Optical coherence tomography-based design for a real-time motion corrected scanning microscope.

While two-photon fluorescence microscopy is a powerful platform for the study of functional dynamics in living cells and tissues, the bulk motion inherent to these applications causes distortions. We have designed a motion tracking module based on spectral domain optical coherence tomography which compliments a laser scanning two-photon microscope with real-time corrective feedback. The module can be added to fluorescent imaging microscopes using a single dichroic and without additional contrast agents. We demonstrate that the system can track lateral displacements as large as 10 µm at 5 Hz with latency under 14 ms and propose a scheme to extend the system to 3D correction with the addition of a remote focusing module. We also propose several ways to improve the module's performance by reducing the feedback latency. We anticipate that this design can be adapted to other imaging modalities, enabling the study of samples subject to motion artifacts at higher resolution.

Neurovascular Uncoupling Is Linked to Microcirculatory Dysfunction in Regions Outside the Ischemic Core Following Ischemic Stroke.

Background Normal brain function depends on the ability of the vasculature to increase blood flow to regions with high metabolic demands. Impaired neurovascular coupling, such as the local hyperemic response to neuronal activity, may contribute to poor neurological outcome after stroke despite successful recanalization, that is, futile recanalization. Methods and Results Mice implanted with chronic cranial windows were trained for awake head-fixation before experiments. One-hour occlusion of the anterior middle cerebral artery branch was induced using single-vessel photothrombosis. Cerebral perfusion and neurovascular coupling were assessed by optical coherence tomography and laser speckle contrast imaging. Capillaries and pericytes were studied in perfusion-fixed tissue by labeling lectin and platelet-derived growth factor receptor ß. Arterial occlusion induced multiple spreading depolarizations over 1 hour associated with substantially reduced blood flow in the peri-ischemic cortex. Approximately half of the capillaries in the peri-ischemic area were no longer perfused at the 3- and 24-hour follow-up (45% [95% CI, 33%-58%] and 53% [95% CI, 39%-66%] reduction, respectively; P<0.0001), which was associated with contraction of an equivalent proportion of peri-ischemic capillary pericytes. The capillaries in the peri-ischemic cortex that remained perfused showed increased point prevalence of dynamic flow stalling (0.5% [95% CI, 0.2%-0.7%] at baseline, 5.1% [95% CI, 3.2%-6.5%] and 3.2% [95% CI, 1.1%-5.3%] at 3- and 24-hour follow-up, respectively; P=0.001). Whisker stimulation at the 3- and 24-hour follow-up led to reduced neurovascular coupling responses in the sensory cortex corresponding to the peri-ischemic region compared with that observed at baseline. Conclusions Arterial occlusion led to contraction of capillary pericytes and capillary flow stalling in the peri-ischemic cortex. Capillary dysfunction was associated with neurovascular uncoupling. Neurovascular coupling impairment associated with capillary dysfunction may be a mechanism that contributes to futile recanalization. Hence, the results from this study suggest a novel treatment target to improve neurological outcome after stroke.

Measuring capillary flow dynamics using interlaced two-photon volumetric scanning.

Two photon microscopy and optical coherence tomography (OCT) are two standard methods for measuring flow speeds of red blood cells in microvessels, particularly in animal models. However, traditional two photon microscopy lacks the depth of field to adequately capture the full volumetric complexity of the cerebral microvasculature and OCT lacks the specificity offered by fluorescent labeling. In addition, the traditional raster scanning technique utilized in both modalities requires a balance of image frame rate and field of view, which severely limits the study of RBC velocities in the microvascular network. Here, we overcome this by using a custom two photon system with an axicon based Bessel beam to obtain volumetric images of the microvascular network with fluorescent specificity. We combine this with a novel scan pattern that generates pairs of frames with short time delay sufficient for tracking red blood cell flow in capillaries. We track RBC flow speeds in 10 or more capillaries simultaneously at 1 Hz in a 237 µm × 237 µm × 120 µm volume and quantified both their spatial and temporal variability in speed. We also demonstrate the ability to track flow speed changes around stalls in capillary flow and measure to 300 µm in depth.

Dynamic capillary stalls in reperfused ischemic penumbra contribute to injury: A hyperacute role for neutrophils in persistent traffic jams.

