Fast two-layer two-photon imaging of neuronal cell populations using an electrically tunable lens

Functional two-photon Ca2+-imaging is a versatile tool to study the dynamics of neuronal populations in brain slices and living animals. However, population imaging is typically restricted to a single two-dimensional image plane. By introducing an electrically tunable lens into the excitation path of a two-photon microscope we were able to realize fast axial focus shifts within 15 ms. The maximum axial scan range was 0.7 mm employing a 40x NA0.8 water immersion objective, plenty for typically required ranges of 0.2–0.3 mm. By combining the axial scanning method with 2D acousto-optic frame scanning and random-access scanning, we measured neuronal population activity of about 40 neurons across two imaging planes separated by 40 μm and achieved scan rates up to 20–30 Hz. The method presented is easily applicable and allows upgrading of existing two-photon microscopes for fast 3D scanning

iDISCO+ for the Study of Neuroimmune Architecture of the Rat Auditory Brainstem

The lower stations of the auditory system display a complex anatomy. The inner ear labyrinth is composed of several interconnecting membranous structures encased in cavities of the temporal bone, and the cerebellopontine angle contains fragile structures such as meningeal folds, the choroid plexus (CP), and highly variable vascular formations. For this reason, most histological studies of the auditory system have either focused on the inner ear or the CNS by physically detaching the temporal bone from the brainstem. However, several studies of neuroimmune interactions have pinpointed the importance of structures such as meninges and CP; in the auditory system, an immune function has also been suggested for inner ear structures such as the endolymphatic duct (ED) and sac. All these structures are thin, fragile, and have complex 3D shapes. In order to study the immune cell populations located on these structures and their relevance to the inner ear and auditory brainstem in health and disease, we obtained a clarified-decalcified preparation of the rat hindbrain still attached to the intact temporal bone. This preparation may be immunolabeled using a clearing protocol (based on iDISCO+) to show location and functional state of immune cells. The observed macrophage distribution suggests the presence of CP-mediated communication pathways between the inner ear and the cochlear nuclei

Transport Coefficients from Large Deviation Functions

We describe a method for computing transport coefficients from the direct evaluation of large deviation function. This method is general, relying on only equilibrium fluctuations, and is statistically efficient, employing trajectory based importance sampling. Equilibrium fluctuations of molecular currents are characterized by their large deviation functions, which is a scaled cumulant generating function analogous to the free energy. A diffusion Monte Carlo algorithm is used to evaluate the large deviation functions, from which arbitrary transport coefficients are derivable. We find significant statistical improvement over traditional Green-Kubo based calculations. The systematic and statistical errors of this method are analyzed in the context of specific transport coefficient calculations, including the shear viscosity, interfacial friction coefficient, and thermal conductivity.Comment: 11 pages, 5 figure

The Prevalence of Cooling Cores in Clusters of Galaxies at z~0.15-0.4

We present a Chandra study of 38 X-ray luminous clusters of galaxies in the ROSAT Brightest Cluster Sample (BCS) that lie at z~0.15-0.4. We find that the majority of clusters at moderate redshift generally have smooth, relaxed morphologies with some evidence for mild substructure perhaps indicative of recent minor merger activity. Using spatially-resolved spectral analyses, cool cores appear to still be common at these redshifts. At a radius of 50 kpc, we find that at least 55 per cent of the clusters in our sample exhibit signs of mild cooling [t(cool)<10 Gyr], while in the central bin at least 34 per cent demonstrate signs of strong cooling [t(cool)<2 Gyr]. These percentages are nearly identical to those found for luminous, low-redshift clusters of galaxies, suggesting little evolution in cluster cores since z~0.4 and that heating and cooling mechanisms may already have stabilised by this epoch. Comparing the central cooling times to central Halpha emission in BCS clusters, we find a strong correspondence between the detection of Halpha and central cooling time. (Abridged)Comment: 10 pages, 8 figures, accepted to MNRA

