HoloTile light engine: new digital holographic modalities and applications.

HoloTile is a patented computer generated holography approach with the aim of reducing the speckle noise caused by the overlap of the non-trivial physical extent of the point spread function in Fourier holographic systems from adjacent frequency components. By combining tiling of phase-only of rapidly generated sub-holograms with a PSF-shaping phase profile, each frequency component-or output 'pixel'- in the Fourier domain is shaped to a desired non-overlapping profile. In this paper, we show the high-resolution, speckle-reduced reconstructions that can be achieved with HoloTile, as well as present new HoloTile modalities, including an expanded list of PSF options with new key properties. In addition, we discuss numerous applications for which HoloTile, its rapid hologram generation, and the new PSF options may be an ideal fit, including optical trapping and manipulation of particles, volumetric additive printing, information transfer and quantum communication.

Emotion Recognition Using a Reduced Set of EEG Channels Based on Holographic Feature Maps.

An important function of the construction of the Brain-Computer Interface (BCI) device is the development of a model that is able to recognize emotions from electroencephalogram (EEG) signals. Research in this area is very challenging because the EEG signal is non-stationary, non-linear, and contains a lot of noise due to artifacts caused by muscle activity and poor electrode contact. EEG signals are recorded with non-invasive wearable devices using a large number of electrodes, which increase the dimensionality and, thereby, also the computational complexity of EEG data. It also reduces the level of comfort of the subjects. This paper implements our holographic features, investigates electrode selection, and uses the most relevant channels to maximize model accuracy. The ReliefF and Neighborhood Component Analysis (NCA) methods were used to select the optimal electrodes. Verification was performed on four publicly available datasets. Our holographic feature maps were constructed using computer-generated holography (CGH) based on the values of signal characteristics displayed in space. The resulting 2D maps are the input to the Convolutional Neural Network (CNN), which serves as a feature extraction method. This methodology uses a reduced set of electrodes, which are different between men and women, and obtains state-of-the-art results in a three-dimensional emotional space. The experimental results show that the channel selection methods improve emotion recognition rates significantly with an accuracy of 90.76% for valence, 92.92% for arousal, and 92.97% for dominance.

In Vivo Submillisecond Two-Photon Optogenetics with Temporally Focused Patterned Light.

To better examine circuit mechanisms underlying perception and behavior, researchers need tools to enable temporally precise control of action-potential generation of individual cells from neuronal ensembles. Here we demonstrate that such precision can be achieved with two-photon (2P) temporally focused computer-generated holography to control neuronal excitability at the supragranular layers of anesthetized and awake visual cortex in both male and female mice. Using 2P-guided whole-cell or cell-attached recordings in positive neurons expressing any of the three opsins ReaChR, CoChR, or ChrimsonR, we investigated the dependence of spiking activity on the opsin's channel kinetics. We found that in all cases the use of brief illumination (≤10 ms) induces spikes of millisecond temporal resolution and submillisecond precision, which were preserved upon repetitive illuminations up to tens of hertz. To reach high temporal precision, we used a large illumination spot covering the entire cell body and an amplified laser at high peak power and low excitation intensity (on average ≤0.2 mW/µm2), thus minimizing the risk for nonlinear photodamage effects. Finally, by combining 2P holographic excitation with electrophysiological recordings and calcium imaging using GCaMP6s, we investigated the factors, including illumination shape and intensity, opsin distribution in the target cell, and cell morphology, which affect the spatial selectivity of single-cell and multicell holographic activation. Parallel optical control of neuronal activity with cellular resolution and millisecond temporal precision should make it easier to investigate neuronal connections and find further links between connectivity, microcircuit dynamics, and brain functions.SIGNIFICANCE STATEMENT Recent developments in the field of optogenetics has enabled researchers to probe the neuronal microcircuit with light by optically actuating genetically encoded light-sensitive opsins expressed in the target cells. Here, we applied holographic light shaping and temporal focusing to simultaneously deliver axially confined holographic patterns to opsin-positive cells in the living mouse cortex. Parallel illumination efficiently induced action potentials with high temporal resolution and precision for three opsins of different kinetics. We extended the parallel optogenetic activation at low intensity to multiple neurons and concurrently monitored their calcium dynamics. These results demonstrate fast and temporally precise in vivo control of a neuronal subpopulation, opening new opportunities for revealing circuit mechanisms underlying brain functions.

