Acousto-optic systems for advanced microscopy

Acoustic waves in an optical medium cause rapid periodic changes in the refraction index, leading to diffraction effects. Such acoustically controlled diffraction can be used to modulate, deflect, and focus light at microsecond timescales, paving the way for advanced optical microscopy designs that feature unprecedented spatiotemporal resolution. In this article, we review the operational principles, optical properties, and recent applications of acousto-optic (AO) systems for advanced microscopy, including random-access scanning, ultrafast confocal and multiphoton imaging, and fast inertia-free light-sheet microscopy. As AO technology is reaching maturity, designing new microscope architectures that utilize AO elements is more attractive than ever, providing new exciting opportunities in fields as impactful as optical metrology, neuroscience, embryogenesis, and high-content screening

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

Variable optical elements for fast focus control

In this Review, we survey recent developments in the emerging field of high-speed variable-z-focus optical elements, which are driving important innovations in advanced imaging and materials processing applications. Three-dimensional biomedical imaging, high-throughput industrial inspection, advanced spectroscopies, and other optical characterization and materials modification methods have made great strides forward in recent years due to precise and rapid axial control of light. Three state-of-the-art key optical technologies that enable fast z-focus modulation are reviewed, along with a discussion of the implications of the new developments in variable optical elements and their impact on technologically relevant applications

Computational Imaging Methods for Improving Resolution in Biological Microscopy

Optical microscopy is an essential tool for biological research, as it allows for non-invasive imaging of small animals. However, optical microscopy has its limits. Due to the low light level, fluorescence microscopy prohibits high speed imaging, making it difficult to study fast dynamic biological processes. In addition, optical blur due to the diffraction of light results in limited spatial resolution, particularly when using objective lenses with low numerical apertures. In this thesis, we propose computational imaging methods to overcome these limitations using a combination of novel image acquisition procedures and reconstruction algorithms.The first part of this thesis deals with improving temporal resolution in fluorescence microscopy to image rapid, repeating processes. We take advantage of multiple acquisitions, each taken with different time delays or temporally modulated illumination patterns, to recover high frequency information that is lost with traditional imaging. We demonstrate our method to image the beating heart in live embryonic zebrafish with reduced motion blur and high resolution in time.The second part of this thesis deals with reducing spatial blur in optical projection tomography, a form of optical microscopy that uses multiple 2D projections to reconstruct a 3D image of an object. We propose a method to reduce the optical distortion (as characterized by the system's optical point spread function) that can be implemented with a scanning acquisition approach combined with a modified filtered backprojection algorithm for reconstruction. We demonstrate our method to image blood vessels in larval zebrafish with high spatial resolution and reduced out-of-focus blur.The final part of this thesis deals with the dimensional limitation of 2D sensors for measuring 3D motion in microscopy. We propose a method to combine two-dimensional motion estimates from multiple views to recover out-of-plane velocity and reconstruct a divergence-free, three-dimensional velocity field. We demonstrate our method to measure, for the first time, dynamic blood flow in 3D inside the beating heart of a live zebrafish using optical microscopy.This thesis provides new tools that integrate custom image acquisition procedures and image reconstruction algorithms to overcome the resolution limitations -- temporal, spatial, and out-of-plane velocity resolution -- in optical microscopy. The methods presented in this thesis, in particular the single camera, active illumination method for temporal superresolution in fluorescence microscopy, will be directly applicable to a broad range of biological studies and will open up new perspectives for imaging small organisms in 3D (and time) with high spatio-temporal resolution

Fast whole-brain imaging of seizures in zebrafish larvae by two-photon light-sheet microscopy

Light-sheet fluorescence microscopy (LSFM) enables real-time whole-brain functional imaging in zebrafish larvae. Conventional one photon LSFM can however induce undesirable visual stimulation due to the use of visible excitation light. The use of two-photon (2P) excitation, employing near-infrared invisible light, provides unbiased investigation of neuronal circuit dynamics. However, due to the low efficiency of the 2P absorption process, the imaging speed of this technique is typically limited by the signal-to-noise-ratio. Here, we describe a 2P LSFM setup designed for non-invasive imaging that enables quintuplicating state-of-the-art volumetric acquisition rate of the larval zebrafish brain (5 Hz) while keeping low the laser intensity on the specimen. We applied our system to the study of pharmacologically-induced acute seizures, characterizing the spatial-temporal dynamics of pathological activity and describing for the first time the appearance of caudo-rostral ictal waves (CRIWs).Comment: Replacement: accepted version of the manuscript, to be published in Biomedical Optics Express. 36 pages, 15 figure

