Digital hologram reconstruction algorithm based on the Fractional Fourier transform in non-telecentric digital holographic microscopy.

A hologram reconstruction algorithm is proposed based on the fractional Fourier transform (FRFT) in non-telecentric digital holographic microscopy. The optimal fractional order representing the recorded hologram is estimated based on an evaluation metric. The FRFT-based hologram reconstruction enables noise robust amplitude and phase imaging with enhanced resolution. The effectiveness of the proposed approach is demonstrated in practical scenarios through both simulation and experimental results.

Microglia Mediate Contact-Independent Neuronal Network Remodeling via Secreted Neuraminidase-3 Associated with Extracellular Vesicles.

Neurons communicate with each other through electrochemical transmission at synapses. Microglia, the resident immune cells of the central nervous system, modulate this communication through a variety of contact-dependent and -independent means. Microglial secretion of active sialidase enzymes upon exposure to inflammatory stimuli is one unexplored mechanism of modulation. Recent work from our lab showed that treatment of neurons with bacterial sialidases disrupts neuronal network connectivity. Here, we find that activated microglia secrete neuraminidase-3 (Neu3) associated with fusogenic extracellular vesicles. Furthermore, we show that Neu3 mediates contact-independent disruption of neuronal network synchronicity through neuronal glycocalyx remodeling. We observe that NEU3 is transcriptionally upregulated upon exposure to inflammatory stimuli and that a genetic knockout of NEU3 abrogates the sialidase activity of inflammatory microglial secretions. Moreover, we demonstrate that Neu3 is associated with a subpopulation of extracellular vesicles, possibly exosomes, that are secreted by microglia upon inflammatory insult. Finally, we demonstrate that Neu3 is necessary and sufficient to both desialylate neurons and decrease neuronal network connectivity. These results implicate Neu3 in remodeling of the glycocalyx leading to aberrant network-level activity of neurons, with implications in neuroinflammatory diseases such as Parkinson's disease and Alzheimer's disease.

State-space modeling approach for fringe pattern demodulation.

A spatial carrier fringe demodulation technique is proposed based on a state-space modeling approach for phase estimation. The fringe background intensity, carrier frequency, and phase quadrature components are considered to be the elements of the state vector, which are estimated simultaneously. The state estimation is performed using the extended Kalman filter. The simulation and experimental results are provided to demonstrate the performance comparison of the proposed method with popular and state-of-the-art methods in terms of noise robustness and phase estimation accuracy.

Microglia mediate contact-independent neuronal pruning via secreted Neuraminidase-3 associated with extracellular vesicles.

Neurons communicate with each other through electrochemical transmission at synapses. Microglia, the resident immune cells of the central nervous system, can prune these synapses through a variety of contact-dependent and -independent means. Microglial secretion of active sialidase enzymes upon exposure to inflammatory stimuli is one unexplored mechanism of pruning. Recent work from our lab showed that treatment of neurons with bacterial sialidases disrupts neuronal network connectivity. Here, we find that activated microglia secrete Neuraminidase-3 (Neu3) associated with fusogenic extracellular vesicles. Furthermore, we show Neu3 mediates contact-independent pruning of neurons and subsequent disruption of neuronal networks through neuronal glycocalyx remodeling. We observe that NEU3 is transcriptionally upregulated upon exposure to inflammatory stimuli, and that a genetic knock-out of NEU3 abrogates the sialidase activity of inflammatory microglial secretions. Moreover, we demonstrate that Neu3 is associated with a subpopulation of extracellular vesicles, possibly exosomes, that are secreted by microglia upon inflammatory insult. Finally, we demonstrate that Neu3 is both necessary and sufficient to both desialylate neurons and decrease neuronal network connectivity. These results implicate Neu3 in remodeling of the glycocalyx leading to aberrant network-level activity of neurons, with implications in neuroinflammatory diseases such as Parkinson's disease and Alzheimer's disease.

Phase-shifting interferometry based on dynamic mode decomposition.

