Fast localized wavefront correction using area-mapped phase-shift interferometry

We propose an innovative method for localized wavefront correction based on area-mapped phase-shift (AMPS) interferometry. In this Letter, we present the theory and then experimentally compare it with a previously demonstrated method based on spot-optimized phase-stepping (SOPS) interferometry. We found that AMPS outperforms SOPS interferometry in terms of speed by threefold, although in noisy environments the improvements may be larger. AMPS yielded similar point-spread functions (PSF) as SOPS for moderate system-induced aberrations, but yielded a slightly less ideal PSF for larger aberrations. The method described in this Letter may prove crucial for applications where the phase-stepping solution does not have sufficient speed

Second harmonic generating (SHG) nanoprobes for in vivo imaging

Fluorescence microscopy has profoundly changed cell and molecular biology studies by permitting tagged gene products to be followed as they function and interact. The ability of a fluorescent dye to absorb and emit light of different wavelengths allows it to generate startling contrast that, in the best cases, can permit single molecule detection and tracking. However, in many experimental settings, fluorescent probes fall short of their potential due to dye bleaching, dye signal saturation, and tissue autofluorescence. Here, we demonstrate that second harmonic generating (SHG) nanoprobes can be used for in vivo imaging, circumventing many of the limitations of classical fluorescence probes. Under intense illumination, such as at the focus of a laser-scanning microscope, these SHG nanocrystals convert two photons into one photon of half the wavelength; thus, when imaged by conventional two-photon microscopy, SHG nanoprobes appear to generate a signal with an inverse Stokes shift like a fluorescent dye, but with a narrower emission. Unlike commonly used fluorescent probes, SHG nanoprobes neither bleach nor blink, and the signal they generate does not saturate with increasing illumination intensity. The resulting contrast and detectability of SHG nanoprobes provide unique advantages for molecular imaging of living cells and tissues

Alterations of the extracellular matrix in ovarian cancer studied by Second Harmonic Generation imaging microscopy

<p>Abstract</p> <p>Background</p> <p>Remodeling of the extracellular matrix (ECM) has been implicated in ovarian cancer, and we hypothesize that these alterations may provide a better optical marker of early disease than currently available imaging/screening methods and that understanding their physical manifestations will provide insight into invasion.</p> <p>Methods</p> <p>For this investigation we use Second Harmonic Generation (SHG) imaging microcopy to study changes in the structure of the ovarian ECM in human normal and malignant ex vivo biopsies. This method directly visualizes the type I collagen in the ECM and provides quantitative metrics of the fibrillar assembly. To quantify these changes in collagen morphology we utilized an integrated approach combining 3D SHG imaging measurements and bulk optical parameter measurements in conjunction with Monte Carlo simulations of the experimental data to extract tissue structural properties.</p> <p>Results</p> <p>We find the SHG emission attributes (directionality and relative intensity) and bulk optical parameters, both of which are related to the tissue structure, are significantly different in the tumors in a manner that is consistent with the change in collagen assembly. The normal and malignant tissues have highly different collagen fiber assemblies, where collectively, our findings show that the malignant ovaries are characterized by lower cell density, denser collagen, as well as higher regularity at both the fibril and fiber levels. This further suggests that the assembly in cancer may be comprised of newly synthesized collagen as opposed to modification of existing collagen.</p> <p>Conclusions</p> <p>Due to the large structural changes in tissue assembly and the SHG sensitivity to these collagen alterations, quantitative discrimination is achieved using small patient data sets. Ultimately these measurements may be developed as intrinsic biomarkers for use in clinical applications.</p

