7 research outputs found
Recommended from our members
Three dimensional two-photon brain imaging in freely moving mice using a miniature fiber coupled microscope with active axial-scanning.
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
Recommended from our members
Two-photon laser scanning microscopy with electrowetting-based prism scanning.
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 μ
Fiber-Coupled Microscopy for 3D Neuronal Imaging
In this dissertation I describe the design and implementation of miniature fiber-coupled microscopes (FCMs) with active focusing for three-dimensional (3D) neuronal imaging. The goal is to provide neuroscience researchers with versatile microscopy tools to perform neuronal optical imaging of awake and mobile mice. This research is motivated by the recent advancements of powerful genetically-encoded optical proteins, including fluorescent activity sensors as well as optogenetic actuators, which permit the functional interrogation of in vivo neuronal circuits. Newly developed miniature microscope tools allow optical imaging in freely-behaving mice, but current designs do not combine optical sectioning capabilities with active focusing for full 3D-imaging. The first three chapters in this dissertation serve as a background for current state of intravital fluorescence microscopy in neuroscience research. I argue for the importance of miniaturizing the microscope technologies that enable high-contrast imaging with optical sectioning, combined with axial focusing, to enable 3D-imaging at the rodent-scale. I present two FCM designs that achieve full 3D-imaging using a coherent imaging fiber bundle (CIFB) for lateral imaging and an electrowetting tunable lens (EWTL) to enable electrically tunable axial focusing. The first design is a confocal FCM (C-FCM) that takes advantage of the optical sectioning capability of the CIFB to acquire high-contrast images. The second design is a two-photon FCM (2P-FCM), in which pre-compensated ultrashort pulses are propagated through the CIFB for two-photon excitation microscopy. In each section, I characterize the 3D optical performance of the FCM. Finally, as a proof-of-principle using the 2P-FCM, I show in vivo 3D-imaging of neurons and Ca 2+-activity in the motor cortex of a freely-behaving mouse
Recommended from our members
Compact diode laser source for multiphoton biological imaging
We demonstrate a compact, pulsed diode laser source suitable for multiphoton microscopy of biological samples. The center wavelength is 976 nm, near the peak of the two-photon cross section of common fluorescent markers such as genetically encoded green and yellow fluorescent proteins. The laser repetition rate is electrically tunable between 66.67 kHz and 10 MHz, with 2.3 ps pulse duration and peak powers \u3e1 kW. The laser components are fiber-coupled and scalable to a compact package. We demonstrate \u3e600 μm depth penetration in brain tissue, limited by laser power
Molecular layer interneurons in the cerebellum encode for valence in associative learning
This study shows that cerebellar molecular layer interneurons (MLIs) develop responses encoding for identity of the stimulus in an associative learning task. Chemogenetic inhibition of MLIs decreased the ability of mice to discriminate stimuli suggesting that MLIs encode for stimulus valence