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

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

Spatial frequency modulated single detector imaging

This thesis presents images of absorbing and fluorescent objects captured by modulating a time varying spatial frequency to an illumination beam. The modulator produces a field intensity with a linear increase in temporal modulation frequency across its spatial extent. The linear temporal modulation is preserved after square law integration over the area of the detector and present in its electronic signal. Recording the temporal signal out of the detector with analog to digital converter and then Fourier transforming recovers the profile of the spatial field intensity distribution on the detector. This imaging modality offers the possibility of relatively simple and high speed imaging of objects with an single element detector. The modulator can be produced at low cost by printing a mask onto a clear CD-ROM substrate. The theory developed explains how the parameters of the modulator and optical system relate to the resolution and number of points in the electrical image. Numerical simulations are used to explore the optical limits of the electrical image in the presence of optical aberrations. Experimental results verify theoretical relations and images are captured of a Air Force test pattern and prepared fluorescent patterns

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

Optical vagus nerve modulation of heart and respiration via heart-injected retrograde AAV.

Vagus nerve stimulation has shown many benefits for disease therapies but current approaches involve imprecise electrical stimulation that gives rise to off-target effects, while the functionally relevant pathways remain poorly understood. One method to overcome these limitations is the use of optogenetic techniques, which facilitate targeted neural communication with light-sensitive actuators (opsins) and can be targeted to organs of interest based on the location of viral delivery. Here, we tested whether retrograde adeno-associated virus (rAAV2-retro) injected in the heart can be used to selectively express opsins in vagus nerve fibers controlling cardiac function. Furthermore, we investigated whether perturbations in cardiac function could be achieved with photostimulation at the cervical vagus nerve. Viral injection in the heart resulted in robust, primarily afferent, opsin reporter expression in the vagus nerve, nodose ganglion, and brainstem. Photostimulation using both one-photon stimulation and two-photon holography with a GRIN-lens incorporated nerve cuff, was tested on the pilot-cohort of injected mice. Changes in heart rate, surface electrocardiogram, and respiratory responses were observed in response to both one- and two-photon photostimulation. The results demonstrate feasibility of retrograde labeling for organ targeted optical neuromodulation

Convergence empirics across economies with (some) capital mobility

This paper uses a model of growth and imperfect capital mobility across multiple economies to characterize the dynamics of (cross-country) in- come distributions. This allows convenient study of the convergence hypothesis, and reveals, where appropriate, polarization and clumping within subgroups. The data show little cross-country convergence; in- stead, the important features are persistence, immobility, and polariza- tion, exemplied by \convergence club" or \twin peaks" dynamic