Our understanding of neural information processing could potentially be advanced by combining flexible three-dimensional (3-D) neuroimaging and stimulation. Recent developments in optogenetics suggest that neurophotonic approaches are in principle highly suited for noncontact stimulation of network activity patterns. In particular, two-photon holographic optical neural stimulation (2P-HONS) has emerged as a leading approach for multisite 3-D excitation, and combining it with temporal focusing (TF) further enables axially confined yet spatially extended light patterns. Here, we study key steps toward bidirectional cell-targeted 3-D interfacing by introducing and testing a hybrid new 2P-TF-HONS stimulation path for accurate parallel optogenetic excitation into a recently developed hybrid multiphoton 3-D imaging system. The system is shown to allow targeted all-optical probing of in vitro cortical networks expressing channelrhodopsin-2 using a regeneratively amplified femtosecond laser source tuned to 905 nm. These developments further advance a prospective new tool for studying and achieving distributed control over 3-D neuronal circuits both in vitro and in vivo.
Temporal focusing (TF) multiphoton systems constitute a powerful solution for cellular resolution optogenetic stimulation and recording in three-dimensional, scattering tissue. Here, we address two fundamental aspects in the design of such systems: first, we examine the design of TF systems with specific optical sectioning by comparatively analyzing previously published results. Next, we develop a solution for obtaining TF in a flexible three-dimensional pattern of cellmatched focal spots. Our solution employs spatio-temporal focusing (SSTF) in a unique optical system design that can be integrated before essentially any multiphoton imaging or stimulation system.
Temporal focusing (TF) nonlinear microscopy enables simultaneous illumination of relatively large areas while
maintaining optical sectioning, by relying on the sensitivity of multiphoton processes to pulse duration. Line
temporal focusing (LITEF) combines temporal focusing in one plane (xz) and spatial focusing in the perpendicular
plane (yz). The additional spatial focusing improves optical sectioning compared to wide field temporal focusing and exhibits improved performance in scattering medium.
Two photon microscopy’s ultimate depth of penetration is limited by out-of-focus excitation. This work explores
whether LITEF can be used to address this limitation. Here, we present experimental results displaying the
feasibility of ultra-deep penetration two-photon excitation in scattering media (<<1mm) using LITEF without
significant distortions or out-of-focus-excitation. Our experimental setup is based on an amplified 800nm ultrafast laser where a dual-prism grating (DPG) is used as a diffractive element, allowing light to propagate on-axis
throughout the optical setup, and providing a high diffraction efficiency. These results present new opportunities for ultra deep, optically sectioned 3D two photon imaging and stimulation within scattering biological tissue, beyond the known out-of-focus excitation limit.
The development of rapid volumetric imaging systems for functional multiphoton microscopy is essential for dynamical
imaging of large-scale neuronal network activity. Here, we introduce a line-illuminating temporal-focusing microscope
capable of rapid three-dimensional imaging at 10-20 volumes/sec, and study the system's characteristics both
theoretically and experimentally. We demonstrate that our system is capable of functional volumetric calcium imaging of
distributed neuronal activity patterns, and introduce a computational strategy for activity reconstruction in strongly
scattering media.
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