Optically gated beating-heart imaging

The constant motion of the beating heart presents an obstacle to clear optical imaging, especially 3D imaging, in small animals where direct optical imaging would otherwise be possible. Gating techniques exploit the periodic motion of the heart to computationally "freeze" this movement and overcome motion artefacts. Optically gated imaging represents a recent development of this, where image analysis is used to synchronize acquisition with the heartbeat in a completely non-invasive manner. This article will explain the concept of optical gating, discuss a range of different implementation strategies and their strengths and weaknesses. Finally we will illustrate the usefulness of the technique by discussing applications where optical gating has facilitated novel biological findings by allowing 3D in vivo imaging of cardiac myocytes in their natural environment of the beating heart

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

Two-photon imaging and analysis of neural network dynamics

The glow of a starry night sky, the smell of a freshly brewed cup of coffee or the sound of ocean waves breaking on the beach are representations of the physical world that have been created by the dynamic interactions of thousands of neurons in our brains. How the brain mediates perceptions, creates thoughts, stores memories and initiates actions remains one of the most profound puzzles in biology, if not all of science. A key to a mechanistic understanding of how the nervous system works is the ability to analyze the dynamics of neuronal networks in the living organism in the context of sensory stimulation and behaviour. Dynamic brain properties have been fairly well characterized on the microscopic level of individual neurons and on the macroscopic level of whole brain areas largely with the help of various electrophysiological techniques. However, our understanding of the mesoscopic level comprising local populations of hundreds to thousands of neurons (so called 'microcircuits') remains comparably poor. In large parts, this has been due to the technical difficulties involved in recording from large networks of neurons with single-cell spatial resolution and near- millisecond temporal resolution in the brain of living animals. In recent years, two-photon microscopy has emerged as a technique which meets many of these requirements and thus has become the method of choice for the interrogation of local neural circuits. Here, we review the state-of-research in the field of two-photon imaging of neuronal populations, covering the topics of microscope technology, suitable fluorescent indicator dyes, staining techniques, and in particular analysis techniques for extracting relevant information from the fluorescence data. We expect that functional analysis of neural networks using two-photon imaging will help to decipher fundamental operational principles of neural microcircuits.Comment: 36 pages, 4 figures, accepted for publication in Reports on Progress in Physic

Quasi-simultaneous multiplane calcium imaging of neuronal circuits

Two-photon excitation fluorescence microscopy is widely used to study the activity of neuronal circuits. However, the fast imaging is typically constrained to a single lateral plane for a standard microscope design. Given that cortical neuronal networks in a mouse brain are complex three-dimensional structures organised in six histologically defined layers which extend over many hundreds of micrometres, there is a strong demand for microscope systems that can record neuronal signalling in volumes. Henceforth, we developed a quasi-simultaneous multiplane imaging technique combining acousto-optic deflector and static remote focusing to provide fast imaging of neurons from different axial positions inside the cortical layers without the need for the mechanical interference of either the objective lens or the specimen. The hardware and the software are easily adaptable to existing two-photon microscopes. Here, we demonstrated that our imaging method can record, at high speed and high image contrast, the calcium dynamics of neurons in two different imaging planes separated axially, with the in-focus and the refocused planes 120 μm and 250 μm below the brain surface respectively

Nonlinear Microscopy for Material Characterization

Making use of femtosecond laser sources, nonlinear microscopy provides access to previously unstudied aspects of materials. By probing third order nonlinear optical signals determined by the nonlinear susceptibility chi(3), which is present in all materials, we gain insight not available by conventional linear or electron microscopy. Third-harmonic (TH) microscopy is applied to supplement laser-induced damage studies of dielectric oxide thin film optical coatings. We present high contrast (S/N\u3e 100 : 1) TH imaging of ~17 nm nanoindentations, individual 10 nm gold nanoparticles, nascent scandia and hafnia films, and laser induced material modification both above and below damage threshold conditions in hafnia thin-films. These results imply that TH imaging is potentially sensitive to laser-induced strain as well as to nanoscale defects or contamination in oxide films. Compared to other sensitive imaging techniques such as Nomarski and dark field, TH imaging exhibits dramatically increased sensitivity to typical material modifications undergone during the formation of optical damage as evidenced by a dynamic range 10^6 : 1. Four-wave mixing (FWM) microscopy is employed to investigate delay dependent FWM signals and their implied characteristic resonant response times in multiple solvents. Mathematical modeling of resonant coherent anti-Stokes Raman scattering (CARS), coherent Stokes Raman scattering (CSRS) and stimulated parametric emission (SPE) processes supplement the FWM studies and suggest a resonant CARS process that accounts for ~95% of the total visible FWM signal which probes a characteristic material response time ~100 fs. This signal enhancement likely indicates the net effects of probing several Raman active C-H stretch bands near 2950 cm^-1. This FWM technique may be applied to characterize the dominant resonant response of the sample under study. Furthermore this technique presents the newfound capability to provide estimates of characteristic material dephasing times in combination with potential spatial resolution ~1 micron. In addition to TH and FWM microscopy, a genetic algorithm is developed and implemented that allows for the synthesis of arbitrary temporal waveforms to maximize the generation of nonlinear optical signals in the focal plane of a microscope without any prior knowledge of the experiment. This algorithm is demonstrated to compensate high order optical dispersion and thereby increase TH microscopy signals ~10x in a fused silica sample

