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

Novel contrasts in photoacoustic tomography

Photoacoustic tomography (PAT) combines rich optical contrast and high ultrasonic resolution in optically scattering tissue at depths. Taking advantage of its 100% sensitivity to optical absorption, PAT has been widely applied to structural, functional and molecular imaging, with both endogenous and exogenous contrasts, at superior depths than pure optical methods. This dissertation explores novel absorption contrast mechanisms of PAT based on optical/thermal patterns, endogenous cellular chromophores, nanoparticles, small-molecule dyes and genetically-encoded proteins. With these novel contrasts, the proof-of-concept applications of PAT have been extended to include homogenous flow measurements, targeted angiogenesis imaging and therapy, label-free white blood cell imaging, 3D-whole-organ cell nuclei imaging with a subcellular resolution, and in vivo neural activity imaging with voltage/calcium-sensitive indicators. Specifically, Chapter 1 introduces photoacoustic microscopy (PAM) and photoacoustic computed tomography (PACT) systems and discuss the motivation of the dissertation. Chapter 2 describes two photoacoustic (PA) flow measurement methods with optical and thermal patterns, which are applicable to homogenous flowing medium. In the first method, a Doppler frequency shift in PA signals of the flow was detected and used to calculate flow speeds. In the second method, unique features in an externally imposed thermal pattern of the flow, captured by repeated B-scans along the flow direction with a PAM system, revealed different flow speeds. Chapter 3 explores the unique PA contrast of macrophages, an important type of white blood cells. Macrophages were imaged by PAM without any label, and their measured PA spectrum was distinctive from the hemoglobin spectrum, so they can be potentially differentiated from red blood cells in the blood stream. Next, with a microtomy-assisted PAM system, cell nuclei distribution in whole organs, including mouse brain and mouse lung, were imaged with subcellular resolution. Chapter 4 introduces a type of target copper nanoparticles, which are less expensive and more biocompatible than its counterpart gold nanoparticles. The PA signals of neovasculature in the mouse flank were enhanced by the ___3-targeted copper nanoparticles. Moreover, the work shows the first example of a systemically targeted antiangiogenic drug delivery with a photoacoustic contrast nanoparticle in vivo. Chapter 5 demonstrates the voltage imaging capability of PA. A voltage sensitive dye with sufficient signal change was discovered and used as a PA voltage indicator for the first time. The mechanism was characterized through both PA imaging and spectroscopic methods. Its use was explored in a mouse epilepsy model and cortical electrical stimulation model in vivo. Finally, the deep imaging potential of PA was realized by imaging the voltage response of cells under 4.5 mm thick slice of rat brain tissue using a PACT system. Chapter 6 proves the neural calcium imaging capability of PA with a genetically encoded calcium indicator. In a fly model, I ambiguously demonstrated for the first time that PA can be used to imaging neural activities in the fly brain without the interference signals from hemoglobin. In the a live-mouse-brain-slice model, I successfully demonstrated the deep imaging capability of PA for calcium imaging by imaging through a 2-mm-thick scattering medium with a PACT system

Neural Field Model of VSD Optical Imaging Signals

In this report we propose a solution to the direct problem of VSD optical imaging based on a neural field model of a cortical area and reproduce optical signals observed in various mammals cortices. We first present a biophysical approach to neural fields and show that these easily integrate the biological knowledge on cortical structure, especially horizontal and vertical connectivity patterns. After having introduced the reader to VSD optical imaging, we propose a biophysical formula expressing the optical imaging signal in terms of the activity of the field. Then, we simulate optical signals that have been observed by experimentalists. We have chosen two experimental sets: the line-motion illusion in the visual cortex of mammals (jancke, chavane, et al. 2004} and the spread of activity in the rat barrel cortex (petersen, grinvald, et al. 2003). We begin with a structural description of both areas, with a focus on horizontal connectivity. Finally we simulate the corresponding neural field equations and extract the optical signal using the direct problem formula developed in the preceding sections. We have been able to reproduce the main experimental results with these models

Spatial summation of individual cones in human color vision.

The human retina contains three classes of cone photoreceptors each sensitive to different portions of the visual spectrum: long (L), medium (M) and short (S) wavelengths. Color information is computed by downstream neurons that compare relative activity across the three cone types. How cone signals are combined at a cellular scale has been more difficult to resolve. This is especially true near the fovea, where spectrally-opponent neurons in the parvocellular pathway draw excitatory input from a single cone and thus even the smallest stimulus projected through natural optics will engage multiple color-signaling neurons. We used an adaptive optics microstimulator to target individual and pairs of cones with light. Consistent with prior work, we found that color percepts elicited from individual cones were predicted by their spectral sensitivity, although there was considerable variability even between cones within the same spectral class. The appearance of spots targeted at two cones were predicted by an average of their individual activations. However, two cones of the same subclass elicited percepts that were systematically more saturated than predicted by an average. Together, these observations suggest both spectral opponency and prior experience influence the appearance of small spots

Microstimulation and multicellular analysis: A neural interfacing system for spatiotemporal stimulation