Analysis of RNA Binding by the Dengue Virus NS5 RNA Capping Enzyme

Flaviviruses are small, capped positive sense RNA viruses that replicate in the cytoplasm of infected cells. Dengue virus and other related flaviviruses have evolved RNA capping enzymes to form the viral RNA cap structure that protects the viral genome and directs efficient viral polyprotein translation. The N-terminal domain of NS5 possesses the methyltransferase and guanylyltransferase activities necessary for forming mature RNA cap structures. The mechanism for flavivirus guanylyltransferase activity is currently unknown, and how the capping enzyme binds its diphosphorylated RNA substrate is important for deciphering how the flavivirus guanylyltransferase functions. In this report we examine how flavivirus NS5 N-terminal capping enzymes bind to the 5′ end of the viral RNA using a fluorescence polarization-based RNA binding assay. We observed that the KD for RNA binding is approximately 200 nM Dengue, Yellow Fever, and West Nile virus capping enzymes. Removal of one or both of the 5′ phosphates reduces binding affinity, indicating that the terminal phosphates contribute significantly to binding. RNA binding affinity is negatively affected by the presence of GTP or ATP and positively affected by S-adensyl methoninine (SAM). Structural superpositioning of the dengue virus capping enzyme with the Vaccinia virus VP39 protein bound to RNA suggests how the flavivirus capping enzyme may bind RNA, and mutagenesis analysis of residues in the putative RNA binding site demonstrate that several basic residues are critical for RNA binding. Several mutants show differential binding to 5′ di-, mono-, and un-phosphorylated RNAs. The mode of RNA binding appears similar to that found with other methyltransferase enzymes, and a discussion of diphosphorylated RNA binding is presented

In Vivo Evaluation of Cervical Stiffness Evolution during Induced Ripening Using Shear Wave Elastography, Histology and 2 Photon Excitation Microscopy: Insight from an Animal Model

Prematurity affects 11% of the births and is the main cause of infant mortality. On the opposite case, the failure of induction of parturition in the case of delayed spontaneous birth is associated with fetal suffering. Both conditions are associated with precocious and/or delayed cervical ripening. Quantitative and objective information about the temporal evolution of the cervical ripening may provide a complementary method to identify cases at risk of preterm delivery and to assess the likelihood of successful induction of labour. In this study, the cervical stiffness was measured in vivo in pregnant sheep by using Shear Wave Elastography (SWE). This technique assesses the stiffness of tissue through the measurement of shear waves speed (SWS). In the present study, 9 pregnant ewes were used. Cervical ripening was induced at 127 days of pregnancy (term: 145 days) by dexamethasone injection in 5 animals, while 4 animals were used as control. Elastographic images of the cervix were obtained by two independent operators every 4 hours during 24 hours after injection to monitor the cervical maturation induced by the dexamethasone. Based on the measurements of SWS during vaginal ultrasound examination, the stiffness in the second ring of the cervix was quantified over a circular region of interest of 5 mm diameter. SWS was found to decrease significantly in the first 4–8 hours after dexamethasone compared to controls, which was associated with cervical ripening induced by dexamethasone (from 1.779 m/s ± 0.548 m/s, p < 0.0005, to 1.291 m/s ± 0.516 m/s, p < 0.000). Consequently a drop in the cervical elasticity was quantified too (from 9.5 kPa ± 0.9 kPa, p < 0.0005, to 5.0 kPa ± 0.8 kPa, p < 0.000). Moreover, SWE measurements were highly reproducible between both operators at all times. Cervical ripening induced by dexamethasone was confirmed by the significant increase in maternal plasma Prostaglandin E2 (PGE2), as evidenced by the assay of its metabolite PGEM. Histological analyses and two-photon excitation microscopy, combining both Second Harmonic Generation (SHG) and Two-photon Fluorescence microscopy (2PF) contrasts, were used to investigate, at the microscopic scale, the structure of cervical tissue. Results show that both collagen and 2PF-active fibrillar structures could be closely related to the mechanical properties of cervical tissue that are perceptible in elastography. In conclusion, SWE may be a valuable method to objectively quantify the cervical stiffness and as a complementary diagnostic tool for preterm birth and for labour induction success

