Three-photon imaging of synthetic dyes in deep layers of the neocortex

Chao J. Liu, Arani Roy, Anthony A. Simons, Deano M. Farinella, Prakash Kara

Research output: Contribution to journalArticlepeer-review

1 Scopus citations


Multiphoton microscopy has emerged as the primary imaging tool for studying the structural and functional dynamics of neural circuits in brain tissue, which is highly scattering to light. Recently, three-photon microscopy has enabled high-resolution fluorescence imaging of neurons in deeper brain areas that lie beyond the reach of conventional two-photon microscopy, which is typically limited to ~ 450 µm. Three-photon imaging of neuronal calcium signals, through the genetically-encoded calcium indicator GCaMP6, has been used to successfully record neuronal activity in deeper neocortical layers and parts of the hippocampus in rodents. Bulk-loading cells in deeper cortical layers with synthetic calcium indicators could provide an alternative strategy for labelling that obviates dependence on viral tropism and promoter penetration, particularly in non-rodent species. Here we report a strategy for visualized injection of a calcium dye, Oregon Green BAPTA-1 AM (OGB-1 AM), at 500–600 µm below the surface of the mouse visual cortex in vivo. We demonstrate successful OGB-1 AM loading of cells in cortical layers 5–6 and subsequent three-photon imaging of orientation- and direction- selective visual responses from these cells.

Original languageEnglish (US)
Article number16351
JournalScientific reports
Issue number1
StatePublished - Oct 1 2020

Bibliographical note

Funding Information:
We thank Austin Leikvoll for technical assistance during mouse surgeries and comments on the manuscript. This research was supported by grants from the NIH (R01 MH111447) and NSF (1707287).

Publisher Copyright:
© 2020, The Author(s).


  • Animals
  • Calcium/metabolism
  • Fluorescent Dyes
  • Mice
  • Microscopy, Fluorescence, Multiphoton/methods
  • Neocortex/physiology
  • Neurons/physiology

PubMed: MeSH publication types

  • Research Support, U.S. Gov't, Non-P.H.S.
  • Journal Article
  • Research Support, N.I.H., Extramural


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