Deep two-photon brain imaging with a red-shifted fluorometric Ca2+ indicator

Proc Natl Acad Sci U S A. 2015 Sep 8;112(36):11377-82. doi: 10.1073/pnas.1514209112. Epub 2015 Aug 24.


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.

Keywords: calcium imaging; mouse cortical circuits; multicolor functional imaging; neuronal activity.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Action Potentials / physiology
  • Animals
  • Calcium / analysis
  • Calcium / metabolism*
  • Cerebral Cortex / cytology
  • Cerebral Cortex / metabolism
  • Dendrites / metabolism
  • Dendrites / physiology
  • Egtazic Acid / analogs & derivatives
  • Egtazic Acid / chemistry
  • Egtazic Acid / metabolism
  • Female
  • Fluorescent Dyes / chemistry
  • Fluorescent Dyes / metabolism
  • Fluorometry / methods*
  • Indicators and Reagents / chemistry
  • Indicators and Reagents / metabolism
  • Male
  • Mice, Inbred C57BL
  • Microscopy, Fluorescence, Multiphoton / methods*
  • Neuroimaging / methods*
  • Neurons / metabolism
  • Neurons / physiology
  • Patch-Clamp Techniques
  • Reproducibility of Results


  • Fluorescent Dyes
  • Indicators and Reagents
  • Egtazic Acid
  • 1,2-bis(2-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid
  • Calcium