Practical Methods for In Vivo Cortical Physiology with 2-Photon Microscopy and Bulk Loading of Fluorescent Calcium Indicator Dyes

  • Stephen D. Van Hooser
  • Elizabeth N. Johnson
  • Ye Li
  • Mark Mazurek
  • Julie H. Culp
  • Arani Roy
  • Rishabh Kasliwal
  • Kelly Flavahan
Part of the Neuromethods book series (NM, volume 92)


In vivo 2-photon imaging of neurons that have been bulk-loaded with fluorescent calcium indicator dyes is permitting many fundamental principles of neural circuit organization and development to be uncovered. In this article, we describe the materials and procedures that we have used in our investigations of ferrets, tree shrews, and mice. Special attention is given to the design and construction of custom stereotaxic devices and the prevention of stray light from entering the 2-photon microscope during vision experiments.

Key words

Head plate Headplate Two-photon 2-photon AM dyes AM calcium dyes Oregon Green BAPTA-1 Light block Optical chamber Epifluorescence 



We thank Prakash Kara, Tom Mrsic-Flogel, Aaron Kerlin, and Clay Reid for their valuable advice as we were learning to perform 2-photon imaging. We also thank David Fitzpatrick and Leonard E. White for support and mentoring. We thank Frank Mello, machinist at Brandeis University, Don Pearce of the Medical Instrument Shop at Duke University Medical Center, and Janet Patterson of the Physics Machine Shop at Duke University for their creative input and expertise in building the stereotaxic devices we describe here. This work was supported by the National Institutes of Health, National Science Foundation, and the John Merck Foundation.


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Copyright information

© Springer Science+Business Media New York 2015

Authors and Affiliations

  • Stephen D. Van Hooser
    • 1
    • 2
  • Elizabeth N. Johnson
    • 2
    • 3
  • Ye Li
    • 2
    • 4
  • Mark Mazurek
    • 2
    • 5
  • Julie H. Culp
    • 2
    • 6
  • Arani Roy
    • 1
  • Rishabh Kasliwal
    • 2
  • Kelly Flavahan
    • 1
  1. 1.Department of BiologyBrandeis UniversityWalthamUSA
  2. 2.Department of NeurobiologyDuke University Medical CenterDurhamUSA
  3. 3.Duke Institute for Brain ScienceDuke UniversityDurhamUSA
  4. 4.Howard Hughes Medical InstituteUniversity of CaliforniaBerkeleyUSA
  5. 5.Department of BiologyMetropolitan State University of DenverDenverUSA
  6. 6.Center for NeuroscienceUniversity of CaliforniaDavisUSA

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