Adaptive Movement Compensation for In Vivo Imaging of Fast Cellular Dynamics within a Moving Tissue

In vivo non-linear optical microscopy has been essential to advance our knowledge of how intact biological systems work. It has been particularly enabling to decipher fast spatiotemporal cellular dynamics in neural networks. The power of the technique stems from its optical sectioning capability that in turn also limits its application to essentially immobile tissue. Only tissue not affected by movement or in which movement can be physically constrained can be imaged fast enough to conduct functional studies at high temporal resolution. Here, we show dynamic two-photon Ca2+ imaging in the spinal cord of a living rat at millisecond time scale, free of motion artifacts using an optical stabilization system. We describe a fast, non-contact adaptive movement compensation approach, applicable to rough and weakly reflective surfaces, allowing real-time functional imaging from intrinsically moving tissue in live animals. The strategy involves enslaving the position of the microscope objective to that of the tissue surface in real-time through optical monitoring and a closed feedback loop. The performance of the system allows for efficient image locking even in conditions of random or irregular movements

Error Resilient Video Coding Using Bitstream Syntax And Iterative Microscopy Image Segmentation

There has been a dramatic increase in the amount of video traffic over the Internet in past several years. For applications like real-time video streaming and video conferencing, retransmission of lost packets is often not permitted. Popular video coding standards such as H.26x and VPx make use of spatial-temporal correlations for compression, typically making compressed bitstreams vulnerable to errors. We propose several adaptive spatial-temporal error concealment approaches for subsampling-based multiple description video coding. These adaptive methods are based on motion and mode information extracted from the H.26x video bitstreams. We also present an error resilience method using data duplication in VPx video bitstreams. A recent challenge in image processing is the analysis of biomedical images acquired using optical microscopy. Due to the size and complexity of the images, automated segmentation methods are required to obtain quantitative, objective and reproducible measurements of biological entities. In this thesis, we present two techniques for microscopy image analysis. Our first method, “Jelly Filling” is intended to provide 3D segmentation of biological images that contain incompleteness in dye labeling. Intuitively, this method is based on filling disjoint regions of an image with jelly-like fluids to iteratively refine segments that represent separable biological entities. Our second method selectively uses a shape-based function optimization approach and a 2D marked point process simulation, to quantify nuclei by their locations and sizes. Experimental results exhibit that our proposed methods are effective in addressing the aforementioned challenges

Selection Rules for All-Optical Magnetic Recording in Iron Garnet

Finding an electronic transition a subtle excitation of which can launch dramatic changes of electric, optical or magnetic properties of media is one of the long-standing dreams in the field of photo-induced phase transitions [1-5]. Therefore the discovery of the magnetization switching only by a femtosecond laser pulse [6-10] triggered intense discussions about mechanisms responsible for these laser-induced changes. Here we report the experimentally revealed selection rules on polarization and wavelengths of ultrafast photo-magnetic recording in Co-doped garnet film and identify the workspace of the parameters (magnetic damping, wavelength and polarization of light) allowing this effect. The all-optical magnetic switching under both single pulse and multiple-pulse sequences can be achieved at room temperature, in narrow spectral ranges with light polarized either along or crystallographic axes of the garnet. The revealed selection rules indicate that the excitations responsible for the coupling of light to spins are d-electron transitions in octahedral and tetrahedral Co-sublattices, respectively

Roadmap on structured light

Structured light refers to the generation and application of custom light fields. As the tools and technology to create and detect structured light have evolved, steadily the applications have begun to emerge. This roadmap touches on the key fields within structured light from the perspective of experts in those areas, providing insight into the current state and the challenges their respective fields face. Collectively the roadmap outlines the venerable nature of structured light research and the exciting prospects for the future that are yet to be realized.Peer ReviewedPostprint (published version