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.
Bibliographical noteFunding 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).
© 2020, The Author(s).
- Fluorescent Dyes
- Microscopy, Fluorescence, Multiphoton/methods
PubMed: MeSH publication types
- Research Support, U.S. Gov't, Non-P.H.S.
- Journal Article
- Research Support, N.I.H., Extramural