For example, MEMS mirrors can be modified to include an axial scanner, but this adds mechanical and optical complexity that limit miniaturizion 16. Although several axial-scanning solutions have been demonstrated for FCMs, these have not been sucessfully translated to in vivo neuronal recording in freely-moving animals due to various drawbacks in these methods.
Axial-scanning requires an additional third degree-of-freedom that can complicate the design of the optics and mechanics of the FCM. Lateral imaging with a FCM is accomplished by a variety of techniques that scan the excitation laser focal spot in two axes at the sample. In order to fully study the spatially complex neuronal interconnections using miniaturized laser-scanning microscopy, it is required to image in 3D. However, all of these devices suffer from lack of active axial-scanning that greatly limits their applications. Furthermore, a variety of miniature laser-scanning fiber-coupled microscopes (FCMs) for confocal and 2PE imaging have been developed 13, 14, 15. Several commercial devices have been developed for head mounted wide-field epifluorescence imaging in freely-behaving mice 11, 12. In the previous decade, there has been a technological push to allow for imaging of neuronal activity of freely-moving animals to study neuronal circuitry in behaviors such as spatial navigation 8, social behavior 9, or prey capture 10. As optical proteins, such as genetically-encoded fluorescence Ca 2+ sensors, have continued to improve 7, 2PE microscopy has been a dominant tool for in vivo imaging in neuroscience research.
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. 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. The 2P-FCM with dynamic axial scanning provides a new capability to record from functionally distinct neuronal layers, opening new opportunities in neuroscience research. The 2P-FCM has a weight of only ~2.5 g and is capable of repeatable and stable head-attachment. 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. 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. 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-color simultaneous imaging of GFP and tdTomato fluorescence is also demonstrated. 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. 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.
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