Three-photon microscopy (3PM) was shown to allow deeper imaging than two-photon microscopy (2PM) in scattering biological tissues, such as the mouse brain, since the longer excitation wavelength reduces tissue scattering and the higher-order non-linear excitation suppresses out-of-focus background fluorescence. Imaging depth and resolution can further be improved by aberration correction using adaptive optics (AO) techniques where a spatial light modulator (SLM) is used to correct wavefront aberrations. Here, we present and analyze a 3PM AO system for in vivo mouse brain imaging. We use a femtosecond source at 1300 nm to generate three-photon (3P) fluorescence in yellow fluorescent protein (YFP) labeled mouse brain and a microelectromechanical (MEMS) SLM to apply different Zernike phase patterns. The 3P fluorescence signal is used as feedback to calculate the amount of phase correction without direct phase measurement. We show signal improvement in the cortex and the hippocampus at greater than 1 mm depth and demonstrate close to diffraction-limited imaging in the cortical layers of the brain, including imaging of dendritic spines. In addition, we characterize the effective volume for AO correction within brain tissues, and discuss the limitations of AO correction in 3PM of mouse brain.
Three-photon imaging of synthetic dyes in deep layers of the neocortex
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.
- Publication Date:
- NSF-PAR ID:
- 10195895
- Journal Name:
- Scientific Reports
- Volume:
- 10
- Issue:
- 1
- ISSN:
- 2045-2322
- Publisher:
- Nature Publishing Group
- Sponsoring Org:
- National Science Foundation
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