Multi frame holograms batched optimization for binary phase spatial light modulators.

Phase retrieval methods used in computer generated holograms such as Gerchberg-Saxton and gradient descent give results which are prone to noise and other defects. This work builds up on the idea of time-averaging multiple hologram frames, first introduced in methods like One-Step Phase-Retrieval and Adaptive One-Step Phase-Retrieval. The proposed technique called Multi-Frame Holograms Batched Optimization uses the L-BFGS optimization algorithm to simultaneously generate a batch of binary phase holograms which result in an average reconstructed image of improved fidelity and fast algorithmic convergence, both in the Fraunhoffer and the Fresnel regimes. The results are compared to One-Step Phase-Retrieval and Adaptive One-Step Phase-Retrieval in simulation and experimentally, proving the superiority of the proposed approach. This technique can be easily extended to other spatial modulation methods.

Scanless two-photon voltage imaging.

Parallel light-sculpting methods have been used to perform scanless two-photon photostimulation of multiple neurons simultaneously during all-optical neurophysiology experiments. We demonstrate that scanless two-photon excitation also enables high-resolution, high-contrast, voltage imaging by efficiently exciting fluorescence in a large fraction of the cellular soma. We present a thorough characterisation of scanless two-photon voltage imaging using existing parallel approaches and lasers with different repetition rates. We demonstrate voltage recordings of high frequency spike trains and sub-threshold depolarizations in intact brain tissue from neurons expressing the soma-targeted genetically encoded voltage indicator JEDI-2P-kv. Using a low repetition-rate laser, we perform recordings from up to ten neurons simultaneously. Finally, by co-expressing JEDI-2P-kv and the channelrhodopsin ChroME-ST in neurons of hippocampal organotypic slices, we perform single-beam, simultaneous, two-photon voltage imaging and photostimulation. This enables in-situ validation of the precise number and timing of light evoked action potentials and will pave the way for rapid and scalable identification of functional brain connections in intact neural circuits.

A flexible two-photon fiberscope for fast activity imaging and precise optogenetic photostimulation of neurons in freely moving mice.

We developed a flexible two-photon microendoscope (2P-FENDO) capable of all-optical brain investigation at near cellular resolution in freely moving mice. The system performs fast two-photon (2P) functional imaging and 2P holographic photostimulation of single and multiple cells using axially confined extended spots. Proof-of-principle experiments were performed in freely moving mice co-expressing jGCaMP7s and the opsin ChRmine in the visual or barrel cortex. On a field of view of 250 µm in diameter, we demonstrated functional imaging at a frame rate of up to 50 Hz and precise photostimulation of selected groups of cells. With the capability to simultaneously image and control defined neuronal networks in freely moving animals, 2P-FENDO will enable a precise investigation of neuronal functions in the brain during naturalistic behaviors.

Advances in computer-generated holography for targeted neuronal modulation.

Genetically encoded calcium indicators and optogenetics have revolutionized neuroscience by enabling the detection and modulation of neural activity with single-cell precision using light. To fully leverage the immense potential of these techniques, advanced optical instruments that can place a light on custom ensembles of neurons with a high level of spatial and temporal precision are required. Modern light sculpting techniques that have the capacity to shape a beam of light are preferred because they can precisely target multiple neurons simultaneously and modulate the activity of large ensembles of individual neurons at rates that match natural neuronal dynamics. The most versatile approach, computer-generated holography (CGH), relies on a computer-controlled light modulator placed in the path of a coherent laser beam to synthesize custom three-dimensional (3D) illumination patterns and illuminate neural ensembles on demand. Here, we review recent progress in the development and implementation of fast and spatiotemporally precise CGH techniques that sculpt light in 3D to optically interrogate neural circuit functions.