Ever since the introduction of thrombolysis and the subsequent expansion of endovascular treatments for acute ischemic stroke, it remains to be identified why the actual outcomes are less favorable despite recanalization. Here, by high spatio-temporal resolution imaging of capillary circulation in mice, we introduce the pathological phenomenon of dynamic flow stalls in cerebral capillaries, occurring persistently in salvageable penumbra after reperfusion. These stalls, which are different from permanent cellular plugs of no-reflow, were temporarily and repetitively occurring in the capillary network, impairing the overall circulation like small focal traffic jams. In vivo microscopy in the ischemic penumbra revealed leukocytes traveling slowly through capillary lumen or getting stuck, while red blood cell flow was being disturbed in the neighboring segments under reperfused conditions. Stall dynamics could be modulated, by injection of an anti-Ly6G antibody specifically targeting neutrophils. Decreased number and duration of stalls were associated with improvement in penumbral blood flow within 2-24 h after reperfusion along with increased capillary oxygenation, decreased cellular damage and improved functional outcome. Thereby, dynamic microcirculatory stall phenomenon can be a contributing factor to ongoing penumbral injury and is a potential hyperacute mechanism adding on previous observations of detrimental effects of activated neutrophils in ischemic stroke.

Epithelial layer unjamming shifts energy metabolism toward glycolysis.

In development of an embryo, healing of a wound, or progression of a carcinoma, a requisite event is collective epithelial cellular migration. For example, cells at the advancing front of a wound edge tend to migrate collectively, elongate substantially, and exert tractions more forcefully compared with cells many ranks behind. With regards to energy metabolism, striking spatial gradients have recently been reported in the wounded epithelium, as well as in the tumor, but within the wounded cell layer little is known about the link between mechanical events and underlying energy metabolism. Using the advancing confluent monolayer of MDCKII cells as a model system, here we report at single cell resolution the evolving spatiotemporal fields of cell migration speeds, cell shapes, and traction forces measured simultaneously with fields of multiple indices of cellular energy metabolism. Compared with the epithelial layer that is unwounded, which is non-migratory, solid-like and jammed, the leading edge of the advancing cell layer is shown to become progressively more migratory, fluid-like, and unjammed. In doing so the cytoplasmic redox ratio becomes progressively smaller, the NADH lifetime becomes progressively shorter, and the mitochondrial membrane potential and glucose uptake become progressively larger. These observations indicate that a metabolic shift toward glycolysis accompanies collective cellular migration but show, further, that this shift occurs throughout the cell layer, even in regions where associated changes in cell shapes, traction forces, and migration velocities have yet to penetrate. In characterizing the wound healing process these morphological, mechanical, and metabolic observations, taken on a cell-by-cell basis, comprise the most comprehensive set of biophysical data yet reported. Together, these data suggest the novel hypothesis that the unjammed phase evolved to accommodate fluid-like migratory dynamics during episodes of tissue wound healing, development, and plasticity, but is more energetically expensive compared with the jammed phase, which evolved to maintain a solid-like non-migratory state that is more energetically economical.

Functional Ultrasound Speckle Decorrelation-Based Velocimetry of the Brain.

A high-speed, contrast-free, quantitative ultrasound velocimetry (vUS) for blood flow velocity imaging throughout the rodent brain is developed based on the normalized first-order temporal autocorrelation function of the ultrasound field signal. vUS is able to quantify blood flow velocity in both transverse and axial directions, and is validated with numerical simulation, phantom experiments, and in vivo measurements. The functional imaging ability of vUS is demonstrated by monitoring the blood flow velocity changes during whisker stimulation in awake mice. Compared to existing Power-Doppler- and Color-Doppler-based functional ultrasound imaging techniques, vUS shows quantitative accuracy in estimating both axial and transverse flow speeds and resistance to acoustic attenuation and high-frequency noise.

Chronic Imaging of Mouse Brain: From Optical Systems to Functional Ultrasound.

Utilization of functional ultrasound (fUS) in cerebral vascular imaging is gaining popularity among neuroscientists. In this article, we describe a chronic surgical preparation method that allows longitudinal studies and therefore is applicable to a wide range of studies, especially on aging, stroke, and neurodegenerative diseases. This method can also be used with awake mice; hence, the deleterious effects of anesthesia on neurovascular responses can be avoided. In addition to fUS imaging, this surgical preparation allows researchers to take advantage of common optical imaging methods to acquire complementary datasets to help increase the technical rigor of studies. © 2020 Wiley Periodicals LLC. Basic Protocol 1: Surgical preparation of mouse chronic cranial windows using polymethylpentene Basic Protocol 2: Imaging of mice with chronic cranial windows.

