Two-dimensional electro-optical multiphoton microscopy.

Significance: The development of genetically encoded fluorescent indicators of neural activity with millisecond dynamics has generated demand for ever faster two-photon (2P) imaging systems, but acoustic and mechanical beam scanning technologies are approaching fundamental limits. We demonstrate that potassium tantalate niobate (KTN) electro-optical deflectors (EODs), which are not subject to the same fundamental limits, are capable of ultrafast two-dimensional (2D) 2P imaging .

Aim: To determine if KTN-EODs are suitable for 2P imaging, compatible with 2D scanning, and capable of ultrafast imaging of genetically encoded indicators with millisecond dynamics.

The Cousa objective: a long-working distance air objective for multiphoton imaging in vivo.

Multiphoton microscopy can resolve fluorescent structures and dynamics deep in scattering tissue and has transformed neural imaging, but applying this technique in vivo can be limited by the mechanical and optical constraints of conventional objectives. Short working distance objectives can collide with compact surgical windows or other instrumentation and preclude imaging. Here we present an ultra-long working distance (20 mm) air objective called the Cousa objective.

Real-time reconstruction of high energy, ultrafast laser pulses using deep learning.

We report a method for the phase reconstruction of an ultrashort laser pulse based on the deep learning of the nonlinear spectral changes induce by self-phase modulation. The neural networks were trained on simulated pulses with random initial phases and spectra, with pulse durations between 8.5 and 65 fs.

Improving laser standards for three-photon microscopy.

Three-photon excitation microscopy has double-to-triple the penetration depth in biological tissue over two-photon imaging and thus has the potential to revolutionize the visualization of biological processes . However, unlike the plug-and-play operation and performance of lasers used in two-photon imaging, three-photon microscopy presents new technological challenges that require a closer look at the fidelity of laser pulses. We implemented state-of-the-art pulse measurements and developed innovative techniques for examining the performance of lasers used in three-photon microscopy.

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.