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.
We report a miniature, lightweight fiber-coupled confocal fluorescence microscope that incorporates an electrowetting variable focus lens to provide axial scanning for full three-dimensional (3D) imaging. Lateral scanning is accomplished by coupling our device to a laser-scanning confocal microscope through a coherent imaging fiber-bundle. The optical components of the device are combined in a custom 3D-printed adapter with an assembled weight of <2 g that can be mounted onto the head of a mouse. Confocal sectioning provides an axial resolution of ~12 µm and an axial scan range of ~80 µm. The lateral field-of-view is 300 µm, and the lateral resolution is 1.8 µm. We determined these parameters by imaging fixed sections of mouse neuronal tissue labeled with green fluorescent protein (GFP) and fluorescent bead samples in agarose gel. To demonstrate viability for imaging intact tissue, we resolved multiple optical sections of ex vivo mouse olfactory nerve fibers expressing yellow fluorescent protein (YFP).
The cerebellum plays a crucial role in sensorimotor and associative learning. However, the contribution of molecular layer interneurons (MLIs) to these processes is not well understood. We used two-photon microscopy to study the role of ensembles of cerebellar MLIs in a go-no go task where mice obtain a sugar water reward if they lick a spout in the presence of the rewarded odorant and avoid a timeout when they refrain from licking for the unrewarded odorant. In naive animals the MLI responses did not differ between the odorants. With learning, the rewarded odorant elicited a large increase in MLI calcium responses, and the identity of the odorant could be decoded from the differential response. Importantly, MLIs switched odorant responses when the valence of the stimuli was reversed. Finally, mice took a longer time to refrain from licking in the presence of the unrewarded odorant and had difficulty becoming proficient when MLIs were inhibited by chemogenetic intervention. Our findings support a role for MLIs in learning valence in the cerebellum.
Abstract:Laser scanners are an integral part of high resolution biomedical imaging systems such as confocal or 2-photon excitation (2PE) microscopes. In this work, we demonstrate the utility of electrowetting on dielectric (EWOD) prisms as a lateral laser-scanning element integrated in a conventional 2PE microscope. To the best of our knowledge, this is the first such demonstration for EWOD prisms. EWOD devices provide a transmissive, low power consuming, and compact alternative to conventional adaptive optics, and hence this technology has tremendous potential. We demonstrate 2PE microscope imaging of cultured mouse hippocampal neurons with a FOV of 130 × 130 μm 2 using EWOD prism scanning. In addition, we show simulations of the optical system with the EWOD prism, to evaluate the effect of propagating a Gaussian beam through the EWOD prism on the imaging quality. Based on the simulation results a beam size of 0.91 mm full width half max was chosen to conduct the imaging experiments, resulting in a numerical aperture of 0.17 of the imaging system.
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