A phase retrieval algorithm in phase-shifting interferometry is presented based on dynamic mode decomposition (DMD). The complex-valued spatial mode obtained from the DMD of phase-shifted interferograms allows the derivation of the phase estimate. At the same time, the oscillation frequency associated with the spatial mode provides the phase step estimate. The performance of the proposed method is compared to methods based on least squares and principle component analysis. The simulation and experimental results demonstrate the improvement in the phase estimation accuracy and noise robustness offered by the proposed method and thus substantiate its practical applicability.

Fringe pattern demodulation using Zernike polynomials and a l1-norm regularized extended Kalman filter.

A novel algorithm for closed fringe demodulation for an absolute phase estimation, to the best of our knowledge, is proposed. The two-dimensional phase is represented as a weighted linear combination of a certain number of Zernike polynomials (ZPs). Essentially, the problem of phase estimation is converted into the estimation of ZP coefficients. The task of ZP coefficient estimation is performed based on a state space model. Due to the nonlinear dependence of the fringe intensity measurement model on the ZP coefficients, the extended Kalman filter (EKF) is used for the state estimation. A pseudo-measurement model is considered based on the state vector sparsity constraint to improve the convergence performance of the EKF. Simulation and experimental results are provided to demonstrate the noise robustness and the practical applicability of the proposed method.

Analyzing nested experimental designs-A user-friendly resampling method to determine experimental significance.

While hierarchical experimental designs are near-ubiquitous in neuroscience and biomedical research, researchers often do not take the structure of their datasets into account while performing statistical hypothesis tests. Resampling-based methods are a flexible strategy for performing these analyses but are difficult due to the lack of open-source software to automate test construction and execution. To address this, we present Hierarch, a Python package to perform hypothesis tests and compute confidence intervals on hierarchical experimental designs. Using a combination of permutation resampling and bootstrap aggregation, Hierarch can be used to perform hypothesis tests that maintain nominal Type I error rates and generate confidence intervals that maintain the nominal coverage probability without making distributional assumptions about the dataset of interest. Hierarch makes use of the Numba JIT compiler to reduce p-value computation times to under one second for typical datasets in biomedical research. Hierarch also enables researchers to construct user-defined resampling plans that take advantage of Hierarch's Numba-accelerated functions.

Objective speckle pattern-based surface roughness measurement using matrix factorization.

A method for the measurement of profile parameters of both isotropic and anisotropic surfaces is presented using the objective laser speckle imaging technique. The surface parameters are characterized in terms of a singular value decomposition method-based metric derived from the initial key contributing singular values of the speckle pattern. A simulation study is performed with random Gaussian anisotropic surfaces generated as a function of the correlation lengths in both x and y directions. In the experimental demonstration, the proposed method is verified with metallic samples having distinct surface roughness processed through widely used machining operations viz., vertical milling, and grinding. A brief discussion about the extent to which the minimum number of singular values that are sufficient to evaluate the profile parameters in the context of experimental results is provided. The method supports the measurement of profile parameters of higher magnitude in the realm of non-contact topographic measurement techniques. The experimental results substantiate the practical applicability of the proposed method.

Improving particle detection and size estimation accuracy in digital in-line holography using autoregressive interpolation.

The accuracy of particle detection and size estimation is limited by the physical size of the digital sensor used to record the hologram in a digital in-line holographic imaging system. In this paper, we propose to utilize the autoregressive (AR) interpolation of the hologram to increase pixel density and, effectively, the quality of hologram reconstruction. Simulation studies are conducted to evaluate the influence of AR interpolation of a hologram on the accuracy of detection and size estimation of single and multiple particles of varying sizes. A comparative study on the performance of different interpolation techniques indicates the advantage of the proposed AR hologram interpolation approach. An experimental result is provided to validate the suitability of the proposed algorithm in practical applications.

Autofocusing in digital holography using eigenvalues.

A new autofocusing algorithm for digital holography is proposed based on the eigenvalues of the images reconstructed at different distances in the measurement volume. An image quality metric evaluated based on the distribution of its eigenvalues is compared in function of the reconstruction distance to identify the location of the focal plane. The proposed automatic focal plane detection algorithm is capable of working with amplitude objects, phase objects, and mixed type objects. A performance comparison of the proposed algorithm with some previously reported representative algorithms is provided. The simulation and experimental results demonstrate the practical applicability of the proposed algorithm.