A nonlinear theory of the parallel firehose and gyrothermal instabilities in a weakly collisional plasma

Weakly collisional plasmas dynamically develop pressure anisotropies with respect to the magnetic field. These anisotropies trigger plasma instabilities at scales just above the ion Larmor radius \rho_i and much below the mean free path \lambda_{mfp}. They have growth rates of a fraction of the ion cyclotron frequency - much faster than either the global dynamics or local turbulence. The instabilities dramatically modify the transport properties and, therefore, the macroscopic dynamics of the plasma. Their nonlinear evolution drives pressure anisotropies towards marginal stability, controlled by the plasma beta \beta_i. Here this nonlinear evolution is worked out for the simplest analytically tractable example - the parallel firehose instability. In the nonlinear regime, both analytical theory and the numerical solution predict secular growth of magnetic fluctuations. They develop a k^{-3} spectrum, extending from scales somewhat larger than \rho_i to the maximum scale that grows secularly with time (~t^{1/2}); the relative pressure anisotropy (\pperp-\ppar)/\ppar tends to the marginal value -2/\beta_i. The marginal state is achieved via changes in the magnetic field, not particle scattering. When a parallel ion heat flux is present, the firehose mutates into the new gyrothermal instability (GTI), which continues to exist up to firehose-stable values of pressure anisotropy, which can be positive and are limited by the heat flux. The nonlinear evolution of the GTI also features secular growth of magnetic fluctuations, but the spectrum is eventually dominated by modes around the scale ~\rho_i l_T/\lambda_{mfp}, where l_T is the scale of the parallel temperature variation. Implications for momentum and heat transport are speculated about. This study is motivated by the dynamics of galaxy cluster plasmas.Comment: 34 pages, replaced with the version published in MNRA

Tissue Clearing and Light Sheet Microscopy: Imaging the Unsectioned Adult Zebra Finch Brain at Cellular Resolution

The inherent complexity of brain tissue, with brain cells intertwining locally and projecting to distant regions, has made three-dimensional visualization of intact brains a highly desirable but challenging task in neuroscience. The natural opaqueness of tissue has traditionally limited researchers to techniques short of single cell resolution such as computer tomography or magnetic resonance imaging. By contrast, techniques with single-cell resolution required mechanical slicing into thin sections, which entails tissue distortions that severely hinder accurate reconstruction of large volumes. Recent developments in tissue clearing and light sheet microscopy have made it possible to investigate large volumes at micrometer resolution. The value of tissue clearing has been shown in a variety of tissue types and animal models. However, its potential for examining the songbird brain remains unexplored. Songbirds are an established model system for the study of vocal learning and sensorimotor control. They share with humans the capacity to adapt vocalizations based on auditory input. Song learning and production are controlled in songbirds by the song system, which forms a network of interconnected discrete brain nuclei. Here, we use the CUBIC and iDISCO+ protocols for clearing adult songbird brain tissue. Combined with light sheet imaging, we show the potential of tissue clearing for the investigation of connectivity between song nuclei, as well as for neuroanatomy and brain vasculature studies

Reflective multi-immersion microscope objectives inspired by the Schmidt telescope

Imaging large, cleared samples requires microscope objectives that combine a large field of view (FOV) with a long working distance (WD) and a high numerical aperture (NA). Ideally, such objectives should be compatible with a wide range of immersion media, which is challenging to achieve with conventional lens-based objective designs. Here we introduce the multi-immersion 'Schmidt objective' consisting of a spherical mirror and an aspherical correction plate as a solution to this problem. We demonstrate that a multi-photon variant of the Schmidt objective is compatible with all homogeneous immersion media and achieves an NA of 1.08 at a refractive index of 1.56, 1.1-mm FOV and 11-mm WD. We highlight its versatility by imaging cleared samples in various media ranging from air and water to benzyl alcohol/benzyl benzoate, dibenzyl ether and ethyl cinnamate and by imaging of neuronal activity in larval zebrafish in vivo. In principle, the concept can be extended to any imaging modality, including wide-field, confocal and light-sheet microscopy

