Three-dimensional scanless holographic optogenetics with temporal focusing (3D-SHOT).

Optical methods capable of manipulating neural activity with cellular resolution and millisecond precision in three dimensions will accelerate the pace of neuroscience research. Existing approaches for targeting individual neurons, however, fall short of these requirements. Here we present a new multiphoton photo-excitation method, termed three-dimensional scanless holographic optogenetics with temporal focusing (3D-SHOT), which allows precise, simultaneous photo-activation of arbitrary sets of neurons anywhere within the addressable volume of a microscope. This technique uses point-cloud holography to place multiple copies of a temporally focused disc matching the dimensions of a neurons cell body. Experiments in cultured cells, brain slices, and in living mice demonstrate single-neuron spatial resolution even when optically targeting randomly distributed groups of neurons in 3D. This approach opens new avenues for mapping and manipulating neural circuits, allowing a real-time, cellular resolution interface to the brain

Flow-Scanning Microfluidic Imaging

The advantages of microfluidics for fast analysis of microscopic suspensions have led to the commercial development of flow cytometers. In this chapter, we propose new microscopy methods that combine controlled motion of micro-organisms in a laminar microfluidic flow, optics, and computation. We propose three new imaging modalities. We first introduce a flow-based version of structured illumination microscopy, where the necessary phase shifts are no longer obtained by controlled displacement of the illumination pattern but by flowing the sample itself. Then, we propose a three-dimensional (3D) deconvolution microscopy method with a microfluidic device for continuous acquisition of gradually defocused images. Finally, we introduce a microfluidic device for phase-space image acquisition, and computational methods for the reconstruction of either phase of intensity, in 3D. The imaging modalities we introduce all retain the benefits of fluid systems for noninvasive bioimaging. The proposed devices can easily be integrated on existing microscopes as a modified microscope slide, or on flow cytometers, and aquatic imagers with minor adjustments. Alternative on-chip implementations are also possible, with lens-free devices, and near-field optical and microfluidic elements directly assembled on the surface of a CCD (Charge-Coupled Device) or CMOS (Complementary metal–oxide–semiconductor) chip

Precise multimodal optical control of neural ensemble activity.

Understanding brain function requires technologies that can control the activity of large populations of neurons with high fidelity in space and time. We developed a multiphoton holographic approach to activate or suppress the activity of ensembles of cortical neurons with cellular resolution and sub-millisecond precision. Since existing opsins were inadequate, we engineered new soma-targeted (ST) optogenetic tools, ST-ChroME and IRES-ST-eGtACR1, optimized for multiphoton activation and suppression. Employing a three-dimensional all-optical read-write interface, we demonstrate the ability to simultaneously photostimulate up to 50 neurons distributed in three dimensions in a 550 × 550 × 100-µm3 volume of brain tissue. This approach allows the synthesis and editing of complex neural activity patterns needed to gain insight into the principles of neural codes

HQA1K Hologram Perceptual Quality Assessment Dataset

The HQA1K dataset was developed for assessing the quality of Computer Generated Holography (CGH) image renderings based on direct human input. HQA1K is comprised of 1,000 pairs of natural images matched to simulated CGH renderings of various quality levels. The result is a diverse set of data for evaluating image quality algorithms and models

Three-dimensional deconvolution microfluidic microscopy using a tilted channel

We present a 3D microfluidic microscope. Focal stacks are recorded by observing samples flowing through a tilted microfluidic channel and then digitally deconvolved. Experimental results are shown on flowing yeast cell

Three-dimensional multi-site random access photostimulation (3D-MAP).

Optical control of neural ensemble activity is crucial for understanding brain function and disease, yet no technology can achieve optogenetic control of very large numbers of neurons at an extremely fast rate over a large volume. State-of-the-art multiphoton holographic optogenetics requires high-power illumination that only addresses relatively small populations of neurons in parallel. Conversely, one-photon holographic techniques can stimulate more neurons with two to three orders lower power, but with limited resolution or addressable volume. Perhaps most problematically, two-photon holographic optogenetic systems are extremely expensive and sophisticated which has precluded their broader adoption in the neuroscience community. To address this technical gap, we introduce a new one-photon light sculpting technique, three-dimensional multi-site random access photostimulation (3D-MAP), that overcomes these limitations by modulating light dynamically, both in the spatial and in the angular domain at multi-kHz rates. We use 3D-MAP to interrogate neural circuits in 3D and demonstrate simultaneous photostimulation and imaging of dozens of user-selected neurons in the intact mouse brain in vivo with high spatio-temporal resolution. 3D-MAP can be broadly adopted for high-throughput all-optical interrogation of brain circuits owing to its powerful combination of scale, speed, simplicity, and cost

UC Berkeley Sculpted Light in the Brain 2017 debates future technologies to communicate with the brain.

This article summarizes presentations at Sculpted Light in the Brain 2017

A micromirror array with annular partitioning for high-speed random-access axial focusing.

Dynamic axial focusing functionality has recently experienced widespread incorporation in microscopy, augmented/virtual reality (AR/VR), adaptive optics and material processing. However, the limitations of existing varifocal tools continue to beset the performance capabilities and operating overhead of the optical systems that mobilize such functionality. The varifocal tools that are the least burdensome to operate (e.g. liquid crystal, elastomeric or optofluidic lenses) suffer from low (≈100 Hz) refresh rates. Conversely, the fastest devices sacrifice either critical capabilities such as their dwelling capacity (e.g. acoustic gradient lenses or monolithic micromechanical mirrors) or low operating overhead (e.g. deformable mirrors). Here, we present a general-purpose random-access axial focusing device that bridges these previously conflicting features of high speed, dwelling capacity and lightweight drive by employing low-rigidity micromirrors that exploit the robustness of defocusing phase profiles. Geometrically, the device consists of an 8.2 mm diameter array of piston-motion and 48-μm-pitch micromirror pixels that provide 2π phase shifting for wavelengths shorter than 1100 nm with 10-90% settling in 64.8 μs (i.e., 15.44 kHz refresh rate). The pixels are electrically partitioned into 32 rings for a driving scheme that enables phase-wrapped operation with circular symmetry and requires <30 V per channel. Optical experiments demonstrated the array's wide focusing range with a measured ability to target 29 distinct resolvable depth planes. Overall, the features of the proposed array offer the potential for compact, straightforward methods of tackling bottlenecked applications, including high-throughput single-cell targeting in neurobiology and the delivery of dense 3D visual information in AR/VR

Three-dimensional scanless holographic optogenetics with temporal focusing (3D-SHOT).

Optical methods capable of manipulating neural activity with cellular resolution and millisecond precision in three dimensions will accelerate the pace of neuroscience research. Existing approaches for targeting individual neurons, however, fall short of these requirements. Here we present a new multiphoton photo-excitation method, termed three-dimensional scanless holographic optogenetics with temporal focusing (3D-SHOT), which allows precise, simultaneous photo-activation of arbitrary sets of neurons anywhere within the addressable volume of a microscope. This technique uses point-cloud holography to place multiple copies of a temporally focused disc matching the dimensions of a neuron's cell body. Experiments in cultured cells, brain slices, and in living mice demonstrate single-neuron spatial resolution even when optically targeting randomly distributed groups of neurons in 3D. This approach opens new avenues for mapping and manipulating neural circuits, allowing a real-time, cellular resolution interface to the brain