BODIPY Fluorophores for Membrane Potential Imaging.

Fluorophores based on the BODIPY scaffold are prized for their tunable excitation and emission profiles, mild syntheses, and biological compatibility. Improving the water-solubility of BODIPY dyes remains an outstanding challenge. The development of water-soluble BODIPY dyes usually involves direct modification of the BODIPY fluorophore core with ionizable groups or substitution at the boron center. While these strategies are effective for the generation of water-soluble fluorophores, they are challenging to implement when developing BODIPY-based indicators: direct modification of BODIPY core can disrupt the electronics of the dye, complicating the design of functional indicators; and substitution at the boron center often renders the resultant BODIPY incompatible with the chemical transformations required to generate fluorescent sensors. In this study, we show that BODIPYs bearing a sulfonated aromatic group at the meso position provide a general solution for water-soluble BODIPYs. We outline the route to a suite of 5 new sulfonated BODIPYs with 2,6-disubstitution patterns spanning a range of electron-donating and -withdrawing propensities. To highlight the utility of these new, sulfonated BODIPYs, we further functionalize them to access 13 new, BODIPY-based, voltage-sensitive fluorophores (VF). The most sensitive of these BODIPY VF dyes displays a 48% ΔF/F per 100 mV in mammalian cells. Two additional BODIPY VFs show good voltage sensitivity (≥24% ΔF/F) and excellent brightness in cells. These compounds can report on action potential dynamics in both mammalian neurons and human stem cell-derived cardiomyocytes. Accessing a range of substituents in the context of a water-soluble BODIPY fluorophore provides opportunities to tune the electronic properties of water-soluble BODIPY dyes for functional indicators.

In Vivo Two-Photon Voltage Imaging with Sulfonated Rhodamine Dyes.

Optical methods that rely on fluorescence for mapping changes in neuronal membrane potential in the brains of awake animals provide a powerful way to interrogate the activity of neurons that underlie neural computations ranging from sensation and perception to learning and memory. To achieve this goal, fluorescent indicators should be bright, highly sensitive to small changes in membrane potential, nontoxic, and excitable with infrared light. We report a new class of fluorescent, voltage-sensitive dyes: sulfonated rhodamine voltage reporters (sRhoVR), synthetic fluorophores with high voltage sensitivity, excellent two-photon performance, and compatibility in intact mouse brains. sRhoVR dyes are based on a tetramethyl rhodamine fluorophore coupled to a phenylenevinylene molecular wire/diethyl aniline voltage-sensitive domain. When applied to cells, sRhoVR dyes localize to the plasma membrane and respond to membrane depolarization with a fluorescence increase. The best of the new dyes, sRhoVR 1, displays a 44% ΔF/F increase in fluorescence per 100 mV change, emits at 570 nm, and possesses excellent two-photon absorption of approximately 200 GM at 840 nm. sRhoVR 1 can detect action potentials in cultured rat hippocampal neurons under both single- and two-photon illumination with sufficient speed and sensitivity to report on action potentials in single trials, without perturbing underlying physiology or membrane properties. The combination of speed, sensitivity, and brightness under two-photon illumination makes sRhoVR 1 a promising candidate for in vivo imaging in intact brains. We show sRhoVR powerfully complements electrode-based modes of neuronal activity recording in the mouse brain by recording neuronal transmembrane potentials from the neuropil of layer 2/3 of the mouse barrel cortex in concert with extracellularly recorded local field potentials (LFPs). sRhoVR imaging reveals robust depolarization in response to whisker stimulation; concurrent electrode recordings reveal negative deflections in the LFP recording, consistent with the canonical thalamocortical response. Importantly, sRhoVR 1 can be applied in mice with chronic optical windows, presaging its utility in dissecting and resolving voltage dynamics using two-photon functional imaging in awake, behaving animals.

hPSC-Derived Striatal Cells Generated Using a Scalable 3D Hydrogel Promote Recovery in a Huntington Disease Mouse Model.