Whole‐brain microscopy reveals distinct temporal and spatial efficacy of anti‐Aβ therapies

Many efforts targeting amyloid-β (Aβ) plaques for the treatment of Alzheimer's Disease thus far have resulted in failures during clinical trials. Regional and temporal heterogeneity of efficacy and dependence on plaque maturity may have contributed to these disappointing outcomes. In this study, we mapped the regional and temporal specificity of various anti-Aβ treatments through high-resolution light-sheet imaging of electrophoretically cleared brains. We assessed the effect on amyloid plaque formation and growth in Thy1-APP/PS1 mice subjected to β-secretase inhibitors, polythiophenes, or anti-Aβ antibodies. Each treatment showed unique spatiotemporal Aβ clearance, with polythiophenes emerging as a potent anti-Aβ compound. Furthermore, aligning with a spatial-transcriptomic atlas revealed transcripts that correlate with the efficacy of each Aβ therapy. As observed in this study, there is a striking dependence of specific treatments on the location and maturity of Aβ plaques. This may also contribute to the clinical trial failures of Aβ-therapies, suggesting that combinatorial regimens may be significantly more effective in clearing amyloid deposition. Keywords: Alzheimer's disease; amyloid-beta; brain; light-sheet microscopy; tissue clearin

Spatially resolved X-ray spectroscopy of cooling clusters of galaxies

We present spatially resolved X-ray spectra taken with the EPIC cameras of XMM-Newton of a sample of 17 cooling clusters and three non-cooling clusters for comparison. The deprojected spectra are analyzed with a multi-temperature model, independent of any a priori assumptions about the physics behind the cooling and heating mechanisms. All cooling clusters show a central decrement of the average temperature, most of them of a factor of ~2. At each radius within the cooling region the gas is non-isothermal. The differential emission measure distribution peaks near the maximum (ambient) temperature, and steeply declines towards lower temperatures, proportional to T^3, or alternatively a cut-off at about a quarter to half of the maximum temperature. In general, we find a poor correlation between radio flux of the central galaxy and the temperature decrement of the cooling flow. This is interpreted as evidence that except for a few cases heating by a central AGN is not the most common cause of weak cooling flows. We investigate the role of heat conduction by electrons and find that the theoretically predicted conductivity rates are not high enough to balance radiation losses. The differential emission measure distribution has remarkable similarities with the predictions from coronal magnetic loop models. Also the physical processes involved (radiative cooling, thermal conduction along the loops, gravity) are similar for clusters loops and coronal loops. If coronal loop models apply to clusters, we find that a few hundred loops per scale height should be present. The typical loop sizes deduced from the observed emission measure distribution are consistent with the characteristic magnetic field sizes deduced from Faraday rotation measurements.Comment: Accepted for publication in Astronomy and Astrophysics, main journal, 25 pages, 12 figure

The Benchtop mesoSPIM: a next-generation open-source light-sheet microscope for large cleared samples

In 2015, we launched the mesoSPIM initiative (www.mesospim.org), an open-source project for making light-sheet microscopy of large cleared tissues more accessible. Meanwhile, the demand for imaging larger samples at higher speed and resolution has increased, requiring major improvements in the capabilities of light-sheet microscopy. Here, we introduce the next-generation mesoSPIM ("Benchtop") with significantly increased field of view, improved resolution, higher throughput, more affordable cost and simpler assembly compared to the original version. We developed a new method for testing objectives, enabling us to select detection objectives optimal for light-sheet imaging with large-sensor sCMOS cameras. The new mesoSPIM achieves high spatial resolution (1.5 μm laterally, 3.3 μm axially) across the entire field of view, a magnification up to 20x, and supports sample sizes ranging from sub-mm up to several centimetres, while being compatible with multiple clearing techniques. The new microscope serves a broad range of applications in neuroscience, developmental biology, and even physics