Improvements in optical techniques to investigate the behavior and neuronal network dynamics over long timescales

Developments in optical technology have produced an important shift in experimental neuroscience from electrophysiological methods for observation and stimulation to all-optical solutions. One expects this trend to continue as future developments continue to deliver, and improve upon, the original promises of the technology: 1) minimally invasive actuation and recording of neurons, and 2) a drastic increase in targets that can be treated simultaneously. Moreover, as the high costs of the technology are reduced, one may expect its larger-scale adoption in the neuroscience community. In this thesis, I describe the development and implementation of two alloptical solutions for the analysis of behavior, neuronal signaling, and stimulation, which improve on previous state-of-the-art: (1) A minimally-invasive, high signal-to-noise twophoton microscopy setup capable of simultaneous, live-imaging of a large subset of sensory neurons post activation, and (2) a low-cost tracking solution to stimulate and record behavior. I begin this thesis with a review of recent advances in optical neuroscience techniques for the study of neuronal networks with the focus on work done in Caenorhabditis elegans. Then, in chapter 2, I describe my implementation of a two-photon temporal focusing microscopy setup and show significant improvements through the use of a high power/ high pulse repetition rate excitation system, enabling live imaging with high resolution for extended periods of time. I model temperature increase during a physiological imaging scenario for different repetition rates at fixed peak intensities and find range centered around 1 MHz to be optimal. Lastly, I describe the low-cost tracking setup with the ability to stimulate and record behavior over the course of hours. The setup is capable of two-color stimulation of optogenetic proteins over the area of the behavioral arena in combination with volatile chemicals. To showcase the utility of the system, I demonstrate behavioral analysis of integration of contradictory cues. In summary, I present a set of techniques for the interrogation of neural networks from animal behavior to neuronal activity, over timescales of potentially hours and days. These techniques can be used to address a new dimension of scientific questions.Okinawa Institute of Science and Technology Graduate Universit

Development of optical methods for real-time whole-brain functional imaging of zebrafish neuronal activity

Each one of us in his life has, at least once, smelled the scent of roses, read one canto of Dante’s Commedia or listened to the sound of the sea from a shell. All of this is possible thanks to the astonishing capabilities of an organ, such as the brain, that allows us to collect and organize perceptions coming from sensory organs and to produce behavioural responses accordingly. Studying an operating brain in a non-invasive way is extremely difficult in mammals, and particularly in humans. In the last decade, a small teleost fish, zebrafish (Danio rerio), has been making its way into the field of neurosciences. The brain of a larval zebrafish is made up of 'only' 100000 neurons and it’s completely transparent, making it possible to optically access it. Here, taking advantage of the best of currently available technology, we devised optical solutions to investigate the dynamics of neuronal activity throughout the entire brain of zebrafish larvae