Huntington disease (HD) is an inherited, progressive neurological disorder characterized by degenerating striatal medium spiny neurons (MSNs). One promising approach for treating HD is cell replacement therapy, where lost cells are replaced by MSN progenitors derived from human pluripotent stem cells (hPSCs). While there has been remarkable progress in generating hPSC-derived MSNs, current production methods rely on two-dimensional culture systems that can include poorly defined components, limit scalability, and yield differing preclinical results. To facilitate clinical translation, here, we generated striatal progenitors from hPSCs within a fully defined and scalable PNIPAAm-PEG three-dimensional (3D) hydrogel. Transplantation of 3D-derived striatal progenitors into a transgenic mouse model of HD slowed disease progression, improved motor coordination, and increased survival. In addition, the transplanted cells developed an MSN-like phenotype and formed synaptic connections with host cells. Our results illustrate the potential of scalable 3D biomaterials for generating striatal progenitors for HD cell therapy.

Diet-induced obesity suppresses cortical bone accrual by a neuropeptide Y-dependent mechanism.

OBJECTIVE: To determine whether age and neuropeptide Y (NPY) were involved in the skeletal response to extended periods of diet-induced obesity. METHODS: Male wild-type (WT) and NPY null (NPYKO) mice were fed a mild (23% fat) high-fat diet for 10 weeks from 6 or 16 weeks of age. Metabolism and bone density were assessed during feeding. Skeletal changes were assessed by microCT and histomorphometry. RESULTS: High-fat feeding in 6-week-old WT mice led to significantly increased body weight, adiposity and serum leptin levels, accompanied with markedly suppressed cortical bone accrual. NPYKO mice were less susceptible to fat accrual but, importantly, displayed a complete lack of suppression of bone accrual or cortical bone loss. In contrast, when skeletally mature (16 week old) mice underwent 10 weeks of fat feeding, the metabolic response to HFD was similar to younger mice, however bone mass was not affected in either WT or NPYKO. Thus, growing mice are particularly susceptible to the detrimental effects of HFD on bone mass, through suppression of bone accrual involving NPY signalling. CONCLUSION: This study provides new insights into the relationship between the opposing processes of a positive weight/bone relationship and the negative 'metabolic' effect of obesity on bone mass. This negative effect is particularly active in growing skeletons, which have heightened sensitivity to changes in obesity. In addition, NPY is identified as a fundamental driver of this negative 'metabolic' pathway to bone.

Phase unwrapping algorithm using polynomial phase approximation and linear Kalman filter.

A noise-robust phase unwrapping algorithm is proposed based on state space analysis and polynomial phase approximation using wrapped phase measurement. The true phase is approximated as a two-dimensional first order polynomial function within a small sized window around each pixel. The estimates of polynomial coefficients provide the measurement of phase and local fringe frequencies. A state space representation of spatial phase evolution and the wrapped phase measurement is considered with the state vector consisting of polynomial coefficients as its elements. Instead of using the traditional nonlinear Kalman filter for the purpose of state estimation, we propose to use the linear Kalman filter operating directly with the wrapped phase measurement. The adaptive window width is selected at each pixel based on the local fringe density to strike a balance between the computation time and the noise robustness. In order to retrieve the unwrapped phase, either a line-scanning approach or a quality guided strategy of pixel selection is used depending on the underlying continuous or discontinuous phase distribution, respectively. Simulation and experimental results are provided to demonstrate the applicability of the proposed method.

Voltage Imaging: Pitfalls and Potential.

Optical methods for interrogating membrane potential changes in neurons promise to revolutionize our ability to dissect the activity of individual cells embedded in neural circuits underlying behavior and sensation. A number of voltage imaging strategies have emerged in the past few years. This Perspective discusses developments in both small-molecule and genetically encoded fluorescent indicators of membrane potential. We survey recent advances in small-molecule fluorescent indicators that rely on photoinduced electron transfer to sense voltage as well as refinements of voltage-sensitive fluorescent proteins and new opsin-based strategies for monitoring voltage changes. We compare the requirements of fluorescent voltage indicators to those for more canonical Ca2+ sensing as a way to illuminate the particular challenges associated with voltage imaging.

