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.

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.

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.

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.

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.

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.

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.

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.

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.

Isomerically Pure Tetramethylrhodamine Voltage Reporters.

We present the design, synthesis, and application of a new family of fluorescent voltage indicators based on isomerically pure tetramethylrhodamines. These new Rhodamine Voltage Reporters, or RhoVRs, use photoinduced electron transfer (PeT) as a trigger for voltage sensing, display excitation and emission profiles in the green to orange region of the visible spectrum, demonstrate high sensitivity to membrane potential changes (up to 47% ΔF/F per 100 mV), and employ a tertiary amide derived from sarcosine, which aids in membrane localization and simultaneously simplifies the synthetic route to the voltage sensors. The most sensitive of the RhoVR dyes, RhoVR 1, features a methoxy-substituted diethylaniline donor and phenylenevinylene molecular wire at the 5'-position of the rhodamine aryl ring, exhibits the highest voltage sensitivity to date for red-shifted PeT-based voltage sensors, and is compatible with simultaneous imaging alongside green fluorescent protein-based indicators. The discoveries that sarcosine-based tertiary amides in the context of molecular-wire voltage indicators prevent dye internalization and 5'-substituted voltage indicators exhibit improved voltage sensitivity should be broadly applicable to other types of PeT-based voltage-sensitive fluorophores.

Closed fringe demodulation using phase decomposition by Fourier basis functions.

We report a new technique for the demodulation of a closed fringe pattern by representing the phase as a weighted linear combination of a certain number of linearly independent Fourier basis functions in a given row/column at a time. A state space model is developed with the weights of the basis functions as the elements of the state vector. The iterative extended Kalman filter is effectively utilized for the robust estimation of the weights. A coarse estimate of the fringe density based on the fringe frequency map is used to determine the initial row/column to start with and subsequently the optimal number of basis functions. The performance of the proposed method is evaluated with several noisy fringe patterns. Experimental results are also reported to support the practical applicability of the proposed method.

Iterative signal separation based multiple phase estimation in digital holographic interferometry.

We propose a new method for signal separation from a multicomponent interference field recorded in a digital holographic interferometry setup. The setup consisting of multiple object illuminating beams results in an interference field containing multiple signal components. The proposed method utilizes an amplitude discrimination criteria established by setting different intensities to the object illuminating beams in order to separate the signal components iteratively. The signal separation is performed in a small block of the interference field at a time. The augmentation of the block matrix with its own rows and columns is performed which has an effect of noise subspace inflation. This operation offers an improved noise robustness to the signal separation capability of the proposed method. The simulation and experimental results are provided to substantiate the applicability of the proposed method in multidimensional deformation measurement.

Phase derivative estimation from a single interferogram using a Kalman smoothing algorithm.

We report a technique for direct phase derivative estimation from a single recording of a complex interferogram. In this technique, the interference field is represented as an autoregressive model with spatially varying coefficients. Estimates of these coefficients are obtained using the Kalman filter implementation. The Rauch-Tung-Striebel smoothing algorithm further improves the accuracy of the coefficient estimation. These estimated coefficients are utilized to compute the spatially varying phase derivative. Stochastic evolution of the coefficients is considered, which allows estimating the phase derivative with any type of spatial variation. The simulation and experimental results are provided to substantiate the noise robustness and applicability of the proposed method in phase derivative estimation.

Three-dimensional displacement measurement from phase signals embedded in a frame in digital holographic interferometry.

A novel technique is proposed for the simultaneous measurement of all three components of displacement from a single recording of the interference field in digital holographic interferometry. The interference field is divided into a number of rectangular segments, and in each of these segments, the interference field is represented as a multicomponent low-order two-dimensional (2D) polynomial phase signal. 2D-product high-order ambiguity function based analysis is applied to obtain an accurate estimation of the 2D polynomial coefficients. These coefficients are further used to compute the interference phases. The simulation and experimental results show the robustness of the proposed method to noise and its effectiveness in multiple phase estimation.

Ron receptor signaling is protective against DSS-induced colitis in mice.

Inflammatory bowel diseases (IBD) are chronic inflammatory disorders of the intestine that result in painful and debilitating complications. Currently no cure exists for IBD, and treatments are primarily aimed at reducing inflammation to alleviate symptoms. Genome-wide linkage studies have identified the Ron receptor tyrosine kinase (TK) and its ligand, hepatocyte growth factor-like protein (HGFL), as genes highly associated with IBD. However, only scant information exists on the role of Ron or HGFL in IBD. Based on the linkage of Ron to IBD, we directly examined the biological role of Ron in colitis. Wild-type mice and mice lacking the TK signaling domain of Ron (TK-/- mice) were utilized in a well-characterized model of chronic colitis induced by cyclic exposure to dextran sulfate sodium. In this model, TK-/- mice were more susceptible to injury as judged by increased mortality compared with control mice and developed more severe colitis. Loss of Ron led to significantly reduced body weights and more aggressive clinical and histopathologies. Ron loss also resulted in a dramatic reduction in colonic epithelial cell proliferation and increased proinflammatory cytokine production, which was associated with alterations in important signaling pathways known to regulate IBD. Examination of human gene expression data further supports the contention that loss of Ron signaling is associated with IBD. In total, our studies point to important functional roles for Ron in IBD by regulating healing of the colonic epithelium and by controlling cytokine secretion.

Digital holographic moiré for the direct and simultaneous estimation of strain and slope fields.

A novel method is proposed for the direct and simultaneous estimation of multiple phase derivatives corresponding to strain and slope fields from a single moiré fringe pattern in digital holographic moiré. The interference field in a given row/column is a multicomponent complex exponential signal and is represented as a spatially-varying autoregressive (SVAR) process. The spatially-varying coefficients of the SVAR model are computed by approximating them as the linear combination of linearly independent basis functions. Further, the spatially varying poles of the transfer function corresponding to the SVAR model are computed which provide the accurate estimation of the multiple phase derivatives. The simulation and experimental results are provided to substantiate the effectiveness of the proposed method.