Advanced optical systems for imaging and fabrication

Advanced optical systems for imaging and fabricatio

Model and learning-based strategies for intensity diffraction tomography

Intensity Diffraction Tomography (IDT) is a recently developed quantitative phase imaging tool with significant potential for biological imaging applications. This modality captures intensity images from a scattering sample under diverse illumination and reconstructs the object's volumetric permittivity contrast using linear inverse scattering models. IDT requires no through-focus sample scans or exogenous contrast agents for 3D object recovery and can be easily implemented with a standard microscope equipped with an off-the-shelf LED array. These factors make IDT ideal for biological research applications where easily implementable setups providing native sample morphological information are highly desirable. Given this modality's recent development, IDT suffers from a number of limitations preventing its widespread adoption: 1) large measurement datasets with long acquisition times limiting its temporal resolution, 2) model-based constraints preventing the evaluation of multiple-scattering samples, and 3) low axial resolution preventing the recovery of fine axial structures such as organelles and other subcellular structures. These factors limit IDT to primarily thin, static objects, and its unknown accuracy and sensitivity metrics cast doubt on the technology's quantitative recovery of morphological features. This thesis addresses the limitations of IDT through advancements provided from model and learning-based strategies. The model-based advancements guide new computational illumination strategies for high volume-rate imaging as well as investigate new imaging geometries, while the learning-based enhancements to IDT present an efficient method for recovering multiple-scattering biological specimens. These advancements place IDT in the optimal position of being an easily implementable, computationally efficient phase imaging modality recovering high-resolution volumes of complex, living biological samples in their native state. We first discuss two illumination strategies for high-speed IDT. The first strategy develops a multiplexed illumination framework based on IDT's linear model enabling hardware-limited 4Hz volume-rate imaging of living biological samples. This implementation is hardware-agnostic, allowing for fast IDT to be added to any existing setup containing programmable illumination hardware. While sacrificing some reconstruction quality, this multiplexed approach recovers high-resolution features in live cell cultures, worms, and embryos highlighting IDT's potential across numerous ranges of biological imaging. Following this illumination scheme, we discuss a hardware-based solution for live sample imaging using ring-geometry LED arrays. Inspired from the linear model, this hardware modification optimally captures the object's information in each LED illumination allowing for high-quality object volumes to be reconstructed from as few as eight intensity images. This small image requirement allows IDT to achieve camera-limited 10Hz volume rate imaging of live biological samples without motion artifacts. We show the capabilities of this annular illumination IDT setup on live worm samples. This low-cost solution for IDT's speed shows huge implications for enabling any biological imaging lab to easily study the form and function of biological samples of interest in their native state. Next, we present a learning-based approach to expand IDT to recovering multiple-scattering samples. IDT's linear model provides efficient computation of an object's 3D volume but fails to recover quantitative information in the presence of highly scattering samples. We introduce a lightweight neural network architecture, trained only on simulated natural image-based objects, that corrects the linear model estimates and improves the recovery of both weakly and strongly scattering samples. This implementation maintains the computational efficiency of IDT while expanding its reconstruction capabilities allowing for more generic imaging of biological samples. Finally, we discuss an investigation of the IDT modality for reflection mode imaging. IDT traditionally captures only low axial resolution information because it cannot capture the backscattered fields from the object that contain rich information regarding the fine details of the object's axial structures. Here, we investigated whether a reflection-mode IDT implementation was possible for recovering high axial resolution structures from this backscattered light. We develop the model, imaging setup, and rigorously evaluate the reflection case in simulation and experiment to show the possibility for reflection IDT. While this imaging geometry ultimately requires a nonlinear model for 3D imaging, we show the technique provides enhanced sensitivity to the object's structures in a complementary fashion to transmission-based IDT

Flow Assessment Using Optical Coherence Microscopy Based Particle Image Velocimetry

Congenital heart diseases (CHDs) are the most common forms of congenital malformation in newborns. Among all types of CHDs, a large portion is contributed by malformation of endocardial cushion malformation during early heart development. Although the etiology of endocardial cushion malformation is unclear, it is a result of interactions between genetic and environmental factors has been confirmed. There is hypothesis indicating that malformation of endocardial cushion is caused by altered shear stress conditions where in cushion forming area the shear stress is supposed to be high compare with other area in congenital heart. However it is difficult to justify due to lack of in vivo imaging modality that is able to monitor structure and hemodynamic conditions simultaneously and over long time period. To address this problem, we present an optical coherence microscopy based particle image velocimetry system. This system is capable of invasively imaging biological sample structures at micrometer resolution and providing velocity information at the same time. With this imaging set up we successfully assessed velocity profile in a microfluidic system with simultaneous structure details demonstration of the microfluidic channel. Both flow measurement and structural information were verified using conventional microscopy. As a result, OCM-based PIV imaging modality not only makes it feasible to study in detail the process of congenital heart remodeling in response to environmental alterations, but also provides new options for measuring fluid flow in live tissue