All-optical inter-layers functional connectivity investigation in the mouse retina.

We developed a multi-unit microscope for all-optical inter-layers circuits interrogation. The system performs two-photon (2P) functional imaging and 2P multiplexed holographic optogenetics at axially distinct planes. We demonstrated the capability of the system to map, in the mouse retina, the functional connectivity between rod bipolar cells (RBCs) and ganglion cells (GCs) by activating single or defined groups of RBCs while recording the evoked response in the GC layer with cell-type specificity and single-cell resolution. We then used a logistic model to probe the functional connectivity between cell types by deriving the "cellular receptive field" describing how RBCs impact each GC type. With the capability to simultaneously image and control neuronal activity at axially distinct planes, the system enables a precise interrogation of multi-layered circuits. Understanding this information transfer is a promising avenue to dissect complex neural circuits and understand the neural basis of computations.

Generation of Ince-Gaussian Beams Using Azocarbazole Polymer CGH.

Ince-Gaussian beams, defined as a solution to a wave equation in elliptical coordinates, have shown great advantages in applications such as optical communication, optical trapping and optical computation. However, to ingress these applications, a compact and scalable method for generating these beams is required. Here, we present a simple method that satisfies the above requirement, and is capable of generating arbitrary Ince-Gaussian beams and their superposed states through a computer-generated hologram of size 1 mm2, fabricated on an azocarbazole polymer film. Other structural beams that can be derived from the Ince-Gaussian beam were also successfully generated by changing the elliptical parameters of the Ince-Gaussian beam. The orthogonality relations between different Ince-Gaussian modes were investigated in order to verify applicability in an optical communication regime. The complete python source code for computing the Ince-Gaussian beams and their holograms are also provided.

Computational optics for high-throughput imaging of neural activity.

Optical microscopy offers a noninvasive way to image neural activity in the mouse brain. To simultaneously record neural activity across a large population of neurons, optical systems that have high spatiotemporal resolution and can access a large volume are necessary. The throughput of a system, that is, the number of resolvable spots acquired by the system at a given time, is usually limited by optical hardware. To overcome this limitation, computation optics that designs optical hardware and computer software jointly becomes a new approach that achieves micronscale resolution, millimeter-scale field-of-view, and hundreds of hertz imaging speed at the same time. This review article summarizes recent advances in computational optics for high-throughput imaging of neural activity, highlighting technologies for three-dimensional parallelized excitation and detection. Computational optics can substantially accelerate the study of neural circuits with previously unattainable precision and speed.

Compact and Scalable Large Vortex Array Generation Using Azocarbazole Polymer and Digital Hologram Printing Technique.

An integrated device capable of generating large number of multiplexed optical vortex beams with arbitrary topological charge is considered as one of the crucial requirement for driving information photonics forward. Here we report a simple method for simultaneous generation of 100 multiplexed optical vortex beams from a polymer film of size 1 mm2 and thickness of 30 µm. This is achieved through a combination of computer-generated holography, digital hologram printing and photoisomeric polymers. When the fabricated sample is illuminated with a collimated laser beam, a pre-determined vortex array with arbitrary topological charge is emitted. The polymer film easy to synthesize and exhibits a diffraction efficiency of 30% with a retention period longer than 50 days.

Single-Cell Resolution Optogenetics Via Expression of Soma-Targeted Rhodopsins.

Optogenetics allows control of neural activity in genetically targeted neuron populations by light. Optogenetic control of individual neurons in neural circuits would enable powerful, causal investigations of neural connectivity and function at single-cell level and provide insights into how neural circuits operate. Such single-cell resolution optogenetics in neuron populations requires precise sculpting of light and subcellular targeting of optogenetic molecules. Here we describe a group of methods for single-cell resolution optogenetics in neuron cultures, in mouse brain slices, and in mouse cortex in-vivo, via patterned light and soma-targeted optogenetic molecules.