Constraining Axion Inflation with Gravitational Waves across 29 Decades in Frequency.

We demonstrate that gravitational waves generated by efficient gauge preheating after axion inflation generically contribute significantly to the effective number of relativistic degrees of freedom N_{eff}. We show that, with existing Planck limits, gravitational waves from preheating already place the strongest constraints on the inflaton's possible axial coupling to Abelian gauge fields. We demonstrate that gauge preheating can completely reheat the Universe regardless of the inflationary potential. Further, we quantify the variation of the efficiency of gravitational wave production from model to model and show that it is correlated with the tensor-to-scalar ratio. In particular, when combined with constraints on models whose tensor-to-scalar ratios would be detected by next-generation cosmic microwave background experiments, r≳10^{-3}, constraints from N_{eff} will probe or rule out the entire coupling regime for which gauge preheating is efficient.

Nonlinear Dynamics of Preheating after Multifield Inflation with Nonminimal Couplings.

We study the postinflation dynamics of multifield models involving nonminimal couplings using lattice simulations to capture significant nonlinear effects like backreaction and rescattering. We measure the effective equation of state and typical timescales for the onset of thermalization, which could affect the usual mapping between predictions for primordial perturbation spectra and measurements of anisotropies in the cosmic microwave background radiation. For large values of the nonminimal coupling constants, we find efficient particle production that gives rise to nearly instantaneous preheating. Moreover, the strong single-field attractor behavior that was previously identified persists until the end of preheating, thereby suppressing typical signatures of multifield models. We therefore find that predictions for primordial observables in this class of models retain a close match to the latest observations.

Departures from the Friedmann-Lemaitre-Robertston-Walker Cosmological Model in an Inhomogeneous Universe: A Numerical Examination.

While the use of numerical general relativity for modeling astrophysical phenomena and compact objects is commonplace, the application to cosmological scenarios is only just beginning. Here, we examine the expansion of a spacetime using the Baumgarte-Shapiro-Shibata-Nakamura formalism of numerical relativity in synchronous gauge. This work represents the first numerical cosmological study that is fully relativistic, nonlinear, and without symmetry. The universe that emerges exhibits an average Friedmann-Lemaître-Robertson-Walker (FLRW) behavior; however, this universe also exhibits locally inhomogeneous expansion beyond that expected in linear perturbation theory around a FLRW background.

Preheating with nonminimal kinetic terms.

We present the first (3+1)-dimensional numerical simulations of scalar fields with nonminimal kinetic terms. As an example, we examine the existence and stability of preheating in the presence of a Dirac-Born-Infeld inflaton coupled to a canonical matter field. The simulations represent the full nonlinear theory in the presence of an expanding universe. We show that parametric resonance in the matter field along with self-resonance in the inflaton repopulate the universe with matter particles as efficiently as in traditional preheating.

Grand unification scale primordial black holes: consequences and constraints.

A population of very light primordial black holes which evaporate before nucleosynthesis begins is unconstrained unless the decaying black holes leave stable relics. We show that gravitons Hawking radiated from these black holes would source a substantial stochastic background of high frequency gravititational waves (10(12) Hz or more) in the present Universe. These black holes may lead to a transient period of matter-dominated expansion. In this case the primordial Universe could be temporarily dominated by large clusters of "Hawking stars" and the resulting gravitational wave spectrum is independent of the initial number density of primordial black holes.

Gravitational wave production at the end of inflation.

We consider gravitational wave production due to parametric resonance at the end of inflation, or "preheating." This leads to large inhomogeneities that source a stochastic background of gravitational waves at scales inside the comoving Hubble horizon at the end of inflation. We confirm that the present amplitude of these gravitational waves need not depend on the inflationary energy scale. We analyze an explicit model where the inflationary energy scale is approximately 10{9} GeV, yielding a signal close to the sensitivity of Advanced Laser Interferometer Gravitational Wave Observatory and Big Bang Observer. This signal highlights the possibility of a new observational "window" into inflationary physics and provides significant motivation for searches for stochastic backgrounds of gravitational waves in the Hz to GHz range, with an amplitude on the order of Omega_{gw}(k)h{2} approximately 10{-11}.