Abstract
We present a miniature head mounted two-photon fiber-coupled microscope (2P-FCM) for neuronal imaging with active axial focusing enabled using a miniature electrowetting lens. We show three-dimensional two-photon imaging of neuronal structure and record neuronal activity from GCaMP6s fluorescence from multiple focal planes in a freely-moving mouse. Two-color simultaneous imaging of GFP and tdTomato fluorescence is also demonstrated. Additionally, dynamic control of the axial scanning of the electrowetting lens allows tilting of the focal plane enabling neurons in multiple depths to be imaged in a single plane. Two-photon imaging allows increased penetration depth in tissue yielding a working distance of 450 μm with an additional 180 μm of active axial focusing. The objective NA is 0.45 with a lateral resolution of 1.8 μm, an axial resolution of 10 μm, and a field-of-view of 240 μm diameter. The 2P-FCM has a weight of only ~2.5 g and is capable of repeatable and stable head-attachment. The 2P-FCM with dynamic axial scanning provides a new capability to record from functionally distinct neuronal layers, opening new opportunities in neuroscience research.
Highlights
Two-photon excitation (2PE) microscopy[1,2] has become a fundamental tool for probing in vivo neuronal circuits because it yields optical access deeper into tissue compared to single-photon fluorescence microscopy. 2PE microscopy allows for optical sectioning while imaging in scattering tissue and, when used in combination with rapid axial-scanning, allows for the retrieval of three-dimensional (3D) representations of neuronal structure and multiple focal plane imaging of neuronal activity[3,4,5,6]
The lateral scanning of the excitation laser is done at the proximal end of the coherent fiber bundle (CFB) using the galvanometric scanner in the 2PM to translate the lateral position of the laser focus through the spatially coherent fiber-cores from the proximal to the distal end
In order to maintain the ultrashort pulse-durations required for efficient 2PE, the laser pulse must be pre-compensated to account for nonlinear spectral narrowing and chromatic dispersion that arise from propagating high intensity light through the CFB cores
Summary
Two-photon excitation (2PE) microscopy[1,2] has become a fundamental tool for probing in vivo neuronal circuits because it yields optical access deeper into tissue compared to single-photon fluorescence microscopy. 2PE microscopy allows for optical sectioning while imaging in scattering tissue and, when used in combination with rapid axial-scanning, allows for the retrieval of three-dimensional (3D) representations of neuronal structure and multiple focal plane imaging of neuronal activity[3,4,5,6]. A variety of miniature laser-scanning fiber-coupled microscopes (FCMs) for confocal and 2PE imaging have been developed[13,14,15] All of these devices suffer from lack of active axial-scanning that greatly limits their applications. Other types of ETLs include shape-changing polymer lenses[19,20], pressure-driven lenses[21], and responsive hydrogel lenses[22] These suffer from not being adaptable to miniaturization, large optical aberrations, and/or being too susceptible to motion and orientation for freely-behaving animal imaging. The 2P-FCM presented here is, to the best of our knowledge, the first device to attain multiple arbitrary focal plane imaging of neuronal activity in the brain of a freely-moving mouse
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