Abstract
3-photon excitation enables in vivo fluorescence microscopy deep in densely labeled and highly scattering samples. To date, 3-photon excitation has been restricted to scanning a single focus, limiting the speed of volume acquisition. Here, for the first time to our knowledge, we implemented and characterized dual-plane 3-photon microscopy with temporal multiplexing and remote focusing, and performed simultaneous in vivo calcium imaging of two planes beyond 600 µm deep in the cortex of a pan-excitatory GCaMP6s transgenic mouse with a per-plane framerate of 7 Hz and an effective 2 MHz laser repetition rate. This method is a straightforward and generalizable modification to single-focus 3PE systems, doubling the rate of volume (column) imaging with off-the-shelf components and minimal technical constraints.
Highlights
Multiphoton laser-scanning fluorescence microscopy has enabled numerous studies of the structure and function of neural circuits in vivo
The idler beam was passed through a single-prism compressor which allowed substantial pre-chirp with robust tunability and a compact footprint. Because this pre-chirp was applied to both the remote focusing and temporal delay paths, it was critical to ensure the pulses in the two paths encountered identical group dispersion delay (GDD) and could be ideally compensated at the microscope focus by the same compressor configuration
Delaying one interferometer arm resulted in fringes in the fluorescence level reflecting the third-order interferometric autocorrelation (TIAC) [24, 25] of the excitation pulse
Summary
Multiphoton laser-scanning fluorescence microscopy has enabled numerous studies of the structure and function of neural circuits in vivo. If scattering is sufficiently strong, such as when attempting to image directly through intact bone [3] or at many scattering lengths deep in brain tissue [4, 5], the second-order nonlinearity becomes insufficient to suppress fluorescence generated by out-of-focus excitation light, inevitably obscuring the in-focus signal. In such cases, three-photon excitation (3PE) microscopy has been shown to penetrate further and enable high-resolution fluorescence imaging beyond the depth limit of 2PE, enabling for example imaging directly through the skull [6] and into subcortical layers [7]. The longer, short-wave IR wavelengths typically used and third-order nonlinearity in 3PE extend the scattering length and further reduce out-of-focus excitation, respectively
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