Region-Specific Microstructure in the Neonatal Ventricles of a Porcine Model

© 2018, Biomedical Engineering Society. The neonate transitions from placenta-derived oxygen, to supply from the pulmonary system, moments after birth. This requires a series of structural developments to divert more blood through the right heart and onto the lungs, with the tissue quickly remodelling to the changing ventricular workload. In some cases, however, the heart structure does not fully develop causing poor circulation and inefficient oxygenation, which is associated with an increase in mortality and morbidity. This study focuses on developing an enhanced knowledge of the 1-day old heart, quantifying the region-specific microstructural parameters of the tissue. This will enable more accurate mathematical and computational simulations of the young heart. Hearts were dissected from 12, 1-day-old deceased Yorkshire piglets (mass: 2.1–2.4kg, length: 0.38–0.51m), acquired from a breeding farm. Evans blue dye was used to label the heart equator and to demarcate the left and right ventricle free walls. Two hearts were used for three-dimensional diffusion-tensor magnetic resonance imaging, to quantify the fractional anisotropy (FA). The remaining hearts were used for two-photon excited fluorescence and second-harmonic generation microscopy, to quantify the cardiomyocyte and collagen fibril structures within the anterior and posterior aspects of the right and left ventricles. FA varied significantly across both ventricles, with the greatest in the equatorial region, followed by the base and apex. The FA in each right ventricular region was statistically greater than that in the left. Cardiomyocyte and collagen fibre rotation was greatest in the anterior wall of both ventricles, with less dispersion when compared to the posterior walls. In defining these key parameters, this study provides a valuable insight into the 1-day-old heart that will provide a valuable platform for further investigation the normal and abnormal heart using mathematical and computational models

Microscale characterization of prostate biopsies tissues using optical coherence elastography and second harmonic generation imaging

© 2018 USCAP, Inc All rights reserved. Photonics, especially optical coherence elastography (OCE) and second harmonic generation (SHG) imaging are novel high-resolution imaging modalities for characterization of biological tissues. Following our preliminary experience, we hypothesized that OCE and SHG imaging would delineate the microstructure of prostate tissue and aid in distinguishing cancer from the normal benign prostatic tissue. Furthermore, these approaches may assist in characterization of the grade of cancer, as well. In this study, we confirmed a high diagnostic accuracy of OCE and SHG imaging in the detection and characterization of prostate cancer for a large set of biopsy tissues obtained from men suspected to have prostate cancer using transrectal ultrasound (TRUS). The two techniques and methods described here are complementary, one depicts the stiffness of tissues and the other illustrates the orientation of collagen structure around the cancerous lesions. The results showed that stiffness of cancer tissue was ∼57.63% higher than that of benign tissue (Young's modulus of 698.43±125.29 kPa for cancerous tissue vs 443.07±88.95 kPa for benign tissue with OCE. Using histology as a reference standard and 600 kPa as a cut-off threshold, the data analysis showed sensitivity and specificity of 89.6 and 99.8%, respectively. Corresponding positive and negative predictive values were 99.5 and 94.6%, respectively. There was a significant difference noticed in terms of Young's modulus for different Gleason scores estimated by OCE (P-value<0.05). For SHG, distinct patterns of collagen distribution were seen for different Gleason grade disease with computed quantification employing a ratio of anisotropic to isotropic (A:I ratio) and this correlated with disease aggressiveness

Target cell-specific synaptic dynamics of excitatory to inhibitory neuron connections in supragranular layers of human neocortex.

Rodent studies have demonstrated that synaptic dynamics from excitatory to inhibitory neuron types are often dependent on the target cell type. However, these target cell-specific properties have not been well investigated in human cortex, where there are major technical challenges in reliably obtaining healthy tissue, conducting multiple patch-clamp recordings on inhibitory cell types, and identifying those cell types. Here, we take advantage of newly developed methods for human neurosurgical tissue analysis with multiple patch-clamp recordings, post-hoc fluorescent in situ hybridization (FISH), machine learning-based cell type classification and prospective GABAergic AAV-based labeling to investigate synaptic properties between pyramidal neurons and PVALB- vs. SST-positive interneurons. We find that there are robust molecular differences in synapse-associated genes between these neuron types, and that individual presynaptic pyramidal neurons evoke postsynaptic responses with heterogeneous synaptic dynamics in different postsynaptic cell types. Using molecular identification with FISH and classifiers based on transcriptomically identified PVALB neurons analyzed by Patch-seq, we find that PVALB neurons typically show depressing synaptic characteristics, whereas other interneuron types including SST-positive neurons show facilitating characteristics. Together, these data support the existence of target cell-specific synaptic properties in human cortex that are similar to rodent, thereby indicating evolutionary conservation of local circuit connectivity motifs from excitatory to inhibitory neurons and their synaptic dynamics