or time consuming staining protocols. Here we present a simple approach that can be readily implemented in combination with standard two-photon microscopes. The method involves an optimized protocol for depth-restricted labeling with the red-shifted fluorescent calcium indicator Cal-590 and benefits from the use of ultra-short laser pulses. The approach allows in vivo functional imaging of neuronal populations with single cell resolution in all six layers of the mouse cortex. We demonstrate that stable recordings in deep cortical layers are not restricted to anesthetized animals but are well feasible in awake, behaving mice. We anticipate that the improved depth penetration will be beneficial for two-photon functional imaging in larger species, such as non-human primates. |
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| Keywords: | Multi-photon microscopy Neuronal activity Calcium signaling Mouse visual cortex |
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