Willfully controlling the focus of an extracellular stimulus remains a significant challenge in the development of neural prosthetics and therapeutic devices. In part, this challenge is due to the vast set of complex interactions between the electric fields induced by the microelectrodes and the complex morphologies and dynamics of the neural tissue. Overcoming such issues to produce methodologies for targeted neural stimulation requires a system that is capable of (1) delivering precise, localized stimuli a function of the stimulating electrodes and (2) recording the locations and magnitudes of the resulting evoked responses a function of the cell geometry and membrane dynamics. In order to improve stimulus delivery, we developed microfabrication technologies that could specify the electrode geometry and electrical properties. Specifically, we developed a closed-loop electroplating strategy to monitor and control the morphology of surface coatings during deposition, and we implemented pulse-plating techniques as a means to produce robust, resilient microelectrodes that could withstand rigorous handling and harsh environments. In order to evaluate the responses evoked by these stimulating electrodes, we developed microscopy techniques and signal processing algorithms that could automatically identify and evaluate the electrical response of each individual neuron. Finally, by applying this simultaneous stimulation and optical recording system to the study of dissociated cortical cultures in multielectode arrays, we could evaluate the efficacy of excitatory and inhibitory waveforms. Although we found that the proximity of the electrode is a poor predictor of individual neural excitation thresholds, we have shown that it is possible to use inhibitory waveforms to globally reduce excitability in the vicinity of the electrode. Thus, the developed system was able to provide very high resolution insight into the complex set of interactions between the stimulating electrodes and populations of individual neurons.Ph.D.Committee Chair: Stephen P. DeWeerth; Committee Member: Bruce Wheeler; Committee Member: Michelle LaPlaca; Committee Member: Robert Lee; Committee Member: Steve Potte

Spectral Unmixing: Analysis of Performance in the Olfactory Bulb In Vivo

Background: The generation of transgenic mice expressing combinations of fluorescent proteins has greatly aided the reporting of activity and identification of specific neuronal populations. Methods capable of separating multiple overlapping fluorescence emission spectra, deep in the living brain, with high sensitivity and temporal resolution are therefore required. Here, we investigate to what extent spectral unmixing addresses these issues. Methodology/Principal Findings: Using fluorescence resonance energy transfer (FRET)-based reporters, and two-photon laser scanning microscopy with synchronous multichannel detection, we report that spectral unmixing consistently improved FRET signal amplitude, both in vitro and in vivo. Our approach allows us to detect odor-evoked FRET transients 180-250 mm deep in the brain, the first demonstration of in vivo spectral imaging and unmixing of FRET signals at depths greater than a few tens of micrometer. Furthermore, we determine the reporter efficiency threshold for which FRET detection is improved by spectral unmixing. Conclusions/Significance: Our method allows the detection of small spectral variations in depth in the living brain, which is essential for imaging efficiently transgenic animals expressing combination of multiple fluorescent proteins

Cell-cell communication enhances the capacity of cell ensembles to sense shallow gradients during morphogenesis

Collective cell responses to exogenous cues depend on cell-cell interactions. In principle, these can result in enhanced sensitivity to weak and noisy stimuli. However, this has not yet been shown experimentally, and, little is known about how multicellular signal processing modulates single cell sensitivity to extracellular signaling inputs, including those guiding complex changes in the tissue form and function. Here we explored if cell-cell communication can enhance the ability of cell ensembles to sense and respond to weak gradients of chemotactic cues. Using a combination of experiments with mammary epithelial cells and mathematical modeling, we find that multicellular sensing enables detection of and response to shallow Epidermal Growth Factor (EGF) gradients that are undetectable by single cells. However, the advantage of this type of gradient sensing is limited by the noisiness of the signaling relay, necessary to integrate spatially distributed ligand concentration information. We calculate the fundamental sensory limits imposed by this communication noise and combine them with the experimental data to estimate the effective size of multicellular sensory groups involved in gradient sensing. Functional experiments strongly implicated intercellular communication through gap junctions and calcium release from intracellular stores as mediators of collective gradient sensing. The resulting integrative analysis provides a framework for understanding the advantages and limitations of sensory information processing by relays of chemically coupled cells.Comment: paper + supporting information, total 35 pages, 15 figure

The role of type 4 phosphodiesterases in generating microdomains of cAMP: Large scale stochastic simulations

Cyclic AMP (cAMP) and its main effector Protein Kinase A (PKA) are critical for several aspects of neuronal function including synaptic plasticity. Specificity of synaptic plasticity requires that cAMP activates PKA in a highly localized manner despite the speed with which cAMP diffuses. Two mechanisms have been proposed to produce localized elevations in cAMP, known as microdomains: impeded diffusion, and high phosphodiesterase (PDE) activity. This paper investigates the mechanism of localized cAMP signaling using a computational model of the biochemical network in the HEK293 cell, which is a subset of pathways involved in PKA-dependent synaptic plasticity. This biochemical network includes cAMP production, PKA activation, and cAMP degradation by PDE activity. The model is implemented in NeuroRD: novel, computationally efficient, stochastic reaction-diffusion software, and is constrained by intracellular cAMP dynamics that were determined experimentally by real-time imaging using an Epac-based FRET sensor (H30). The model reproduces the high concentration cAMP microdomain in the submembrane region, distinct from the lower concentration of cAMP in the cytosol. Simulations further demonstrate that generation of the cAMP microdomain requires a pool of PDE4D anchored in the cytosol and also requires PKA-mediated phosphorylation of PDE4D which increases its activity. The microdomain does not require impeded diffusion of cAMP, confirming that barriers are not required for microdomains. The simulations reported here further demonstrate the utility of the new stochastic reaction-diffusion algorithm for exploring signaling pathways in spatially complex structures such as neurons