Optimized Protocol for Imaging Cleared Neural Tissues Using Light Microscopy

Understanding physical and chemical processes at an organismal scale is a fundamental goal in biology. While science is adept at explaining biological phenomena at both molecular and cellular levels, understanding how these processes translate to organismal functions remains a challenging problem. This issue is particularly significant for the nervous system where cell signaling and synaptic activities function in the context of broad neural networks. Recent progress in tissue clearing technologies lessens the barriers that previously prevented the study of large tissue samples while maintaining molecular and cellular resolution. While these new methods open vast opportunities and exciting new questions, the logistics of analyzing cellular processes in intact tissue have to be carefully considered. In this protocol, we outline a procedure to rapidly image intact brain tissue up to thousands of cubic millimeters. This experimental pipeline involves three steps: tissue clearing, tissue imaging, and data analysis. In an attempt to streamline the process for researchers entering this field, we address important considerations for each of these stages and describe an integrated solution to image intact biological tissues. Hopefully, this optimized protocol will lower the barrier of implementing high-resolution tissue imaging and facilitate the investigations of mesoscale questions at molecular and cellular resolution

No Evidence of Intrinsic Optical/Near-Infrared Linear Polarization for V404 Cygni During its Bright Outburst in 2015: Broadband Modeling and Constraint on Jet Parameters

We present simultaneous optical and near-infrared (NIR) polarimetric results for the black hole binary V404 Cygni spanning the duration of its 7-day long optically-brightest phase of its 2015 June outburst. The simultaneous R and Ks-band light curves showed almost the same temporal variation except for the isolated (~30 min duration) orphan Ks-band flare observed at MJD 57193.54. We did not find any significant temporal variation of polarization degree (PD) and position angle (PA) in both R and Ks bands throughout our observations, including the duration of the orphan NIR flare. We show that the observed PD and PA are predominantly interstellar in origin by comparing the V404 Cyg polarimetric results with those of the surrounding sources within the 7'x7' field-of-view. The low intrinsic PD (less than a few percent) implies that the optical and NIR emissions are dominated by either disk or optically-thick synchrotron emission, or both. We also present the broadband spectra of V404 Cyg during the orphan NIR flare and a relatively faint and steady state by including quasi-simultaneous Swift/XRT and INTEGRAL fluxes. By adopting a single-zone synchrotron plus inverse-Compton model as widely used in modeling of blazars, we constrained the parameters of a putative jet. Because the jet synchrotron component cannot exceed the Swift/XRT disk/corona flux, the cutoff Lorentz factor in the electron energy distribution is constrained to be <10^2, suggesting particle acceleration is less efficient in this microquasar jet outburst compared to AGN jets. We also suggest that the loading of the baryon component inside the jet is inevitable based on energetic arguments.Comment: 8 pages, 6 figures, 2 tables. Accepted by Ap

Spectropolarimetry of R Coronae Borealis in 1998--2003: Discovery of Transient Polarization at Maximum Brightness

We present an extended optical spectropolarimetry of R CrB from 1998 January to 2003 September. The polarization was almost constant in the phase of maximum brightness, being consistent with past observations. We detected, however, temporal changes of polarization ($\sim 0.5$ %) in 2001 March and August, which were the first detection of large polarization variability in R CrB near maximum brightness. The amplitude and the position angle of the `transient polarization' were almost constant with wavelength in both two events. There was a difference by about 20 degrees in the position angle between the two events. Each event could be explained by light scattering due to short-lived dust puff occasionally ejected off the line of sight. The flatness of the polarization against the wavelength suggests that the scatterer is a mixture of dust grains having various sizes. The rapid growth and fading of the transient polarization favors the phenomenological model of dust formation near the stellar photosphere (e.g., within two stellar radii) proposed for the time evolution of brightness and chromospheric emission lines during deeply declining periods, although the fading timescale can hardly be explained by a simple dispersal of expanding dust puff with a velocity of $\sim 200-350$ km s $^{-1}$. Higher expansion velocity or some mechanism to destroy the dust grains should be needed.Comment: 22 pages, 10 figures, accepted for publication in A

Reversing Blood Flows Act through klf2a to Ensure Normal Valvulogenesis in the Developing Heart

Heart valve anomalies are some of the most common congenital heart defects, yet neither the genetic nor the epigenetic forces guiding heart valve development are well understood. When functioning normally, mature heart valves prevent intracardiac retrograde blood flow; before valves develop, there is considerable regurgitation, resulting in reversing (or oscillatory) flows between the atrium and ventricle. As reversing flows are particularly strong stimuli to endothelial cells in culture, an attractive hypothesis is that heart valves form as a developmental response to retrograde blood flows through the maturing heart. Here, we exploit the relationship between oscillatory flow and heart rate to manipulate the amount of retrograde flow in the atrioventricular (AV) canal before and during valvulogenesis, and find that this leads to arrested valve growth. Using this manipulation, we determined that klf2a is normally expressed in the valve precursors in response to reversing flows, and is dramatically reduced by treatments that decrease such flows. Experimentally knocking down the expression of this shear-responsive gene with morpholine antisense oligonucleotides (MOs) results in dysfunctional valves. Thus, klf2a expression appears to be necessary for normal valve formation. This, together with its dependence on intracardiac hemodynamic forces, makes klf2a expression an early and reliable indicator of proper valve development. Together, these results demonstrate a critical role for reversing flows during valvulogenesis and show how relatively subtle perturbations of normal hemodynamic patterns can lead to both major alterations in gene expression and severe valve dysgenesis

Implications For The Origin Of GRB 051103 From LIGO Observations

We present the results of a LIGO search for gravitational waves (GWs) associated with GRB 051103, a short-duration hard-spectrum gamma-ray burst (GRB) whose electromagnetically determined sky position is coincident with the spiral galaxy M81, which is 3.6 Mpc from Earth. Possible progenitors for short-hard GRBs include compact object mergers and soft gamma repeater (SGR) giant flares. A merger progenitor would produce a characteristic GW signal that should be detectable at the distance of M81, while GW emission from an SGR is not expected to be detectable at that distance. We found no evidence of a GW signal associated with GRB 051103. Assuming weakly beamed gamma-ray emission with a jet semi-angle of 30 deg we exclude a binary neutron star merger in M81 as the progenitor with a confidence of 98%. Neutron star-black hole mergers are excluded with > 99% confidence. If the event occurred in M81 our findings support the the hypothesis that GRB 051103 was due to an SGR giant flare, making it the most distant extragalactic magnetar observed to date.Comment: 8 pages, 3 figures. For a repository of data used in the publication, go to: https://dcc.ligo.org/cgi-bin/DocDB/ShowDocument?docid=15166 . Also see the announcement for this paper on ligo.org at: http://www.ligo.org/science/Publication-GRB051103/index.ph