Insulin controls food intake and energy balance via NPY neurons.

OBJECTIVES: Insulin signaling in the brain has been implicated in the control of satiety, glucose homeostasis and energy balance. However, insulin signaling is dispensable in energy homeostasis controlling AgRP or POMC neurons and it is unclear which other neurons regulate these effects. Here we describe an ancient insulin/NPY neuronal network that governs energy homeostasis across phyla. METHODS: To address the role of insulin action specifically in NPY neurons, we generated a variety of models by selectively removing insulin signaling in NPY neurons in flies and mice and testing the consequences on energy homeostasis. RESULTS: By specifically targeting the insulin receptor in both fly and mouse NPY expressing neurons, we found NPY-specific insulin signaling controls food intake and energy expenditure, and lack of insulin signaling in NPY neurons leads to increased energy stores and an obese phenotype. Additionally, the lack of insulin signaling in NPY neurons leads to a dysregulation of GH/IGF-1 axis and to altered insulin sensitivity. CONCLUSIONS: Taken together, these results suggest that insulin actions in NPY neurons is critical for maintaining energy balance and an impairment of this pathway may be causally linked to the development of metabolic diseases.

Engineered hydrogels increase the post-transplantation survival of encapsulated hESC-derived midbrain dopaminergic neurons.

Cell replacement therapies have broad biomedical potential; however, low cell survival and poor functional integration post-transplantation are major hurdles that hamper clinical benefit. For example, following striatal transplantation of midbrain dopaminergic (mDA) neurons for the treatment of Parkinson's disease (PD), only 1-5% of the neurons typically survive in preclinical models and in clinical trials. In general, resource-intensive generation and implantation of larger numbers of cells are used to compensate for the low post-transplantation cell-survival. Poor graft survival is often attributed to adverse biochemical, mechanical, and/or immunological stress that cells experience during and after implantation. To address these challenges, we developed a functionalized hyaluronic acid (HA)-based hydrogel for in vitro maturation and central nervous system (CNS) transplantation of human pluripotent stem cell (hPSC)-derived neural progenitors. Specifically, we functionalized the HA hydrogel with RGD and heparin (hep) via click-chemistry and tailored its stiffness to encourage neuronal maturation, survival, and long-term maintenance of the desired mDA phenotype. Importantly, ∼5 times more hydrogel-encapsulated mDA neurons survived after transplantation in the rat striatum, compared to unencapsulated neurons harvested from commonly used 2D surfaces. This engineered biomaterial may therefore increase the therapeutic potential and reduce the manufacturing burden for successful neuronal implantation.

Voltage-sensitive rhodol with enhanced two-photon brightness.

We have designed, synthesized, and applied a rhodol-based chromophore to a molecular wire-based platform for voltage sensing to achieve fast, sensitive, and bright voltage sensing using two-photon (2P) illumination. Rhodol VoltageFluor-5 (RVF5) is a voltage-sensitive dye with improved 2P cross-section for use in thick tissue or brain samples. RVF5 features a dichlororhodol core with pyrrolidyl substitution at the nitrogen center. In mammalian cells under one-photon (1P) illumination, RVF5 demonstrates high voltage sensitivity (28% ΔF/F per 100 mV) and improved photostability relative to first-generation voltage sensors. This photostability enables multisite optical recordings from neurons lacking tuberous sclerosis complex 1, Tsc1, in a mouse model of genetic epilepsy. Using RVF5, we show that Tsc1 KO neurons exhibit increased activity relative to wild-type neurons and additionally show that the proportion of active neurons in the network increases with the loss of Tsc1. The high photostability and voltage sensitivity of RVF5 is recapitulated under 2P illumination. Finally, the ability to chemically tune the 2P absorption profile through the use of rhodol scaffolds affords the unique opportunity to image neuronal voltage changes in acutely prepared mouse brain slices using 2P illumination. Stimulation of the mouse hippocampus evoked spiking activity that was readily discerned with bath-applied RVF5, demonstrating the utility of RVF5 and molecular wire-based voltage sensors with 2P-optimized fluorophores for imaging voltage in intact brain tissue.