Phase-controlled, speckle-free holographic projection with applications in precision optogenetics.

Holographic speckle is a major impediment to computer-generated holographic (CGH) projections in applications ranging from display, optical tweezers, and machining to optogenetic neural control. We present an iterative phase retrieval algorithm that allows the projection of amplitude-controlled speckle-free one-dimensional patterns with a high degree of pattern uniformity. The algorithm, termed the weighted Gerchberg-Saxton with phase-control (GSW-PC), is shown to have the ability to simultaneously control both the phase and amplitude of projected patterns with high diffraction efficiencies. Furthermore, we show that the framework can address the challenge of projecting volumetric phase and amplitude-controlled patterns, by incorporating GSW-PC with the angular spectrum method. The algorithms' performance is numerically and experimentally tested, and further compared with conventional and modern CGH techniques.

Emerging Applications of Virtual Reality in Cardiovascular Medicine.

Recently, rapid development in the mobile computing arena has allowed extended reality technologies to achieve performance levels that remove longstanding barriers to medical adoption. Importantly, head-mounted displays have become untethered and are light enough to be worn for extended periods of time, see-through displays allow the user to remain in his or her environment while interacting with digital content, and processing power has allowed displays to keep up with human perception to prevent motion sickness. Across cardiology, many groups are taking advantage of these advances for education, pre-procedural planning, intraprocedural visualization, and patient rehabilitation. Here, we detail these applications and the advances that have made them possible.

Temperature Rise under Two-Photon Optogenetic Brain Stimulation.

In recent decades, optogenetics has been transforming neuroscience research, enabling neuroscientists to drive and read neural circuits. The recent development in illumination approaches combined with two-photon (2P) excitation, either sequential or parallel, has opened the route for brain circuit manipulation with single-cell resolution and millisecond temporal precision. Yet, the high excitation power required for multi-target photostimulation, especially under 2P illumination, raises questions about the induced local heating inside samples. Here, we present and experimentally validate a theoretical model that makes it possible to simulate 3D light propagation and heat diffusion in optically scattering samples at high spatial and temporal resolution under the illumination configurations most commonly used to perform 2P optogenetics: single- and multi-spot holographic illumination and spiral laser scanning. By investigating the effects of photostimulation repetition rate, spot spacing, and illumination dependence of heat diffusion, we found conditions that make it possible to design a multi-target 2P optogenetics experiment with minimal sample heating.

Linking Neurons to Network Function and Behavior by Two-Photon Holographic Optogenetics and Volumetric Imaging.

We introduce a flexible method for high-resolution interrogation of circuit function, which combines simultaneous 3D two-photon stimulation of multiple targeted neurons, volumetric functional imaging, and quantitative behavioral tracking. This integrated approach was applied to dissect how an ensemble of premotor neurons in the larval zebrafish brain drives a basic motor program, the bending of the tail. We developed an iterative photostimulation strategy to identify minimal subsets of channelrhodopsin (ChR2)-expressing neurons that are sufficient to initiate tail movements. At the same time, the induced network activity was recorded by multiplane GCaMP6 imaging across the brain. From this dataset, we computationally identified activity patterns associated with distinct components of the elicited behavior and characterized the contributions of individual neurons. Using photoactivatable GFP (paGFP), we extended our protocol to visualize single functionally identified neurons and reconstruct their morphologies. Together, this toolkit enables linking behavior to circuit activity with unprecedented resolution.

Computer Generated Holography with Intensity-Graded Patterns.

