title: Deep two-photon brain imaging with a red-shifted fluorometric Ca2+ indicator.
abstract: In vivo Ca2+ imaging of neuronal populations in deep cortical layers has remained a major challenge, as the recording depth of two-photon microscopy is limited because of the scattering and absorption of photons in brain tissue. A possible strategy to increase the imaging depth is the use of red-shifted fluorescent dyes, as scattering of photons is reduced at long wavelengths. Here, we tested the red-shifted fluorescent Ca2+ indicator Cal-590 for deep tissue experiments in the mouse cortex in vivo. In experiments involving bulk loading of neurons with the acetoxymethyl (AM) ester version of Cal-590, combined two-photon imaging and cell-attached recordings revealed that, despite the relatively low affinity of Cal-590 for Ca2+ (Kd=561 nM), single-action potential-evoked Ca2+ transients were discernable in most neurons with a good signal-to-noise ratio. Action potential-dependent Ca2+ transients were recorded in neurons of all six layers of the cortex at depths of up to -900 µm below the pial surface. We demonstrate that Cal-590 is also suited for multicolor functional imaging experiments in combination with other Ca2+ indicators. Ca2+ transients in the dendrites of an individual Oregon green 1,2-bis(o-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid-1 (OGB-1)-labeled neuron and the surrounding population of Cal-590-labeled cells were recorded simultaneously on two spectrally separated detection channels. We conclude that the red-shifted Ca2+ indicator Cal-590 is well suited for in vivo two-photon Ca2+ imaging experiments in all layers of mouse cortex. In combination with spectrally different Ca2+ indicators, such as OGB-1, Cal-590 can be readily used for simultaneous multicolor functional imaging experiments.
authors: Institute for Neuroscience, Technische Universität München, 80802 Munich, Germany; Munich Cluster for Systems Neurology, 80802 Munich, Germany; Center for Integrated Protein Sciences, 80802 Munich, Germany.
authors: Carsten
authors: C
authors: Tischbirek
authors: Institute for Neuroscience, Technische Universität München, 80802 Munich, Germany; Munich Cluster for Systems Neurology, 80802 Munich, Germany; Center for Integrated Protein Sciences, 80802 Munich, Germany.
authors: Antje
authors: A
authors: Birkner
authors: Institute for Neuroscience, Technische Universität München, 80802 Munich, Germany; Munich Cluster for Systems Neurology, 80802 Munich, Germany; Center for Integrated Protein Sciences, 80802 Munich, Germany.
authors: Hongbo
authors: H
authors: Jia
authors: Institute for Neuroscience, Technische Universität München, 80802 Munich, Germany; bsakmann@neuro.mpg.de arthur.konnerth@tum.de.
authors: Bert
authors: B
authors: Sakmann
authors: Institute for Neuroscience, Technische Universität München, 80802 Munich, Germany; Munich Cluster for Systems Neurology, 80802 Munich, Germany; Center for Integrated Protein Sciences, 80802 Munich, Germany bsakmann@neuro.mpg.de arthur.konnerth@tum.de.
authors: Arthur
authors: A
authors: Konnerth
mesh_terms: D000200:Action Potentials / Q000502:physiology; D000818:Animals; D002118:Calcium / Q000032:analysis / Q000378:metabolism*; D002540:Cerebral Cortex / Q000166:cytology / Q000378:metabolism; D003712:Dendrites / Q000378:metabolism / Q000502:physiology; D004533:Egtazic Acid / Q000031:analogs & derivatives / Q000737:chemistry / Q000378:metabolism; D005260:Female; D005456:Fluorescent Dyes / Q000737:chemistry / Q000378:metabolism; D005470:Fluorometry / Q000379:methods*; D007202:Indicators and Reagents / Q000737:chemistry / Q000378:metabolism; D008297:Male; D008810:Mice, Inbred C57BL; D036641:Microscopy, Fluorescence, Multiphoton / Q000379:methods*; D059906:Neuroimaging / Q000379:methods*; D009474:Neurons / Q000378:metabolism / Q000502:physiology; D018408:Patch-Clamp Techniques; D015203:Reproducibility of Results
chemical_list: D005456:Fluorescent Dyes; D007202:Indicators and Reagents; D004533:Egtazic Acid; C025603:1,2-bis(2-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid; D002118:Calcium
keywords: calcium imaging; mouse cortical circuits; multicolor functional imaging; neuronal activity