Computer Generated Holography achieves patterned illumination at the sample plane through phase modulation of the laser beam at the objective back aperture. This is obtained by using liquid crystal-based spatial light modulators (LC-SLMs), which modulate the spatial phase of the incident laser beam. A variety of algorithms is employed to calculate the phase modulation masks addressed to the LC-SLM. These algorithms range from simple gratings-and-lenses to generate multiple diffraction-limited spots, to iterative Fourier-transform algorithms capable of generating arbitrary illumination shapes perfectly tailored on the base of the target contour. Applications for holographic light patterning include multi-trap optical tweezers, patterned voltage imaging and optical control of neuronal excitation using uncaging or optogenetics. These past implementations of computer generated holography used binary input profile to generate binary light distribution at the sample plane. Here we demonstrate that using graded input sources, enables generating intensity graded light patterns and extend the range of application of holographic light illumination. At first, we use intensity-graded holograms to compensate for LC-SLM position dependent diffraction efficiency or sample fluorescence inhomogeneity. Finally we show that intensity-graded holography can be used to equalize photo evoked currents from cells expressing different levels of chanelrhodopsin2 (ChR2), one of the most commonly used optogenetics light gated channels, taking into account the non-linear dependence of channel opening on incident light.

Superresolving dendritic spine morphology with STED microscopy under holographic photostimulation.

Emerging all-optical methods provide unique possibilities for noninvasive studies of physiological processes at the cellular and subcellular scale. On the one hand, superresolution microscopy enables observation of living samples with nanometer resolution. On the other hand, light can be used to stimulate cells due to the advent of optogenetics and photolyzable neurotransmitters. To exploit the full potential of optical stimulation, light must be delivered to specific cells or even parts of cells such as dendritic spines. This can be achieved with computer generated holography (CGH), which shapes light to arbitrary patterns by phase-only modulation. We demonstrate here in detail how CGH can be incorporated into a stimulated emission depletion (STED) microscope for photostimulation of neurons and monitoring of nanoscale morphological changes. We implement an original optical system to allow simultaneous holographic photostimulation and superresolution STED imaging. We present how synapses can be clearly visualized in live cells using membrane stains either with lipophilic organic dyes or with fluorescent proteins. We demonstrate the capabilities of this microscope to precisely monitor morphological changes of dendritic spines after stimulation. These all-optical methods for cell stimulation and monitoring are expected to spread to various fields of biological research in neuroscience and beyond.

Two-Photon Holographic Stimulation of ReaChR.

Optogenetics provides a unique approach to remotely manipulate brain activity with light. Reaching the degree of spatiotemporal control necessary to dissect the role of individual cells in neuronal networks, some of which reside deep in the brain, requires joint progress in opsin engineering and light sculpting methods. Here we investigate for the first time two-photon stimulation of the red-shifted opsin ReaChR. We use two-photon (2P) holographic illumination to control the activation of individually chosen neurons expressing ReaChR in acute brain slices. We demonstrated reliable action potential generation in ReaChR-expressing neurons and studied holographic 2P-evoked spiking performances depending on illumination power and pulse width using an amplified laser and a standard femtosecond Ti:Sapphire oscillator laser. These findings provide detailed knowledge of ReaChR's behavior under 2P illumination paving the way for achieving in depth remote control of multiple cells with high spatiotemporal resolution deep within scattering tissue.

Computer-generated holography enhances voltage dye fluorescence discrimination in adjacent neuronal structures.

Voltage-sensitive fluorescence indicators enable tracking neuronal electrical signals simultaneously in multiple neurons or neuronal subcompartments difficult to access with patch electrodes. However, efficient widefield epifluorescence detection of rapid voltage fluorescence transients necessitates that imaged cells and structures lie sufficiently far from other labeled structures to avoid contamination from out of focal plane and scattered light. We overcame this limitation by exciting dye fluorescence with one-photon computer-generated holography shapes contoured to axons or dendrites of interest, enabling widefield detection of voltage fluorescence with high spatial specificity. By shaping light onto neighboring axons and dendrites, we observed that dendritic back-propagating action potentials were broader and slowly rising compared with axonal action potentials, differences not measured in the same structures illuminated with a large "pseudowidefield" (pWF) spot of the same excitation density. Shaped illumination trials showed reduced baseline fluorescence, higher baseline noise, and fractional fluorescence transient amplitudes two times greater than trials acquired with pWF illumination of the same regions.